Regulators of methyl modification enzymes, compositions and uses thereof

Abstract

Provided are novel compounds of formula (I): and pharmaceutically acceptable salts thereof, which can be used to treat various diseases, disorders or conditions associated with methyl modification enzymes. Also provided are pharmaceutical compositions comprising the novel compounds of formula (I), pharmaceutically acceptable salts thereof, and methods for using the pharmaceutical compositions to treat one or more diseases, disorders or conditions associated with methyl modification enzymes.

Description

[0001] Related Applications

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 659,408, filed on April 18, 2018, the contents of which are incorporated herein by reference. Background Art

[0003] 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 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.

[0004] 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.

[0005] 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).

[0006] 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

[0007] It has been found that the compounds described herein and pharmaceutically acceptable compositions thereof can modulate the activity of EZH2 (see, e.g., Table 5). These compounds include compounds of structural formula I:

[0008]

[0009] or a pharmaceutically acceptable salt thereof, wherein R 1 , R 2 , R 3 , R 4 , R 5 and X are each as defined herein.

[0010] In one aspect, certain compounds described herein have also been found to have increased residence time (e.g., >100 hours) and enhanced permeability. Increases in residence time (i.e., slower off-rates) and / or permeability have been found to correlate with increased cellular potency.

[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 halogen, -S(C1-C4)alkyl, -S(C3-C7)cycloalkyl or -S[halo(C1-C4)alkyl];

[0017] X is CH or N;

[0018] R 2 is hydrogen, halogen, (C1-C4)alkyl or halogenated (C1-C4)alkyl;

[0019] R 3 is halogen, (C1-C4)alkyl or halogenated (C1-C4)alkyl;

[0020] R 4 is (C3-C7)cycloalkyl or 4 to 7 membered heterocyclyl, each of which is optionally substituted by 1 to 3 groups selected from halogen, (C1-C4)alkyl, halo(C1-C4)alkyl, (C1-C4)alkoxy, halo(C1-C4)alkoxy and -NR a R b The group substitution;

[0021] R a is hydrogen, (C1-C4)alkyl or halogenated (C1-C4)alkyl;

[0022] R b is (C1-C4)alkyl, halo(C1-C4)alkyl or 4- to 7-membered heterocyclyl, wherein the heterocyclyl is optionally substituted by 1 to 3 groups selected from halogen, (C1-C4)alkyl, halo(C1-C4)alkyl, (C1-C4)alkoxy and halo(C1-C4)alkoxy; or

[0023] R a and R b Together with the nitrogen atom to which it is attached, it forms a radical optionally substituted by 1 to 3 radicals selected from halogen, (C1-C4)alkyl, halo(C1-C4)alkyl and -OR c A 4- to 7-membered heterocyclic group substituted with a group;

[0024] R c is (C1-C4)alkyl, halo(C1-C4)alkyl or (C3-C7)cycloalkyl; and

[0025] R 5 It is halogen, (C1-C4)alkyl or halogenated (C1-C4)alkyl.

[0026] 2. definition

[0027] When used to describe a chemical group that may have multiple points of attachment, the hyphen (-) indicates the point of attachment of this group to the variable defining it. For example, -S(C3-C7)cycloalkyl and -S[halo(C1-C4)alkyl] indicate that the point of attachment of this group is at the sulfur atom.

[0028] As used herein, the term "halo" or "halogen" refers to an atom selected from fluorine (fluoro, -F), chlorine (chloro, -Cl), bromine (bromo, -Br) and iodine (iodo, -I).

[0029] As used herein, the term "alkyl" refers to a monovalent saturated, straight or branched hydrocarbon radical having 1 to 10 carbon atoms, unless otherwise specified. Examples of alkyl radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, sec-pentyl, isopentyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, sec-hexyl, and the like.

[0030] The term "haloalkyl" embraces monohaloalkyl, polyhaloalkyl and perhaloalkyl wherein the halogen is independently selected from fluorine, chlorine, bromine and iodine.

[0031] "Alkoxy" is an alkyl group attached to another moiety through an oxygen linker (-O(alkyl)). Non-limiting examples include methoxy, ethoxy, propoxy, and butoxy.

[0032] "Haloalkoxy" is a haloalkyl group attached to another moiety through an oxygen atom such as, for example but not limited to, -OCHCF2 or -OCF3.

[0033] The term "cycloalkyl" refers to a fully saturated 3 to 12 yuan (e.g., 3 to 7 yuan) monocyclic, bicyclic (e.g., bridged or spirocyclic bicyclic) or polycyclic (e.g., tricyclic) hydrocarbon ring system. Monocyclic cycloalkyl includes, but is not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. Bridged bicyclic cycloalkyl includes, but is not limited to, bicyclo [3.2.1] octane, bicyclo [2.2.1] heptane, bicyclo [3.1.0] hexane, bicyclo [1.1.1] pentane, etc. Spiro bicyclic cycloalkyl includes, for example, spiro [3.6] decane, spiro [4.5] decane, etc. Fused cycloalkyl rings include, for example, decahydronaphthalene, octahydrocycloolefin, etc. When specified as optionally substituted or substituted, the substituent on the cycloalkyl (e.g., in the case of an optionally substituted cycloalkyl) can be present in any substitutable position, and includes, for example, the position to which the cycloalkyl is attached.

[0034] The term "heterocyclyl" refers to a 3 to 12-membered (e.g., 4-membered, 5-membered, 6-membered, and 7-membered) saturated or partially unsaturated heterocyclic ring containing 1 to 4 heteroatoms independently selected from N, O, and S. It can be monocyclic, bicyclic (e.g., bridged, fused, or spirocyclic bicyclic) or tricyclic. The terms "heterocycle", "heterocyclyl", "heterocyclyl ring", "heterocyclyl", "heterocyclic moiety", and "heterocyclic radical" are used interchangeably herein. The heterocyclyl ring can be attached to its side group at any heteroatom or carbon atom that forms a stable structure. Examples of such saturated heterocyclic radicals or partially unsaturated heterocyclic radicals include, but are not limited to, tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, pyrrolidinyl, pyridyl, pyrrolidinyl, piperidinyl, oxazolidinyl, piperazinyl, dioxacycloalkyl, dioxolane, morpholinyl, dihydrofuranyl, dihydropyranyl, dihydropyridyl, tetrahydropyridyl, dihydropyrimidinyl, oxetanyl, azetidinyl and tetrahydropyrimidinyl. The heterocyclic radical can be monocyclic or bicyclic. The term "heterocyclic radical" also includes, for example, an unsaturated heterocyclic radical fused to another unsaturated heterocyclic radical or an aryl or heteroaryl ring, such as tetrahydronaphthyridine, indolinone, dihydropyrrolotriazole, imidazopyrimidine, quinolinone, dioxospirane. When designated as optionally substituted or unsubstituted, substituents on the heterocyclyl (eg, 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.

[0035] The term "spirocycle" refers to two rings that share one ring atom (eg, carbon).

[0036] The term "fused" refers to two rings that share two adjacent ring atoms with each other.

[0037] The term "bridged" refers to two rings that share three or more ring atoms with each other.

[0038] As used herein, "geometric isomer" refers to isomers that differ in the orientation of substituent atoms relative to the cycloalkyl ring, i.e., cis-isomers 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 includes 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.

[0039] 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 enantiomer depicted or named 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.

[0040] Unless otherwise indicated, when a disclosed compound is named or depicted by a structure without indication of stereochemistry, and the compound has at least one chiral center or at least one geometric isomer, or both, the name or structure encompasses one enantiomer or geometric isomer of the compound free of the corresponding optical isomer or geometric isomer, racemic mixtures of the compounds, and mixtures enriched in one enantiomer or geometric isomer relative to its corresponding optical isomer or geometric isomer.

[0041] Unless otherwise indicated, when only some stereochemical centers in a disclosed compound are described or named according to the structure, the described or named configuration is enriched, for example, by at least 60%, 70%, 80%, 90%, 99% or 99.9% molar excess relative to the remaining configurations. For example, the following structure:

[0042] This means that the configuration of the chiral carbon is stereochemically enriched in R (e.g., enriched in at least 60%, 70%, 80%, 90%, 99% or 99.9% molar excess), and the geometry of the cyclohexyl group can be cis or trans, or a mixture thereof.

[0043] In some cases, disclosed compounds are described by structure without indicating stereochemistry, but are identified as stereochemically enriched (e.g., "single enantiomer; single geometric isomer" or "single enantiomer" where geometric isomers are not possible). For example, unless otherwise indicated

[0044] Indicates that the configuration of the chiral dioxolane carbon is stereochemically enriched in R (e.g., enriched in at least 60%, 70%, 80%, 90%, 99%, or 99.9% molar excess) or is stereochemically enriched in S (e.g., enriched in at least 60%, 70%, 80%, 90%, 99%, or 99.9% molar excess), and the geometry of the cyclohexyl ring is enriched in cis (e.g., enriched in at least 60%, 70%, 80%, 90%, 99%, or 99.9% molar excess) or is enriched in trans (e.g., enriched in at least 60%, 70%, 80%, 90%, 99%, or 99.9% molar excess).

[0045] 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.

[0046] 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.

[0047] 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.

[0048] Disease, disorder and condition are used interchangeably herein.

[0049] 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.

[0050] The terms "inhibit", "inhibition" and "inhibiting" encompass a decrease in the baseline activity of a biological activity or process.

[0051] 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).

[0052] 3. Description of Exemplary Compounds

[0053] In some embodiments, the present disclosure provides a compound of Formula I:

[0054]

[0055] or a pharmaceutically acceptable salt thereof, wherein the variables are the same as those of Formula I above.

[0056] In a second embodiment, in the compound of formula I,

[0057] R 1 is halogen or -S(C1-C4)alkyl;

[0058] R 2 It is a halogen;

[0059] R 3 is (C1-C4)alkyl;

[0060] R 4 is (C3-C7)cycloalkyl or (C4-C7)heterocyclyl, each of which is optionally substituted by 1 to 2 groups selected from halo(C1-C4)alkyl and -NR a R b The group substitution;

[0061] R a is hydrogen or (C1-C4)alkyl;

[0062] R b is (C1-C4)alkyl or (C4-C7)heterocyclyl, wherein the heterocyclyl is optionally substituted with halo(C1-C4)alkyl; or

[0063] R a and R b Together with the nitrogen atom to which it is attached, it forms a cA 4- to 7-membered nitrogen-containing heterocyclic group substituted with a group;

[0064] R c is (C1-C4)alkyl, halo(C1-C4)alkyl or (C3-C7)cycloalkyl; and

[0065] R 5 It is halogen or (C1-C4) alkyl.

[0066] In a third embodiment, the compound of formula I has formula II:

[0067]

[0068] or a pharmaceutically acceptable salt thereof, wherein the remaining variables are the same as described above for Formula I or the Second Example.

[0069] In a fourth embodiment, R in the compound of Formula I or Formula II is 1 is chloro, wherein the remaining variables are as described above for Formula I or the Second Example.

[0070] In a fifth embodiment, R in the compound of Formula I or Formula II 1 is -SCH3, wherein the remaining variables are the same as described above for Formula I or the second embodiment.

