EA042914B1 - MODULATORS OF METHYL-MODIFYING ENZYMES, COMPOSITIONS AND THEIR APPLICATIONS - Google Patents

Description

Cross-reference to related applications

This application claims priority of U.S. Provisional Application No. 62/659408, filed April 18, 2018, which is hereby incorporated by reference in its entirety.

State of the art

Eukaryotic chromatin is made up of macromolecular complexes called nucleosomes. The nucleosome has 147 base pairs of DNA wrapped around a protein octamer containing two subunits of each of the H2A, H2B, H3, and H4 histone proteins. Histone proteins are subject to post-translational modifications, which in turn affect chromatin structure and gene expression. One type of post-translational histone modification is the methylation of lysine and arginine residues. Histone methylation plays a critical role in the regulation of gene expression in eukaryotes. Methylation affects chromatin structure and is associated with both activation and repression of transcription (Zhang and Reinberg, Genes Dev. 15: 2343-2360, 2001). Enzymes that catalyze the attachment and removal of methyl groups from histones are involved in gene silencing, embryonic development, cell proliferation, and other processes.

One class of histone methylases is characterized by the presence of a SET domain containing about 130 amino acids. EZH2 is an example of a human methylase containing a SET domain. EZH2 associates with EED (embryonic ectoderm development) and SUZ12 (zeste homologue suppressor 12) to form a complex known as PRC2 (Polycomb Group Repressive Complex 2), which has the ability to trimethylate histone H3 at lysine 27 (Cao and Zhang, Mol. Cell 15: 57-67, 2004). PRC2 complexes may also include RBAP46 and RBAP48 subunits. Another example is the related methylase EZH1.

The oncogenic activity of EZH2 has been shown in a number of studies on various types of cancer. ~1520% of GCB-DLBCLs carry an increase in function mutation in EZH2 (residue Y641), and these cells are hypersensitive to EZH2 inhibition both in vitro and in vivo (McCabe et al., 2012; Bradley et al., 2014). In cell line experiments, EZH2 overexpression induces cell invasion, soft agar growth, and motility, while EZH2 knockdown inhibits cell proliferation and cell invasion (Kleer et al., 2003, Nat. Acad. Sci. USA 100: 11606-11611 ; Varambally et al., (2002), The polycomb group protein EZH2 is involved in the progression of prostate cancer, Nature 419, 624-629). EZH2 has been shown to repress the expression of several tumor suppressors including E-cadherin, DAB2IP and RUNX3 among others. In xenograft models, EZH2 knockdown suppresses tumor growth and metastasis. Inhibitory modulation of EZH2 has been shown to block prostate cancer metastasis 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). It has recently been demonstrated that EZH2 is overexpressed in neuroendocrine tumors, and inhibition of EZH2 in mouse tumors restores androgen dependence (Ku et al, Science, 355, 2017). Overexpression of EZH2 is associated with the aggressiveness of certain cancers such as breast cancer (Kleer et al., Proc. Nat. Acad. Sci. USA 100: 11606-11611, 2003). Recent studies also suggest that the prostate cancer-specific TMPRSS2-ERG oncogenic fusion gene induces repressive epigenetic programs through direct activation of 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).

Considering their role in the