[0071] In a sixth embodiment, R in the compound of Formula I or Formula II 4 is cyclohexyl or piperidinyl, each of which is optionally substituted by 1 to 2 groups selected from halo(C1-C4)alkyl and -NR a R b wherein the remaining variables are as described above for Formula I or the second, fourth or fifth embodiment.

[0072] In a seventh embodiment, the compound of Formula I or Formula II has Formula III:

[0073]

[0074] or a pharmaceutically acceptable salt thereof, wherein R 6 is halo(C1-C4)alkyl, and wherein the remaining variables are as described above for Formula I or the second, fourth or fifth embodiment.

[0075] In an eighth embodiment, the compound of Formula I, Formula II or Formula III has Formula IV:

[0076]

[0077] or a pharmaceutically acceptable salt thereof, wherein the remaining variables are the same as described above for Formula I or the second, fourth, fifth or seventh embodiment.

[0078] In a ninth embodiment, the compound of Formula I or Formula II has Formula V:

[0079]

[0080] or a pharmaceutically acceptable salt thereof, wherein the remaining variables are the same as described above for Formula I or the second, fourth or fifth embodiment.

[0081] In a tenth embodiment, the compound of Formula I, Formula II or Formula V has Formula VI or Formula VII:

[0082]

[0083]

[0084] or a pharmaceutically acceptable salt thereof, wherein the remaining variables and features are the same as described above for Formula I or the second, fourth or fifth embodiment.

[0085] In an eleventh embodiment, Rb in the compounds of Formula I, Formula II, Formula V, Formula VI and Formula VII is (C1-C4)alkyl or oxetanyl, wherein the oxetanyl is optionally substituted with a halo (C1-C4)alkyl group; or R a and R b Together with the nitrogen atom to which it is attached, it forms a c Substituted azetidinyl wherein the remainder of the variables and features are as described above for Formula I or the second, fourth or fifth embodiment.

[0086] In the twelfth embodiment, R in the compounds of Formula I, Formula II, Formula V, Formula VI and Formula VII a is hydrogen or methyl; and R b is methyl or oxetanyl, wherein the oxetanyl is optionally substituted with -CH2F or -CF3, and wherein the remaining variables and features are the same as described above for Formula I or the second, fourth, fifth or seventh embodiment.

[0087] In the thirteenth embodiment, R in the compounds of Formula I, Formula II, Formula V, Formula VI and Formula VII a and R b Together with the nitrogen atom to which it is attached, it forms an optionally fluorinated or -OR c substituted azetidinyl; and R cis -CH3, -CHF2 or cyclopropyl, wherein the remaining variables and features are the same as described above for Formula I or the second, fourth, fifth or eleventh embodiment.

[0088] In a fourteenth embodiment, the 1,3-dioxolanyl and NR a R b The configuration of the radical is trans-oriented around the cyclohexyl group, wherein the remaining variables and features are the same as described above for Formula I or the second, fourth, fifth, sixth, eleventh, twelfth or thirteenth embodiment. Alternatively, the 1,3-dioxolanyl and NR a R b The radical configuration is cis-oriented about the cyclohexyl group, with the remaining features being the same as described above for Formula I or the second, fourth, fifth, sixth, eleventh, twelfth or thirteenth embodiment.

[0089] In a fifteenth embodiment, the stereochemical configuration of the chiral center of the 1,3-dioxolanyl group of any one of Formula I, Formula II, Formula III, Formula IV, Formula V, Formula VI, and Formula VII is R, wherein the remaining features are as described above for Formula I or the second, fourth, fifth, sixth, eleventh, twelfth, thirteenth, or fourteenth embodiment. Alternatively, the stereochemical configuration of the chiral center of the 1,3-dioxolanyl group of any one of Formula I, Formula II, Formula III, Formula IV, Formula V, Formula VI, and Formula VII is S, wherein the remaining features are as described above for Formula I or the second, fourth, fifth, sixth, eleventh, twelfth, thirteenth, or fourteenth embodiment.

[0090] Specific examples of compounds are provided in the Examples section and are included herein as part of the fourteenth embodiment. Pharmaceutically acceptable salts and neutral forms of these compounds are also included. In one aspect, the present disclosure includes racemic forms of any compound described herein.

[0091] 4. Use, preparation and application:

[0092] 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.

[0093] 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.

[0094] In some embodiments, the composition is administered orally.

[0095] 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 compound described herein in the composition will also depend on the specific compound in the composition.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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, cartilaginous hamartoma, 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. ;

[0102] 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, 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 Tumor, 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, rhabdomyoma, rhabdomyosarcoma, Richter's transformation (Richter's transformation), colorectal 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, pseudomyxoma peritonei, renal cell carcinoma, renal medullary carcinoma, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter's transformation, colorectal 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 tumorWilms' tumor.

[0103] 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.

[0104] 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.

[0105] Also provided is the use of a compound described herein or a pharmaceutically acceptable salt thereof or a composition comprising a 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 a disclosed compound or a pharmaceutically acceptable salt thereof for treating a condition described herein.

[0106] example

[0107] The following representative examples are intended to help illustrate the invention and are not intended to, and should not be construed to, limit the scope of the invention.

[0108] Preparation of intermediates

[0109] Intermediate 1: 3-(Aminomethyl)-6-methyl-4-(methylthio)pyridin-2(1H)-one (hydrochloride)

[0110]

[0111] Step 1: Synthesis of sodium 3-oxobut-1-ene-1,1-bis(thioate)

[0112] A mixture of sodium tert-butoxide (16.6 g, 172 mmol) in toluene (30 mL) was degassed under vacuum and purged with nitrogen (3 cycles). Acetone (5.0 g, 6.4 mL, 86 mmol) was then added at 0 ° C, followed by the slow addition of carbon disulfide (6.6 g, 5.24 mL, 86 mmol). The resulting mixture was stirred at 0 ° C for 4 hours and then filtered. The filter cake was dried under vacuum to give the title compound (15.4 g, crude product) as a yellow solid, which was used in the next step without further purification.

[0113] Step 2: Synthesis of 4,4-bis(methylthio)but-3-en-2-one

[0114] Iodomethane (24.5 g, 10.7 mL, 173 mmol) was slowly added to a methanol (90 mL) solution of sodium 3-oxobut-1-ene-1,1-bis(thioate) (15.4 g, 86.4 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).

[0115] Step 3: Synthesis of 6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridine-3-carbonitrile

[0116] 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, 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. 1H NMR (400 MHz, dimethyl sulfoxide-d6) δ 6.27 (s, 1H), 2.56 (s, 3H), 2.25 (s, 3H).

[0117] Step 4: Synthesis of 3-(aminomethyl)-6-methyl-4-(methylthio)pyridin-2(1H)-one

[0118] 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 before the reaction mixture was warmed to 70 ° C and stirred for 2 hours. Methanol (15 mL) was slowly added at 0 ° C before the mixture was concentrated under reduced pressure 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.

[0119] Step 5: Synthesis of tert-butyl ((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)carbamate

[0120] Triethylamine (5.9 g, 8.1 mL, 59 mmol) was added to a solution of 3-(aminomethyl)-6-methyl-4-(methylthio)pyridin-2(1H)-one (3.6 g, 20 mmol) in tetrahydrofuran (80 mL). The mixture was stirred for 30 minutes, then di-tert-butyl dicarbonate (6.4 g, 29 mmol) was added, and the reaction was stirred at 25 ° C for 12 hours. 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 ethyl 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-d6) δ 6.05 (s, 1H), 4.03-4.00 (m, 2H), 2.42 (s, 3H), 2.15 (s, 3H), 1.39 (s, 9H).

[0121] Step 6: Synthesis of 3-(aminomethyl)-6-methyl-4-(methylthio)pyridin-2(1H)-one (hydrochloride)

[0122] 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 (400MHz, D2O) δ6.31(s,1H),4.03(s,2H),2.41(s,3H),2.18(s,3H).

[0123] Intermediate 2: 5-(Aminomethyl)-2-methyl-6-(methylthio)pyrimidin-4(3H)-one (hydrochloride)

[0124]

[0125] Step 1: Synthesis of methyl 2-cyano-3,3-bis(methylthio)acrylate

[0126] Before dropwise adding methyl cyanoacetate (8.90mL, 101mmol), the suspension of sodium hydride (60% dispersion in mineral oil) (4.23g, 106mmol) in tetrahydrofuran (400mL) was cooled to 0 ℃. The reaction mixture was stirred vigorously for 15 minutes, then dropwise added carbon disulfide (6.1mL, 101mmol), while keeping the reaction temperature lower than 20 ℃ (white semi-solid becomes yellow). Then drip iodomethane (15.7mL, 252mmol), and the reaction mixture was at room temperature stirred for 15 minutes. Then under reduced pressure concentrated reaction and residue was poured into ice water. Filter gained precipitation, vacuum drying, obtain the title compound (11.9g, productive rate is 58%) of yellow / brown solid form, it is used for next step without further purification. 1 H NMR (400 MHz, CHLOROFORM-d) δ 3.83 (s, 3H), 2.76 (s, 3H), 2.61 (s, 3H).

[0127] Step 2: Synthesis of 2-methyl-4-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile

[0128] Methyl 2-cyano-3,3-bis(methylthio)acrylate (11.9 g, 58.5 mmol) was dissolved in ethanol (1000 mL). Acetamide hydrochloride (5.53 g, 58.5 mmol) and triethylamine (36.7 mL, 263 mmol) were added to the mixture and stirred overnight under reflux. The reactants were then concentrated under reduced pressure and the residue was washed with 10% hydrochloric acid. The precipitate was filtered, the filter cake was washed with water and ether, and then dried under vacuum to give the title compound (5 g, 47% yield) in the form of a solid, which was used in the next step without further purification. LCMS [M + H] + m / z: calculated: 182.03; found: 182.10. 1 H NMR (400 MHz, dimethyl sulfoxide-d6) δ 2.57 (s, 3H), 2.37 (s, 3H).

[0129] Step 3: Synthesis of tert-butyl ((2-methyl-4-(methylthio)-6-oxo-1,6-dihydropyrimidin-5-yl)methyl)carbamate

[0130] To a suspension of 2-methyl-4-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile (1.5 g, 8.27 mmol) in tetrahydrofuran (50 mL) at 0°C was added a solution of lithium aluminum hydride in tetrahydrofuran (2 M, 6.2 mL, 12.4 mmol). The reaction was slowly warmed to room temperature and immediately quenched with an aqueous solution of sodium hydroxide (1 M, 33 mL, 33.1 mmol). To this mixture were added 1,4-dioxane (50 mL) and di-tert-butyl dicarbonate (3.61 g, 16.6 mmol) in sequence. The biphasic mixture was stirred vigorously until the amino group was fully protected. The biphasic mixture was acidified to neutral pH with acetic acid, separated, and the aqueous layer was extracted with dichloromethane. The combined organic layers were dried over magnesium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was triturated with dichloromethane, and the filtrate (containing the desired product) was concentrated to dryness under reduced pressure. The resulting solid was purified by reverse phase flash chromatography (C18 column, gradient from 5% to 95% acetonitrile in water, buffered with 0.1% trifluoroacetic acid) and then by normal phase flash chromatography (silica gel, gradient from 30% to 90% ethyl acetate in dichloromethane) to afford the title compound as a white solid (2.57 g, 26% yield, purity >99%). LCMS [M-(C4H8)+H] + m / z: calculated: 230.1; found: 230.0. 1H NMR (400 MHz, CHLOROFORM-d) δ 13.20 (s, 1H), 5.37 (t, J = 5.5 Hz, 1H), 4.26 (d, J = 5.6 Hz, 2H), 2.51 (s, 3H), 2.44 (s, 3H), 1.44 (s, 9H).

[0131] Step 4. Synthesis of 5-(aminomethyl)-2-methyl-6-(methylthio)pyrimidin-4(3H)-one (hydrochloride)

[0132] To a suspension of tert-butyl ((2-methyl-4-(methylthio)-6-oxo-1,6-dihydropyrimidin-5-yl)methyl)carbamate (300 mg, 1.05 mmol) in 1,4-dioxane (7.2 mL) at 23°C was added a solution of hydrogen chloride in 1,4-dioxane (4M, 2.10 mL, 8.40 mmol). The reaction was stirred at 23°C for 3 hours, then more hydrogen chloride in 1,4-dioxane (4M, 2.10 mL, 8.40 mmol) was added. After 14 hours, the reaction was concentrated to dryness under reduced pressure to give the title compound (254 mg, 94% yield) as a white solid, which was used in the next step without further purification. LCMS [M+Na] + (Free base) m / z: calculated: 208.1; found: 208.1.

[0133] Intermediate 3: 3-(Aminomethyl)-6-chloro-4-(methylthio)pyridin-2(1H)-one (hydrochloride)

[0134]

[0135] Step 1: Synthesis of 2-(benzyloxy)-6-chloronicotinic acid

[0136] To a suspension of sodium hydride (60% mineral oil dispersion) (11.5 g, 286 mmol) in tetrahydrofuran (500 mL) at 0°C was added benzyl alcohol (14 mL, 143 mmol). The reaction mixture was stirred for 20 minutes, then a solution of 2,6-dichloropyridine-3-carboxylic acid (25 g, 130 mmol) in tetrahydrofuran (500 mL) was added, and the reaction mixture was stirred at 23°C for 4 hours. The reaction was then quenched with 10% hydrochloric acid (300 mL), treated with solid NaHCO3 to a pH of about 8, acidified with acetic acid (50% acetic acid in water), and finally extracted with ethyl acetate (200 mL×3). The combined organic layers were washed with brine (200 mL), dried over magnesium sulfate, filtered, and concentrated to dryness. The residue was purified by flash chromatography (silica gel, gradient from 5% to 50% ethyl acetate in dichloromethane) to give the title compound (33.68 g, 98% yield).

[0137] Step 2: Synthesis of 2-(benzyloxy)-6-chloronicotinamide

[0138] To a 0°C solution of 2-(benzyloxy)-6-chloronicotinic acid (33.68 g, 128.2 mmol) in dichloromethane (500 mL) was added N,N-dimethylformamide (1 mL) followed by oxalyl chloride (11.9 mL, 141 mmol). The reactants were stirred at 23°C for 2 hours and then concentrated to dryness. The residue was dissolved in dichloromethane (200 mL) and slowly added to a concentrated solution of ammonium hydroxide (50 mL) at 0°C. After 1 hour, the organic layer was collected and the aqueous phase was extracted with dichloromethane (50 mL×3). The combined organic layers were dried over magnesium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by flash chromatography (silica gel, gradient of 0% to 50% ethyl acetate in dichloromethane) to give the title compound as a solid (quantitative yield).

[0139] Step 3: Synthesis of 2-(benzyloxy)-6-chloro-4-(methylthio)nicotinamide

[0140] To a solution of 2,2,6,6-tetramethylpiperidine (29.45 mL, 175.1 mmol) in tetrahydrofuran (300 mL) at -78°C was added a solution of n-butyllithium in hexane (2.5 M, 70 mL, 175.1 mmol). The reaction mixture was warmed to 0°C and maintained for 30 minutes, then cooled to -78°C. A solution of 2-(benzyloxy)-6-chloronicotinamide (11.5 g, 43.8 mmol) in tetrahydrofuran (100 mL) was then added, while maintaining the internal temperature below -60°C. The reaction mixture was stirred at -78°C for 1 hour, then dimethyl disulfide (15.77 mL, 175.1 mmol) was added. The reaction was stirred at -78°C for an additional 2 hours, then quenched with an aqueous solution of acetic acid (50% v / v) at -78°C. The desired product was extracted with ethyl acetate (100 mL x 3), and the combined organic layers were washed with water (20 mL), saturated aqueous sodium bicarbonate solution (20 mL), brine (20 mL), dried over magnesium sulfate, filtered and concentrated to dryness under reduced pressure. The residue was purified by flash chromatography (silica gel, gradient of 0% to 20% ethyl acetate in dichloromethane) to give the title compound (4.86 g, 36% yield).

[0141] Step 4: Synthesis of (2-(benzyloxy)-6-chloro-4-(methylthio)pyridin-3-yl)methanamine

[0142] To a solution of 2-(benzyloxy)-6-chloro-4-(methylthio)nicotinamide (4.86 g, 15.7 mmol) in tetrahydrofuran (100 mL) at 23°C was added a solution of borane in tetrahydrofuran (1 M, 63 mL, 63 mmol). The reaction mixture was stirred at reflux for 15 hours and then quenched by the slow addition of methanol at room temperature. The reaction mixture was concentrated to dryness under reduced pressure and the residue was purified by flash chromatography (silica gel, gradient of 0% to 30% methanol in dichloromethane) to give the title compound (710 mg, 15% yield) as a white solid.

[0143] Step 5: Synthesis of 3-(aminomethyl)-6-chloro-4-(methylthio)pyridin-2(1H)-one (hydrochloride)

[0144] To a flask containing (2-(benzyloxy)-6-chloro-4-(methylthio)pyridin-3-yl)methanamine (710 mg, 2.41 mmol) was added concentrated hydrochloric acid (12 M, 5.0 mL, 60 mmol). The reaction mixture was stirred at 23 ° C for 2 hours and then concentrated to dryness under reduced pressure. The residue was recrystallized from a mixture of methanol and diethyl ether to give the title compound as a white solid (400 mg, 69% yield).

[0145] Step 6: Synthesis of tert-butyl ((6-chloro-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)carbamate

[0146] To a suspension of 3-(aminomethyl)-6-chloro-4-(methylthio)pyridin-2(1H)-one hydrochloride (1.575 g, 6.53 mmol) in dichloromethane (50 mL) was added di-tert-butyl dicarbonate (2.85 g, 13.1 mmol) at room temperature followed by N,N-diisopropylethylamine (3.4 mL, 19.6 mmol). The reaction was stirred at room temperature for 3 hours (until all material dissolved) and then quenched with water. The organic layer was separated, dried over magnesium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by flash chromatography (silica gel, gradient of 0% to 10% methanol in dichloromethane) to give the title compound as a white solid (1.5 g, 75% yield). LCMS [M+H] + m / z: calculated: 305.1; found: 305.1.

[0147] Step 7: Synthesis of 3-(aminomethyl)-6-chloro-4-(methylthio)pyridin-2(1H)-one (hydrochloride)

[0148] To a suspension of tert-butyl ((6-chloro-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)carbamate (300 mg, 984 μmol) in 1,4-dioxane (4.9 mL) was added a solution of hydrogen chloride in 1,4-dioxane (4M, 1.96 mL, 7.87 mmol) at room temperature. The reaction was stirred at room temperature for 3 hours, then more hydrogen chloride in 1,4-dioxane (4M, 1.96 mL, 7.87 mmol) was added. After 14 hours, the reaction was concentrated to dryness under reduced pressure to give the title compound (253 mg, quantitative yield) as a white solid, which was used in the next step without further purification. LCMS [M+Na] + (Free base) m / z: Calculated: 227.0; Found: 227.0.

[0149] Intermediate 4: 7-chloro-2,4-dimethyl-2-(4-oxocyclohexyl)benzo[d][1,3]dioxole-5-carboxylic acid methyl ester

[0150]

[0151] Step 1: Synthesis of methyl 5-chloro-3,4-dihydroxy-2-methylbenzoate

[0152] To a solution of methyl 3,4-dihydroxy-2-methylbenzoate (5.11 g, 27.9 mmol) in tetrahydrofuran (199 mL) at -20°C was added sulfonyl chloride (2.45 mL, 30.6 mmol) dropwise. 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×3). The combined organic layers were washed with brine (25 mL), dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, gradient of 0% to 60% ethyl acetate 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).

[0153] Step 2: Synthesis of methyl 7-chloro-2,4-dimethyl-2-(4-oxocyclohexyl)-2H-1,3-benzodioxole-5-carboxylate

[0154] A mixture of 5-chloro-3,4-dihydroxy-2-methylbenzoic acid methyl ester (1.2g, 5.53mmol), triruthenium dodecacarbonyl (176mg, 276μmol) and triphenylphosphine (145mg, 553μmol) was degassed under vacuum and purged with nitrogen (3 cycles). Toluene (8.1mL) was added and the reaction mixture was heated to reflux for 30 minutes. A solution of 4-ethynylcyclohexane-1-one (1.34g, 11.0mmol) in toluene (17mL) was then added dropwise, and the reactants were 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, gradient of heptane containing 0% to 60% ethyl acetate) to obtain the title compound (1.327g, 70% yield) in the form of a yellow oil. LCMS[M+Na] + m / z: calculated: 361.1; found: 361.1 (Cl isotope pattern).

[0155] Step 3: Separation of methyl (R)-7-chloro-2,4-dimethyl-2-(4-oxocyclohexyl)benzo[d][1,3]dioxole-5-carboxylate and methyl (S)-7-chloro-2,4-dimethyl-2-(4-oxocyclohexyl)benzo[d][1,3]dioxole-5-carboxylate

[0156] The 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 dissolved by preparative SFC [column: ChiralPak AY (250 mm×50 mm ID, 10 μm) from Daicel Chemical Industries. Mobile phase A: CO2 / mobile phase B: 0.1% NH4OH in methanol. Isocratic (85% mobile phase A and 15% mobile phase B). Flow rate: 80 ml / min. Column temperature: 40°C]. Intermediate 4 (peak 1) (unwanted enantiomer / minor enantiomer): retention time = 6.2 minutes. 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 4 (peak 2) (desired enantiomer / isomeric): retention time = 7.0 minutes. Recovery = 1.1 g, 3.08 mmol, yield 23.75%, 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 analysis: [Column: ChiralPak AY-3 (150×4.6 mm ID, 3 μm). Mobile phase A: CO2 / mobile phase B: 0.05% iPrOH containing Et2NH. Gradient: 5% to 40% mobile phase B (over 5.5 minutes). Flow rate: 2.5 ml / min. Column temperature: 40°C]. Intermediate 4 (peak 1—unwanted enantiomer / minor enantiomer): retention time = 2.853 minutes. Intermediate 4 (Peak 2 - desired enantiomer / eutomer): Retention time = 2.979 minutes.

[0157] Intermediate 5: 7-chloro-2,4-dimethyl-2-(trans-4-((3-(trifluoromethyl)oxetane-3-yl)amino)cyclohexyl)benzo[d][1,3]dioxole-5-carboxylic acid methyl ester

[0158]

[0159] A mixture of 1-chloro-N-[3-(trifluoromethyl)oxetane-3-yl]hydroamine (678 mg, 3.82 mmol) and N,N-diisopropylethylamine (698 μL, 4.01 mmol) in dichloroethane (9.54 mL) was stirred at room temperature for 30 minutes, then acetic acid (218 μL, 3.82 mmol) was added, followed by 7-chloro-2,4-dimethyl-2-(4-oxocyclohexyl)benzo[d][1,3]dioxole-5-carboxylic acid methyl ester (650 mg, 1.91 mmol). After 5 minutes at room temperature, the mixture became clear (yellow / brown), and sodium triacetoxyborohydride (404 mg, 1.91 mmol) was added. The reaction was stirred at room temperature for 3 hours, then quenched with a saturated aqueous solution of sodium bicarbonate (30 mL). The desired product was extracted with dichloromethane (30 mL x 3), and the combined organic layers were dried over sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by reverse phase flash chromatography (C18 column, acetonitrile / water 1:1, containing 0.1% trifluoroacetic acid) to obtain two geometric isomers (cis and trans). The first eluting peak corresponds to geometric isomer 1.

[0160] Analytical LCMS Method (for Geometric Isomers)

[0161] [Column: Zorbax SB-C8 (75×4.6 mm ID, 3.5 μm). Mobile phase A: 0.1% trifluoroacetic acid in acetonitrile / mobile phase B: trifluoroacetic acid in water. Gradient: 5% to 100% mobile phase B (over 3.0 minutes), then 100% mobile phase B (4.5 minutes). Flow rate: 1.5 ml / min. Column temperature: 20° C.] Geometric isomer 1 (desired): retention time = 4.073 minutes (278 mg, 31%). Geometric isomer 2 (undesired): retention time = 4.277 minutes (253 mg, 29%).

[0162] The enantiomers of geometric isomer 1 (as required based on the LCMS method) were separated by preparative SFC [column: Chiralcel OX-H (250×21 mm I.D.). Mobile phase A: CO2 / mobile phase B: 1:1 isopropanol / hexane mixture containing 0.25% isopropylamine. Isocratic (85% mobile phase A and 15% mobile phase B). Flow rate: 80 g / min. Column temperature: 25° C.]. Intermediate 5 (peak 1): retention time = 1.84 minutes. LCMS [M+H] + m / z: calculated: 464.15; found: 464. Intermediate 5 (peak 2): retention time = 2.1 minutes. LCMS [M+H] + m / z: calculated: 464.15; found: 464.2.

[0163] Intermediate 6: 3-(Aminomethyl)-4-chloro-6-methylpyridin-2(1H)-one

[0164]

[0165] Step 1: Synthesis of 2,4-dichloro-6-methylnicotinonitrile

[0166] A solution of 2,4-dihydroxy-6-methylnicotinonitrile (80 g, 533.3 mmol) in phosphorus oxychloride (150 mL) was stirred at 120 ° C for 2 hours, then quenched with a saturated aqueous sodium bicarbonate solution (until pH = 8). It was distributed between water (2000 mL) and ethyl acetate (1000 mL), and the organic layer was dried over sodium sulfate, filtered and concentrated to dryness under reduced pressure to give the title compound (85 g, 86% yield) in the form of a brown solid, which was used in the next step without further purification. LCMS [M + H] + m / z: calculated: 186.98; found: 186.6.

[0167] Step 2: Synthesis of 2-chloro-4-hydroxy-6-methylnicotinonitrile

[0168] To a solution of 2,4-dichloro-6-methylnicotinonitrile (10 g, 53 mmol) in N,N-dimethylformamide (50 mL) was added cesium acetate (30.8 g, 160 mmol) at room temperature. The reactant was stirred at 80 ° C overnight and then quenched with water (800 mL). The desired product was extracted with ethyl acetate (800 mL), and the organic layer was dried over sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The title compound (8.8 g, 98% yield) was obtained as a brown solid, which was used in the next step without further purification. LCMS [M + H] + m / z: calculated: 169.01; found: 168.8.

[0169] Step 3: Synthesis of 4-hydroxy-2-methoxy-6-methylnicotinonitrile

[0170] A mixture of 2-chloro-4-hydroxy-6-methylnicotinonitrile (8.8 g, 52.2 mmol) and sodium methoxide (14.1 g, 261 mmol) in methanol (50 mL) was stirred at 60 ° C overnight. The mixture was quenched with 1M hydrochloric acid solution until the pH was 5. It was partitioned between water (500 mL) and ethyl acetate (500 mL), and the organic layer was dried over sodium sulfate and concentrated to give the title compound (8.0 g, 93% yield) as a brown solid, which was used in the next step without further purification. LCMS [M + H] + m / z: calculated: 165.1; found: 164.8.

[0171] Step 4: 4-Chloro-2-methoxy-6-methylnicotinonitrile

[0172] A mixture of 4-hydroxy-2-methoxy-6-methylnicotinonitrile (8.0 g, 48.7 mmol), phosphorus pentachloride (20.3 g, 97.5 mmol), phosphorus oxychloride (14.9 g, 9.09 mL, 97.5 mmol) and dimethylformamide (5 mL) in chloroform (100 mL) was stirred at 60 ° C for 30 minutes. The reaction was quenched with saturated aqueous sodium bicarbonate solution (until pH 8). It was distributed between water (1000 mL) and ethyl acetate (1000 mL), the organic layer was dried over sodium sulfate, and concentrated to dryness to give the title compound (8.0 g, 90% yield) as a brown solid, which was used directly in the next step without further purification. LCMS [M + H] + m / z: calculated: 183.0; found: 182.8.

[0173] Step 5: Synthesis of (4-chloro-2-methoxy-6-methylpyridin-3-yl)methanamine

[0174] Borane dimethyl sulfide complex (10M, 5.3mL, 53mmol) was added to a solution of 4-chloro-2-methoxy-6-methylnicotinonitrile (8.0g, 43.8mmol) in tetrahydrofuran (50mL). The mixture was stirred at 60°C for 2 hours and then quenched with methanol (10mL) at 0°C. The mixture was concentrated to dryness under reduced pressure to give the title (7.0g, 93% yield) in the form of a brown solid, which was used directly in the next step without further purification. LCMS [M+H] + m / z: calculated: 187.1; found: 187.1.

[0175] Step 6: tert-Butyl ((4-chloro-2-methoxy-6-methylpyridin-3-yl)methyl)carbamate

[0176] A mixture of (4-chloro-2-methoxy-6-methylpyridin-3-yl)methylamine (7.0 g, 37.5 mmol), di-tert-butyl dicarbonate (15.2 g, 75.0 mmol) and triethylamine (11.4 g, 15.7 mL, 113 mmol) in tetrahydrofuran (50 mL) was stirred at 20 ° C for 16 hours and then quenched with water (500 mL). The desired product was extracted with ethyl acetate (500 mL), and the organic layer was dried over sodium sulfate, filtered and concentrated to dryness under reduced pressure. The residue was purified by flash column (silica gel column, petroleum ether: ethyl acetate: 40: 1) to give the title compound (3.0 g, 28% yield) as a colorless oil. LCMS [M + H] +m / z: calculated: 287.1; found: 286.9.

[0177] Step 7: 3-(Aminomethyl)-4-chloro-6-methylpyridin-2(1H)-one

[0178] To an aqueous solution of hydrogen chloride (4M, 10 mL, 10 mmol) was added tert-butyl ((4-chloro-2-methoxy-6-methylpyridin-3-yl)methyl)carbamate (3.0 g, 10.5 mmol) at room temperature. The reaction was heated at 100°C for 2 hours and then concentrated to dryness under reduced pressure to give the title compound as a yellow solid (1.7 g, 94% yield). LCMS [M+H] + m / z: calculated: 173.04; found: 173.1. 1 H NMR (400 MHz, methanol-d4): δ 6.38 (s, 1H), 4.15 (s, 2H), 2.32 (s, 3H).

[0179] Intermediate 7: 7-chloro-2-(4-(3,3-difluoroazetidin-1-yl)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxylic acid methyl ester

[0180]

[0181] Step 1: Synthesis of methyl 7-chloro-2-(4-(3,3-difluoroazetidin-1-yl)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxylate

[0182] A solution of 7-chloro-2,4-dimethyl-2-(4-oxocyclohexyl)benzo[d][1,3]dioxole-5-carboxylic acid methyl ester (Intermediate 4—racemic mixture) (550 mg, 1.62 mmol), 3,3-difluoroazetidine hydrochloride (839 mg, 6.48 mmol) and triethylamine (895 μL, 6.64 mmol) in methanol (5 mL) and tetrahydrofuran (5 mL) was stirred at room temperature overnight. The next day, the reaction was cooled to -78 °C and lithium borohydride solution (2M in tetrahydrofuran, 1.21 mL, 2.43 mmol) was added dropwise. The viscous yellow mixture was gradually warmed to room temperature and then quenched with saturated aqueous sodium carbonate solution at 0 °C. The desired product was extracted with dichloromethane (three times), and the combined organic layers were dried over sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified twice by flash chromatography (silica gel KP-NH column, gradient from 0% to 20% ethyl acetate in heptane) to afford the title compound (354 mg, 53% yield) as a racemic mixture of a single geometric isomer (cis or trans). LCMS [M+H]+ m / z: calculated: 416.9; found: 416.2.

[0183] Step 2: Separation of (2R)-7-chloro-2-(4-(3,3-difluoroazetidin-1-yl)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxylic acid methyl ester and (2S)-7-chloro-2-(4-(3,3-difluoroazetidin-1-yl)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxylic acid methyl ester

[0184] A racemic mixture of methyl 7-chloro-2-(4-(3,3-difluoroazetidin-1-yl)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxylate (560 mg) was subjected to preparative SFC [column: Chiralpak AD-H from Daicel Chemical Industries. Mobile phase A: CO2 / mobile phase B: a mixture of hexane and ethanol (1{}:1) containing 0.25% isopropylamine. Isocratic (90% mobile phase A and 10% mobile phase B). Flow rate: 80 g / min. Column temperature: ] dissolved. SFC analysis: Column: Chiralpak AD-H (100 mm × 4.6 mm) from Daicel Chemical Industries. Mobile phase A: CO2 / mobile phase B: a mixture of hexane and ethanol (3:1) containing 0.1% isopropylamine. Isocratic (85% mobile phase A and 15% mobile phase B). Flow rate: 4 ml / min. Column temperature: 40°C. Intermediate 7 (peak 1): retention time = 1.02 min (SFC analysis). Recovery = 173 mg, yield 15%, 96% ee. 1 H NMR (400 MHz, chloroform-d) δ7.48 (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 7 (peak 2): retention time = 1.16 minutes (SFC analysis). Recovery = 150 mg, yield 13%, 97% ee. LCMS [M+H] + m / z: calculated: 416.86; found: 416.2.

[0185] Intermediate 8: 7-Chloro-2,4-dimethyl-2-(1-(2,2,2-trifluoroethyl)piperidin-4-yl)benzo[d][1,3]dioxole-5-carboxylic acid perfluorophenyl ester

[0186]

[0187] Step 1: Synthesis of tert-butyl 4-(7-chloro-5-(methoxycarbonyl)-2,4-dimethylbenzo[d][1,3]dioxol-2-yl)piperidine-1-carboxylate

[0188] In a 100 mL pear-shaped flask equipped with a condenser, a mixture of methyl 5-chloro-3,4-dihydroxy-2-methylbenzoate (1.219 g, 5.58 mmol), triphenylphosphine (146 mg, 0.558 mmol) and triruthenium dodecacarbonyl (178 mg, 0.279 mmol) was purged with nitrogen cycle / vacuum cycle (4 cycles), then toluene (12 mL) was added, purged with nitrogen / vacuum cycle, and then stirred at 120 ° C for 30 minutes. A mixture of tert-butyl 4-ethynylpiperidine-1-carboxylate (2.32 g, 11.1 mmol) in toluene (10 mL) was added to the dark mixture. The resulting orange solution was stirred at 120 ° C for another 2 hours. The reaction mixture was concentrated under reduced pressure and then purified by flash chromatography (silica gel, gradient from 0% to 100% ethyl acetate in heptane) to give the title compound (2.33 g, 98%) as a yellow oil. LCMS [M+Na] + m / z: calcd 448.2; actual value: 448.2.

[0189] Step 2: Synthesis of methyl 7-chloro-2,4-dimethyl-2-(piperidin-4-yl)benzo[d][1,3]dioxole-5-carboxylate (trifluoroacetate)

[0190] To a yellow solution of tert-butyl 4-[7-chloro-5-(methoxycarbonyl)-2,4-dimethyl-2H-1,3-benzodioxol-2-yl]piperidine-1-carboxylate (2.33 g, 5.47 mmol) in dichloromethane (2 mL) was added TFA (1 mL). After 30 minutes, the reaction mixture was concentrated under reduced pressure to give the title compound (2.40 g, quantitative) in the form of a gum, which was used in the next step without further purification. LCMS [M+H] + m / z: calculated: 326.79; found: 326.2.

[0191] Step 3: Synthesis of methyl 7-chloro-2,4-dimethyl-2-(1-(2,2,2-trifluoroethyl)piperidin-4-yl)benzo[d][1,3]dioxole-5-carboxylate

[0192] To a solution of 7-chloro-2,4-dimethyl-2-(piperidin-4-yl)-2H-1,3-benzodioxole-5-carboxylic acid methyl ester, trifluoroacetate (2.40 g, 5.45 mmol) in toluene (20 mL) and acetonitrile (10 mL) was added triethylamine (8 mL, 57.6 mmol) and 2,2,2-trifluoroethyl trifluoromethanesulfonate (3.0 g, 12.9 mmol). After 3 hours, the reaction mixture was concentrated under reduced pressure. Water (50 mL) was added, and the mixture was extracted with EtOAc (30 mL×3). The combined organic layers were washed with brine (50 mL×2), dried over sodium sulfate, filtered and concentrated under reduced pressure, and then purified by flash chromatography (silica gel, gradient of 0% to 50% ethyl acetate in heptane) to obtain the title compound (2.04 g, 92%) as a yellow oil. LCMS [M+H] + m / z: calculated: 408.81; found: 408.2.

[0193] Step 4: Synthesis of 7-chloro-2,4-dimethyl-2-(1-(2,2,2-trifluoroethyl)piperidin-4-yl)benzo[d][1,3]dioxole-5-carboxylic acid

[0194] To a solution of 7-chloro-2,4-dimethyl-2-[1-(2,2,2-trifluoroethyl)piperidin-4-yl]-2H-1,3-benzodioxole-5-carboxylic acid methyl ester (691 mg, 1.69 mmol) in methanol (3 mL) was added a 6M aqueous solution of sodium hydroxide (1 mL, 6.00 mmol) and heated at 60°C. After 25 minutes, the reaction mixture was concentrated under reduced pressure to remove most of the methanol. The mixture was then diluted with water, cooled to 0°C, and then neutralized with 1M hydrochloric acid to pH 7. The mixture was extracted with dichloromethane (3 times), and the combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure to give the title compound as a white solid, which was used in the next step without further purification. LCMS [M + H] + m / z: calculated: 394.79; found: 394.2.

[0195] Step 5: Synthesis of perfluorophenyl 7-chloro-2,4-dimethyl-2-(1-(2,2,2-trifluoroethyl)piperidin-4-yl)benzo[d][1,3]dioxole-5-carboxylate

[0196] To a stirred solution of 7-chloro-2,4-dimethyl-2-[1-(2,2,2-trifluoroethyl)piperidin-4-yl]-2H-1,3-benzodioxole-5-carboxylic acid (665 mg, 1.68 mmol) in dichloromethane (5 mL) was added pyridine (1 mL, 12.4 mmol) followed by 2,2,2-trifluoroacetic acid 2,3,4,5,6-pentafluorophenyl ester (500 μL, 2.90 mmol). After 20 minutes, the reaction mixture was concentrated under reduced pressure to give the title compound, which was used in the next step without further purification. LCMS [M+H] + m / z: calculated: 560.84; found: 560.2.

[0197] Intermediate 9: 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxylic acid

[0198]

[0199] Step 1: Synthesis of methyl 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxylate

[0200] 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 stirred at room temperature for 30 minutes, and then another solution of 7-chloro-2,4-dimethyl-2-(4-oxocyclohexyl)-1,3-benzodioxole-5-carboxylic acid methyl ester (Intermediate 4-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 the starting material was completely consumed by TLC, ethyl acetate / methanol: 5:1]. Next, the two batches of reactions were combined, quenched with a saturated aqueous solution of ammonium chloride (120 mL) at 0 ° C, and the desired product was extracted with dichloromethane (200 mL × 3). The combined organic layer was dried over sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The residue was purified by flash chromatography (silica gel, gradient of dichloromethane containing 0% to 14% methanol) to give the title compound (8.05 g, 67% yield, 83% purity) as a light yellow oil. The 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; found: 410.1. 1 H NMR (400 MHz, methanol-d4) δ 7.39 (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, 8.0 Hz, 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).

[0201] Step 2: Synthesis of 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxylic acid

[0202] To a solution of 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxylic acid methyl ester (4g, 9.75mmol) in methanol (48mL) was added a solution of lithium hydroxide hydrate (4.03g, 96.06mmol) in water (12mL). The reactants were stirred at 70°C for 2 hours, then the two batches were combined and concentrated under reduced pressure. Water (50mL) was added, and the pH was adjusted to 6 with a saturated aqueous citric acid solution at 0°C. The desired product was extracted with a 3:1 mixture of dichloromethane and isopropanol (300mL×5). The combined organic layers were dried over sodium sulfate, filtered, and concentrated to dryness under reduced pressure to give the title compound (6.1g, crude) as an off-white solid, which was used in the next step without further purification. LCMS[M+H] + m / z: calculated: 396.2; found: 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).

[0203] Example #1: 7-chloro-2-(4-(3-(difluoromethoxy)azetidin-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

[0204]

[0205] Step 1: Synthesis of methyl 7-chloro-2-(4-(3-(difluoromethoxy)azetidin-1-yl)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxylate

[0206] A solution of a mixture of methanol (2mL) and tetrahydrofuran (2mL) containing 7-chloro-2,4-dimethyl-2-(4-oxocyclohexyl)benzo[d][1,3]dioxole-5-carboxylic acid methyl ester (intermediate 4-peak 2) (224mg, 0.661mmol), 3-(difluoromethoxy)azetidine (336mg, 2.72mmol) and triethylamine hydrochloride (373mg, 2.71mmol) was stirred at room temperature for 1.5 hours and cooled to -78°C. A tetrahydrofuran solution of lithium borohydride (2M, 500μL, 1mmol) was added dropwise at -78°C, and the reaction was then warmed to room temperature for 15 minutes. Next, the mixture was cooled to 0°C, then quenched with a saturated aqueous solution of sodium bicarbonate, diluted with dichloromethane, and warmed to room temperature. The desired product was extracted from the aqueous layer with dichloromethane (three times), and the combined organic layers were dried over a hydrophobic filter and concentrated to dryness under reduced pressure. The residue was purified by flash chromatography (silica gel, gradient from 10% to 100% ethyl acetate in heptane, then 0% to 100% ethanol in ethyl acetate) to give a single geometric isomer (cis or trans) of the title compound (205 mg, 70% yield). LCMS [M+H] + m / z: calculated: 445.9; found: 446.2.

[0207] Step 2: Synthesis of 7-chloro-2-(4-(3-(difluoromethoxy)azetidin-1-yl)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxylic acid

[0208] To a solution of methyl 7-chloro-2-(4-(3-(difluoromethoxy)azetidin-1-yl)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxylate (205 mg, 0.460 mmol) in methanol (3 mL) was added an aqueous solution of sodium hydroxide (6 M, 1 mL, 6.00 mmol). The reactants were heated at 60 ° C for 20 minutes, then diluted with water, cooled to 0 ° C, acidified to pH = 2 with 1 M hydrochloric acid, and then neutralized to pH = 7 with 1 M aqueous sodium hydroxide solution. The desired product was extracted with dichloromethane (three times), dried with a hydrophobic filter, and concentrated to dryness under reduced pressure to give the title compound as a white solid (176 mg, 89% yield), which was used in the next step without further purification. LCMS [M + H] + m / z: calculated: 431.9; found: 432.2.

[0209] Step 3: Synthesis of perfluorophenyl 7-chloro-2-((1r,4R)-4-(3-(difluoromethoxy)azetidin-1-yl)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxylate

[0210] To a solution of 7-chloro-2-(4-(3-(difluoromethoxy)azetidin-1-yl)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxylic acid (176 mg, 0.408 mmol) in dichloromethane (0.5 mL) was added pyridine (1.0 mL, 12.4 mmol) at room temperature, followed by 2,2,2-trifluoroacetic acid 2,3,4,5,6-pentafluorophenyl ester (150 μL, 0.871 mmol). After 20 minutes, the reaction mixture was concentrated to dryness under reduced pressure to give a mixture of the title compound (100% theoretical yield = 243 mg) and by-products. The crude mixture was used in the next step without further purification. LCMS [M+H] + m / z: calculated: 597.91; found: 598.2.

[0211] Step 4: Synthesis of 7-chloro-2-(4-(3-(difluoromethoxy)azetidin-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

[0212] To a solution of perfluorophenyl 7-chloro-2-(4-(3-(difluoromethoxy)azetidin-1-yl)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxylate (≤243 mg, ≤0.406 mmol) in dimethyl sulfoxide (1 mL) was added 3-(aminomethyl)-6-methyl-4-(methylthio)pyridin-2(1H)-one (hydrochloride) (Intermediate 1) (222 mg, 1.20 mmol) and N,N-diisopropylethylamine (0.30 mL, 1.71 mmol). The reaction mixture was stirred at 60 °C for 1 hour and then concentrated to dryness overnight under a strong stream of nitrogen. The residue was purified twice by reverse phase flash chromatography (C18 column, gradient from 0% to 100% acetonitrile in water with 0.1% trifluoroacetic acid). The residue was diluted with dichloromethane and saturated sodium bicarbonate solution. The aqueous layer was washed with dichloromethane. The combined organic layers were dried over sodium sulfate and concentrated under reduced pressure to give the title compound (166 mg, 68% yield in two steps) as a single geometric isomer (cis or trans). LCMS [M+H] + m / z: calculated: 598.1; found: 598.3. 1 H NMR (400MHz, methanol-d4) δ6.88(s,1H),6.26(s,1H),6.38(t,J=74.3Hz,2H),4.71 (quin,J=6.0Hz,1H),4.48(s,2H),3.69-3.60(m,2H),3.18-3.11(m,2H),2.52 (s,3H),2.29(s,3H),2.18(s,3H),2.10(tt,J=3.5,11.2Hz,1H),1.98-1.86( m,4H),1.85-1.78(m,1H),1.60(s,3H),1.32-1.21(m,2H),1.06-0.92(m,2H).

[0213] Table 1: The following compounds were prepared in a similar manner to Example 1 using appropriate starting materials.

[0214]

[0215] Example #3: (R)-7-Chloro-N-((6-chloro-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-(trans-4-(dimethylamino)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxamide

[0216]

[0217] To a solution of (R)-7-chloro-2-(trans-4-(dimethylamino)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxylic acid (prepared according to the procedure described in patent application US2017 / 0073335A1) (48 mg, 0.1356 mmol) in dimethyl sulfoxide (0.5 mL) was added triethylamine (56.6 μL, 0.407 mmol) and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (41.2 mg, 0.108 mmol) at room temperature. The formation of the activated ester was detected by LCMS ([M+H]) after 5 minutes. + m / z: 368.2) was incomplete, so additional dimethyl sulfoxide (1 mL) containing hexafluorophosphate O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium ester (16 mg, 0.042 mmol) was added twice. After acid activation was complete, a suspension of 3-(aminomethyl)-6-chloro-4-(methylsulfonyl)-1,2-dihydropyridin-2-one hydrochloride (65.3 mg, 0.271 mmol) and triethylamine (56.6 μL, 0.407 mmol) in dimethyl sulfoxide (0.5 mL) was added at room temperature. The reaction mixture was heated at 60°C for 45 minutes and then diluted with dichloromethane and water. The organic layer was dried using a hydrophobic filter and concentrated to dryness under reduced pressure. The residue was purified by flash chromatography [silica gel KP-NH column, gradient from 0% to 100% ethyl acetate in heptane, then 0% to 100% ethanol in dichloromethane, then 0% to 100% methanol (containing 20% ​​ammonium hydroxide) in ethyl acetate] to give the title compound as a white solid (30 mg, 41% yield). LCMS [M+H] + m / z: calculated: 540.5; found: 540.2. 1 H NMR (400MHz, methanol-d4) δ6.89(s,1H),6.68(s,1H),4.50(s,2H),3.20(tt,J=3.2,11.9Hz,1H),2.83(s,6H),2.53(s,3H ),2.18(s,3H),2.16-2.08(m,4H),2.02-1.94(m,1H),1.62(s,3H),1.54(dq,J=3.4,12.4Hz,2H),1.47-1.37(m,2H).

[0218] Table 2: The following compounds were prepared in a similar manner to Example 3 using appropriate starting materials.

[0219]

[0220] Example #6 (Enantiomer 1): 7-Chloro-N-((6-chloro-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-(4-(dimethylamino)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxamide

[0221]

[0222] Step 1: Synthesis of 7-chloro-2,4-dimethyl-2-(4-((3-(trifluoromethyl)oxetan-3-yl)amino)cyclohexyl)benzo[d][1,3]dioxole-5-carboxylic acid

[0223] To a solution of 7-chloro-2,4-dimethyl-2-[4-{[3-(trifluoromethyl)oxetane-3-yl]amino}cyclohexyl]-2H-1,3-benzodioxole-5-carboxylic acid methyl ester (Intermediate 5—Peak 1) (81 mg, 0.174 mmol) in methanol (1 mL) was added sodium hydroxide (72 mg, 1.79 mmol). The reaction mixture was heated at 60°C for 20 minutes, cooled to 0°C, and then acidified to pH=2 with 1M hydrochloric acid. The desired product was then extracted with dichloromethane (three times), and the combined organic layers were dried with a hydrophobic filter. The filtrate was concentrated to dryness under reduced pressure to give the title compound (70 mg, 89% yield) as a white solid, which was used in the next step without further purification. LCMS[M+H] + m / z: calculated: 450.1; found: 450.2.

[0224] Step 2: Synthesis of perfluorophenyl 7-chloro-2,4-dimethyl-2-(4-((3-(trifluoromethyl)oxetan-3-yl)amino)cyclohexyl)benzo[d][1,3]dioxole-5-carboxylate

[0225] To a solution of 7-chloro-2,4-dimethyl-2-(4-((3-(trifluoromethyl)oxetane-3-yl)amino)cyclohexyl)benzo[d][1,3]dioxole-5-carboxylic acid (70 mg, 155 μmol) in dichloromethane (0.5 mL) was added pyridine (24.9 μL, 310 μmol) and 2,2,2-trifluoroacetic acid 2,3,4,5,6-pentafluorophenyl ester (39.8 μL, 232 μmol) at room temperature. After 15 minutes, the reactant was concentrated under reduced pressure to give a mixture of the title compound and by-products (68 mg, crude product). The crude mixture was used in the next step without further purification. LCMS[M+H] + m / z: calculated: 616.1; found: 616.2.

[0226] Step 3: Synthesis of 7-chloro-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-(4-((3-(trifluoromethyl)oxetan-3-yl)amino)cyclohexyl)benzo[d][1,3]dioxole-5-carboxamide

[0227] To a solution of 2,3,4,5,6-pentafluorophenyl 7-chloro-2,4-dimethyl-2-[4-{[3-(trifluoromethyl)oxetan-3-yl]amino}cyclohexyl]-2H-1,3-benzodioxole-5-carboxylate (≤68 mg, ≤110 μmol) in N,N-dimethylformamide (1 mL) was added 3-(aminomethyl)-6-methyl-4-(methylsulfonyl)-1,2-dihydropyridin-2-one (free base) (30.4 mg, 0.165 mmol) and N,N-diisopropylamine (0.1 mL, 574 mol) at room temperature. The reaction was stirred at 50°C for 30 minutes and then added directly to a C18 column for purification by reverse phase flash chromatography (C18 column, gradient from 5% to 50% acetonitrile in water, containing 0.1% trifluoroacetic acid) to give the trifluoroacetate salt of the title compound. The salt was dissolved in dichloromethane and the organic layer was washed with saturated aqueous sodium bicarbonate solution. Finally, the organic layer was concentrated to dryness under reduced pressure to give the title compound (free base) as a white solid (42 mg, 62% yield). LCMS [M+H] + m / z: calcd 616.19; actual value: 616.3. 1 H NMR (400MHz, chloroform-d) δ12.62-12.44(m,1H),7.16(t,J=5.1Hz,1H),6.91(s,1H),6.04(s,1H),4.76(s,3H),4.62-4.46(m,4H),2.7 2-2.63(m,1H),2.49(s,3H),2.32(s,3H),2.27(s,3H),1.92(d,J=5.9Hz,4H),1.80(br.s.,1H),1.60(s,3H),1.36-1.10(m,6H).

[0228] Table 3: The following compounds were prepared in a similar manner to Example 6 using appropriate starting materials.

[0229]

[0230] Example 8: 7-chloro-N-((4-chloro-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-(4-(3-cyclopropyloxyazetidin-1-yl)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxamide

[0231]

[0232] Step 1: Synthesis of methyl 7-chloro-2-(4-(3-cyclopropyloxyazetidin-1-yl)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxylate

[0233] A solution of 3-(cyclopropyloxy)azetidine hydrochloride (1.9 g, 12.7 mmol) and N,N-diisopropylethylamine (2.7 mL, 15.5 mmol) in methanol (10 mL) was stirred at room temperature for 1 hour, and then another solution of 7-chloro-2,4-dimethyl-2-(4-oxocyclohexyl)-1,3-benzodioxole-5-carboxylic acid methyl ester (Intermediate 4-peak 2) (860 mg, 2.54 mmol) in tetrahydrofuran (30 mL) was added. The reaction mixture was stirred at room temperature for 1.5 hours and then cooled to -70 ° C. Lithium borohydride (120 mg, 5.51 mmol) was added, and the reaction was stirred at -70 ° C for 30 minutes [or until the starting material was completely consumed by TLC, ethyl acetate / methanol: 5: 1]. Then, the reaction was quenched with a saturated aqueous solution of ammonium chloride (100 mL), and the desired product was extracted with ethyl acetate (50 mL×2). The combined organic layers were washed with brine (30 mL), dried over sodium sulfate, filtered and concentrated to dryness under reduced pressure. The residue was purified by flash chromatography (silica gel, gradient from 50% to 100% ethyl acetate in petroleum ether) to give the title compound (760 mg, 65% yield, 95% purity) as a yellow oil. LCMS [M+H] + m / z: calculated: 436.1; found: 436.0.

[0234] Step 2: Synthesis of 7-chloro-2-(4-(3-cyclopropyloxyazetidin-1-yl)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxylic acid

[0235] To a solution of 7-chloro-2-(4-(3-cyclopropyloxyazetidin-1-yl)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxylic acid methyl ester (760 mg, 1.74 mmol) in methanol (15 mL) and water (3 mL) was added lithium hydroxide hydrate (962 mg, 22.93 mmol). The reactants were stirred at 70 ° C for 15 hours. Then concentrated under reduced pressure. Water (15 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 mixture of 10:1 dichloromethane and isopropanol (30 mL×3). The combined organic layers were washed with brine (150 mL×2), dried over sodium sulfate, filtered, and concentrated to dryness under reduced pressure to give the title compound (720 mg, crude) as a yellow solid, which was used in the next step without further purification. LCMS[M+H] + m / z: calculated: 422.1; found: 422.0.

[0236] Step 3: Synthesis of 7-chloro-N-((4-chloro-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-(4-(3-cyclopropyloxyazetidin-1-yl)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxamide

[0237] To a solution of 7-chloro-2-[4-[3-(cyclopropyloxy)azetidin-1-yl]cyclohexyl]-2,4-dimethyl-1,3-benzodioxole-5-carboxylic acid (360 mg, 0.853 mmol) in N,N-dimethylformamide (4 mL) was added O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (500 mg, 1.31 mmol) and N,N-diisopropylethylamine (1 mL, 5.74 mmol). The mixture was stirred at 15 ° C for 30 minutes, and then 3-(aminomethyl)-4-chloro-6-methyl-1H-pyridin-2-one hydrochloride (intermediate 6) (250 mg, 1.2 mmol) was added. The reaction mixture was stirred at 15 ° C for another 4 hours and then filtered. The filtrate was purified by preparative HPLC [column: Waters Xbridge (150 mm×25 mm, 5 μm). Mobile phase A: water (0.05% ammonium hydroxide v / v / mobile phase B: acetonitrile. Gradient (65% to 55% mobile phase A / 35% to 65% mobile phase B, over 9.5 minutes). Column temperature: 30° C.] to give the title compound (209 mg, 42% yield) as a white solid. LCMS [M+H] + m / z: calculated: 576.2; found: 576.2. 1H NMR (400MHz, methanol-d4) δ6.90(s,1H),6.30(s,1H),4.52(s,2H),4.22(quin,J=6.0 Hz,1H),3.61(dd,J=6.4,8.6Hz,2H),3.31-3.25(m,1H),2.99(dd,J=6.3,8.4Hz, 2H),2.28(s,3H),2.20(s,3H),2.12-2.04(m,1H),1.99-1.85(m,5H),1.62(s,3 H),1.33-1.24(m,2H),1.06-0.95(m,2H),0.56-0.51(m,2H),0.51-0.44(m,2H).

[0238] Table 4: The following compounds were prepared in a similar manner to Example 8 using appropriate starting materials.

[0239]

[0240]

[0241] Example 14: (R)-7-Chloro-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-(1-(2,2,2-trifluoroethyl)piperidin-4-yl)benzo[d][1,3]dioxole-5-carboxamide and (S)-7-Chloro-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-(1-(2,2,2-trifluoroethyl)piperidin-4-yl)benzo[d][1,3]dioxole-5-carboxamide

[0242]

[0243] Step 1: Synthesis of 7-chloro-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-(1-(2,2,2-trifluoroethyl)piperidin-4-yl)benzo[d][1,3]dioxole-5-carboxamide

[0244] To a solution of 2,3,4,5,6-pentafluorophenyl 7-chloro-2,4-dimethyl-2-[1-(2,2,2-trifluoroethyl)piperidin-4-yl]-2H-1,3-benzodioxole-5-carboxylate (Intermediate 8) (752 mg, 1.34 mmol) in dimethyl sulfoxide (4 mL) was added 3-(aminomethyl)-6-methyl-4-(methylsulfonyl)-1,2-dihydropyridin-2-one (Intermediate 1) (740 mg, 4.02 mmol) and N,N-diisopropylamine (932 μL, 5.36 mmol).

[0245] The reaction mixture was heated at 60° C. and after 1 hour it was added to a C18 column for purification. The desired product was purified twice by reverse phase flash chromatography (C18 column, gradient 0% to 100% acetonitrile in water, 0.1% trifluoroacetic acid) to give the title compound (289 mg, 38% yield) as a racemic mixture as a gum.

[0246] Step 2: (R)-7-chloro-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-(1-(2,2,2-trifluoroethyl)piperidin-4-yl)benzo[d][1,3]dioxole-5-carboxamide and (S)-7-chloro-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-(1-(2,2,2-trifluoroethyl)piperidin-4-yl)benzo[d][1,3]dioxole-5-carboxamide

[0247] The racemic mixture of 7-chloro-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-(1-(2,2,2-trifluoroethyl)piperidin-4-yl)benzo[d][1,3]dioxole-5-carboxamide (289 mg) was dissolved by preparative SFC [column: ChromegaChiral CC4 (250 mm x 20 mm ID) from ES Industries. Mobile phase A: CO2 / mobile phase B: methanol containing 0.25% isopropylamine. Isocratic (55% mobile phase A and 45% mobile phase B). Flow rate: 80 g / min. Column temperature: 25°C]. SFC analysis: column: Chiralcel OX-H (100 mm x 4.6 mm ID) from Chiral Technologies. Mobile phase A: CO2 / mobile phase B: methanol containing 0.1% isopropylamine. Isocratic (75% mobile phase A and 25% mobile phase B). Flow rate: 4 ml / min. Column temperature: 40°C. Example 16 (Enantiomer 1) (desired enantiomer / isomeric): retention time = 3.74 minutes (SFC analysis). Recovery = 90 mg, yield 12%, 99% ee (yellow solid). LCMS [M+H] + m / z: calculated: 560.2; found: 560.2. 1 H NMR (400 MHz, methanol-d4) δ 6.89 (s, 1H), 6.27 (s, 1H), 4.49 (s, 2H), 3.08-2.98 (m, 4H), 2.52 (s, 3H), 2.34 (br t, J = 11.0 Hz, 2H), 2.29 (s, 3H), 2.20 (s, 3H), 1.93-1.84 (m, 1H), 1.83-1.76 (m, 2H), 1.62 (s, 3H), 1.60-1.47 (m, 2H). Example 16 (enantiomer 2) (unwanted enantiomer / minor enantiomer): retention time = 4.25 minutes (SFC analysis). Recovery = 101 mg g, yield 13%, 98% ee, (yellow solid). LCMS [M+H] + m / z: calculated: 560.2; found: 560.2. 1H NMR (400 MHz, methanol-d4) δ 6.89 (s, 1H), 6.27 (s, 1H), 4.49 (s, 2H), 3.08-2.98 (m, 4H), 2.52 (s, 3H), 2.34 (br t, J=11.0 Hz, 2H), 2.29 (s, 3H), 2.20 (s, 3H), 1.93-1.84 (m, 1H), 1.83-1.76 (m, 2H), 1.62 (s, 3H), 1.60-1.47 (m, 2H).

[0248] Example 15: 7-Chloro-N-((4-chloro-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxamide

[0249]

[0250] To a solution of 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxylic acid (900 mg, 2.27 mmol) in N,N-dimethylformamide (5 mL) was added O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.04 g, 2.73 mmol) and N,N-diisopropylethylamine (2.38 mL, 13.6 mmol). The mixture was stirred at 25 °C for 30 minutes, then 3-(aminomethyl)-4-chloro-6-methyl-1H-pyridin-2-one hydrochloride (Intermediate 6) (710 mg, 3.4 mmol) was added. The reaction mixture was stirred at room temperature for another 1.5 hours, then filtered. The filtrate was subjected to preparative HPLC [column: YMC-Actus Triart C18 (100 mm×30 mm, 5 μm). Mobile phase A: water (0.05% hydrochloric acid) / mobile phase B: acetonitrile. Gradient (85% to 55% mobile phase A / 15% to 45% mobile phase B, over 10 minutes). Column temperature: and column: Xtimate C18 (150 mm×25 mm, 5 μm). Mobile phase A: water (0.05% ammonium hydroxide v / v) / mobile phase B: acetonitrile. Gradient (69% to 39% mobile phase A / 31% to 61% mobile phase B, over 7 minutes). Column temperature: 30°C] Purified twice to give the title compound as a white solid (533 mg, 43% yield, purity>99%). LCMS [M+H] + m / z: calculated: 550.2; found: 550.1. 1H NMR (400MHz, chloroform-d) δ11.97(br,1H),7.06-7.03(m,1H),6.88(s,1H),6.19(s,1H),4.65(d,J=6.0Hz,2H),4.05-4.01(m,1H),3. 66-3.62(m,2H),3.25(s,3H),2.93-2.90(m,2H),2.28(s,3H),2.25(s,3H),1.93-1.81(m,6H),1.60(s,3H),1.25-1.02(m,4H).

[0251] Example 16: (R)-7-Chloro-N-((4-chloro-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2,4-dimethyl-2-(1-(2,2,2-trifluoroethyl)piperidin-4-yl)benzo[d][1,3]dioxole-5-carboxamide and (S)-7-Chloro-N-((4-chloro-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2,4-dimethyl-2-(1-(2,2,2-trifluoroethyl)piperidin-4-yl)benzo[d][1,3]dioxole-5-carboxamide

[0252]

[0253] Step 1: Synthesis of 7-chloro-N-((4-chloro-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2,4-dimethyl-2-(1-(2,2,2-trifluoroethyl)piperidin-4-yl)benzo[d][1,3]dioxole-5-carboxamide

[0254] 3-(Aminomethyl)-4-chloro-6-methyl-1,2-dihydropyridin-2-one hydrochloride (Intermediate 6) (70.2 mg, 0.336 mmol) and N,N-diisopropylamine (233 μL, 1.34 mmol) were added to a solution of 7-chloro-2,4-dimethyl-2-[1-(2,2,2-trifluoroethyl)piperidin-4-yl]-2H-1,3-benzodioxole-5-carboxylic acid 2,3,4,5,6-pentafluorophenyl ester (Intermediate 8) (188 mg, 0.3358 mmol) in dimethyl sulfoxide (1 mL). The reaction mixture was heated at 60° C. and added to a C18 column for purification after 1 hour. The desired product was purified twice by reverse phase flash chromatography (C18 column, gradient 0% to 100% acetonitrile in water with 0.1% trifluoroacetic acid) to give the title compound as a racemic mixture as a gum (180 mg, 98% yield).

[0255] Step 2: Separation of (R)-7-chloro-N-((4-chloro-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2,4-dimethyl-2-(1-(2,2,2-trifluoroethyl)piperidin-4-yl)benzo[d][1,3]dioxole-5-carboxamide and (S)-7-chloro-N-((4-chloro-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2,4-dimethyl-2-(1-(2,2,2-trifluoroethyl)piperidin-4-yl)benzo[d][1,3]dioxole-5-carboxamide

[0256] The racemic mixture of 7-chloro-N-((4-chloro-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2,4-dimethyl-2-(1-(2,2,2-trifluoroethyl)piperidin-4-yl)benzo[d][1,3]dioxole-5-carboxamide (180 mg) was dissolved by preparative SFC [column: ChromegaChiral CC4 (250 mm×20 mm ID) from ES Industries. Mobile phase A: CO2 / mobile phase B: methanol containing 0.25% isopropylamine. Isocratic (65% mobile phase A and 35% mobile phase B). Flow rate: 80 g / min. Column temperature: 25°C]. SFC analysis: column: Chiralcel OZ-H (100 mm×4.6 mm ID, xx μm) from Chiral Technologies. Mobile phase A: CO2 / Mobile phase B: Methanol with 0.1% isopropylamine. Isocratic (65% mobile phase A and 35% mobile phase B). Flow rate: 4 ml / min. Column temperature: 40°C. Example 17 (Enantiomer 1) (desired enantiomer / isoenantiomer): Retention time = 0.73 min (SFC analysis). Recovery = 61 mg, 33% yield, 100% ee (yellow solid). Example 17 (Enantiomer 2) (undesired enantiomer / isoenantiomer): Retention time = 0.98 min (SFC analysis). Recovery = 63 mg, 34% yield, 97 ee (yellow solid). LCMS [M+H] + m / z: calculated: 548.1; found: 548.2. 1 H NMR (400 MHz, methanol-d4) δ 6.89 (s, 1H), 6.29 (s, 1H), 4.52 (s, 2H), 3.04 (q, J = 9.8 Hz, 4H), 2.35 (brt, J = 11.0 Hz, H), 2.27 (s, 3H), 2.19 (s, 3H), 1.95-1.85 (m, 1H), 1.85-1.75 (m, 2H), 1.63 (s, 3H), 1.62-1.50 (m, 2H).

[0257] Example 17: 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

[0258]

[0259] To a solution of 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxole-5-carboxylic acid (Intermediate 9 - 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 minutes, 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 another 1.5 hours, 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 minutes). 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; found: 562.2. 1 H NMR (400MHz, methanol-d4) δ6.91(s,1H),6.29(s,1H),4.50(s,2H),4.01(quin,J=6Hz,1H),3.58(dd,J=8.8,6.4Hz,2H),3.26(s,3H),2.92-3.02 (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).

[0260] EZH2 assay

[0261] Measuring IC of inhibitors using EZH2 50

[0262] 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 (H2N-RKQLATKAAR(Kme3)SAPATGGVKKP-amide) and compound (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. Reactions were initiated with biotinylated H3 substrate peptide (H2N-RKQLATKAAR(Kme1)SAPATGGVKKP-NTPEGBiot) as a 2 μM stock in 12.5 μl buffer and allowed to react for 18-22 hours at room temperature. Quenching was accomplished by adding 20 μl of 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 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).

[0263] EC of inhibitors in HeLa cell assay 50 Measurement

[0264] H3K27me3 Alpha Hela 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; PerkinElmer, Waltham, MA). Hela cells grown in culture were trypsinized and incubated using Cells were counted using a cell counter (Catalog No. C10281; Life Technologies, Grand Island, NY). Cells were diluted to 67,000 cells per ml in 10% DMEM (Catalog No. 10569-010, Life Technologies, Grand Island, NY) and plated using a Biotek MicroFlo TM A dispenser (BioTek Instruments, Inc. Vermont, USA) was selected to seed 15 μL (1,000 cells) into each well. The plates were incubated at 37°C / 5% CO2 for 72 hours. 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, PerkinElmer, Waltham, MA) per well was added to the plate processed for AlphaLISA and this plate was incubated at RT on a plate shaker at low speed for 30 minutes (Model: 4625-Q, Thermo Fisher Scientific, Waltham, MA). Then, 10 μL of histone extraction buffer (Catalog No.: AL009F2; PerkinElmer, Waltham, MA) was added to each well and the plate was incubated at RT on a plate shaker at low speed for 20 minutes. 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).

[0265] 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).

[0266] GI of inhibitors in Karpas-422 viability assay 50 Measurement

[0267] 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 5.

[0268] Table 5:

[0269]

[0270]

[0271]

[0272]

[0273]

[0274] Dwell time measurement

[0275] EZH2 Retention Time Assay: Compound retention time was assessed by monitoring the recovery of enzyme activity following a 100-fold dilution of the preformed enzyme-inhibitor complex (dilution reaction) and comparing it to the activity of an undiluted control in which all reagents were at the same final concentration (control reaction). 3H-SAM incorporation into biotinylated H3 peptide was used to measure enzyme activity. For dilution reactions, 20 nM PRC2 containing wt EZH2 (pentameric complex prepared in-house) was mixed with 1 μM activation peptide (H2N-RKQLATKAAR(Kme3)SAPATGGVKKP-amide) and K i Compounds that are 600-fold more potent than the PRC2 were pre-incubated for 2 hours, then diluted 100-fold and added by transferring 1.4 μL to a 138.6 μL volume containing 1 μM activation peptide, 5.05 μM substrate peptide (H2N-RKQLATKAARKSAPATGGVKKP-NTPEGbiot), 1.01 μM SAM and 1.01 μM 3 For the control reaction, 0.202 nM PRC2 was mixed with 1 μM activation peptide, 1.01 μM SAM, 1.01 μM 3 H-SAM and K i The compound, which is 6.06 times more potent than the PRC2, was pre-incubated in 138.6 μL buffer for 2 hours, and then the reaction was initiated by adding 1.4 μL 500 μM substrate peptide in water. The reaction was quenched at different time points up to 10 hours by transferring 8 μL aliquots from the reaction vessel to a plate containing 8 μL STOP solution (50 mM Tris pH 8.5, 200 mM EDTA, 2 mM SAH) per well. After the last time point, 12 μL of the quenched solution was transferred to a streptavidin-coated FlashPlate (PerkinElmer) containing 40 μL STOP solution per well, incubated for 1 to 8 hours, washed, and read in a TopCount plate reader (PerkinElmer).

[0276] Use custom scripts to control the fitting of reaction progress data to a straight line and the dilution of reaction progress data:

[0277]

[0278] where y is the product formed, t is the reaction time, v i is the initial velocity, v s is the steady-state velocity, and k obs is the rate constant for the transition from the initial velocity phase of the curve to the steady-state velocity phase of the curve. When fitting dilution reaction progress data, v s is constrained to be the slope of the straight line fitted to the control reaction progress data. The fitted value k obs Converted to residence time:

[0279]

[0280] Where τ is the residence time, K i is the inhibition constant, [EI] is the calculated equilibrium concentration of the enzyme-inhibitor complex, [E] is the calculated equilibrium concentration of the free enzyme, and [I] is the calculated equilibrium concentration of the free inhibitor.

[0281] Cell permeability measurements of inhibitors in the MDCK assay

[0282] Cell culture: MDCK II cells (obtained from Piet Borst, Netherlands Cancer Institute) were seeded at 2.5 x 105 cells / mL onto polyethylene membrane (PET) in a 96-well BD insert system until 4-7 days to form a confluent cell monolayer.

[0283] Experimental Procedure: Test and reference compounds (nadolol, metoprolol, and digoxin) were diluted from stock solutions to a concentration of 2 μM (<1% DMSO) in transport buffer (HBSS containing 10 mM Hepes, pH 7.4) and applied to the apical (A) or basolateral (B) side of the cell monolayer. Permeation of test compounds from the A to B direction or from the B to A direction was determined in duplicate with a P-gp inhibitor (GF120918, 10 μM). 10 μM digoxin was also tested in the A to B direction or the B to A direction with / without the P-gp inhibitor (GF120918, 10 μM), while 2 μM nadolol and metoprolol were tested in duplicate without the P-gp inhibitor (GF120918, 10 μM) in the A to B direction. The plates were incubated in a CO2 incubator at 37±1°C for 2.5 hours at 5% CO2 under saturated humidity without shaking. In addition, the efflux ratio of each compound was determined. The test and reference compounds were quantified by LC / MS / MS analysis based on the peak area ratio of analyte / IS. After the transport assay, the lucifer yellow exclusion assay was applied to determine the cell monolayer integrity. The buffer was removed from the apical and basolateral chambers, and then 75 μL of 100 μM lucifer yellow was added to the transport buffer and 250 μL of transport buffer was added to the apical and basolateral chambers, respectively. The plates were incubated at 37°C for 30 minutes at 5% CO2 and 95% relative humidity without shaking. After incubation for 30 minutes, 20 μL of lucifer yellow sample was removed from the apical side, and 60 μL of transport buffer was added. Then 80 μL of lucifer yellow sample was removed from the basolateral side. The relative fluorescence units (RFU) of lucifer yellow were measured at 425 / 528 nm (excitation / emission) using a molecular device M2e plate reader.

[0284] Data analysis: The apparent permeability coefficient Papparent (cm / s) is calculated using the following formula:

[0285] P 表观 =(C r / dt)×V r / (A×C0)

[0286] where dC r / dt is the cumulative concentration of the compound in the receptor chamber over time (μM / s); V r is the volume of solution in the receptor compartment (0.075 mL on the apical side and 0.25 mL on the basolateral side); A is the surface area for transport, i.e., the area of ​​the monolayer is 0.0804 cm2; C0 is the initial concentration in the donor compartment (μM).

[0287] The outflow ratio is calculated using the following equation:

[0288] Outflow ratio = P 表观 (BA) / P 表观 (AB)

[0289] Calculate the percent recovery using the following equation:

[0290] Recovery % = 100 × [(V r ×C r )+(V d ×C d )] / (V d ×C0)

[0291] Where V d is the volume of the donor chamber (0.075 mL on the apical side and 0.25 mL on the basolateral side); C d and C r are the final concentrations of the transported compound in the donor and acceptor compartments, respectively.

[0292] The percentage of Lucifer Yellow in the basolateral wells was calculated using the following equation:

[0293]

[0294] RFU 顶端 and RFU 基底外侧 V are the relative fluorescence units of fluorescein in the apical pore and basolateral pore, respectively; 顶端 and V 基底外侧 The volumes of the apical and basolateral pores are respectively (0.075 mL and 0.25 mL). The percentage of fluorescein should be less than 2. The data are shown in Table 6.

[0295] Table 6

[0296]

[0297]

[0298]

Claims

1. A compound of formula I: or a pharmaceutically acceptable salt thereof, wherein: R 1 Yes – SCH3; X is CH; R 2 It is a halogen; R 3 is (C1-C4)alkyl; R 4 Is selected from 1 to 2 -NR a R b A cyclohexyl group substituted with: R a and R b Together with the nitrogen atom to which it is attached, it forms an optionally fluorinated or -OR c Substituted azetidinyl; R c is -CH3, -CHF2 or cyclopropyl; and R 5 It is a (C1-C4) alkyl group.

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

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

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

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

6. A compound according to any one of claims 1 to 5, wherein the group and NR a R b It is trans oriented about the cyclohexyl group.

7. A compound according to any one of claims 1 to 5, wherein the group and NR a R b The cyclohexyl group is cis-oriented.

8. The compound according to any one of claims 1 to 5, wherein the stereochemical configuration of the chiral center of the 1,3-dioxolanyl group is R.

9. The compound according to any one of claims 1 to 5, wherein the stereochemical configuration of the chiral center of the 1,3-dioxolanyl group is S.

10. The compound according to any one of claims 1 to 5, wherein the compound has the following formula: or a pharmaceutically acceptable salt thereof.

11. A pharmaceutical composition comprising the compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.

12. Use of a compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof or a composition according to claim 11 in the preparation of a medicament for treating cancer, wherein the cancer is selected from bladder cancer, blastoma, bronchial cancer, bone cancer, breast cancer, brain cancer, cervical cancer, colorectal cancer, esophageal cancer, glioblastoma multiforme, gallbladder cancer, gastric cancer, head and neck cancer, kidney cancer, laryngeal cancer, leukemia, lung cancer, lymphoma, liver cancer, mesothelioma, multiple myeloma, melanoma, eye cancer, optic nerve tumor, oral cancer, ovarian cancer, pituitary tumor, prostate cancer, pancreatic cancer, pharyngeal cancer, renal cell carcinoma, sarcoma, skin cancer, spinal tumor, testicular cancer, thyroid cancer, laryngeal cancer, urothelial carcinoma, uterine cancer, vaginal cancer and Wilms' tumor.

13. The use according to claim 12, wherein the cancer is selected from breast cancer, prostate cancer, colon cancer, renal cell carcinoma, glioblastoma multiforme, bladder cancer, melanoma, bronchial carcinoma, lymphoma, cholangiosarcoma, multiple myeloma, lung cancer, ovarian cancer and liver cancer.

14. The use according to claim 12, wherein the cancer is selected from adenocarcinoma, adult T-cell leukemia / lymphoma, intestinal cancer, malignant epithelial tumors, and genitourinary cancer.

15. The use according to claim 12, wherein the cancer is selected from the group consisting of colorectal cancer, small intestinal cancer, glioma and acute myeloid leukemia.

16. The use according to claim 12, wherein the cancer is selected from cholangiosarcoma, myeloid sarcoma, gastrointestinal cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, small cell lung cancer, non-small cell lung cancer, diffuse large B-cell lymphoma, primary central nervous system lymphoma, and T-cell lymphoma.

Images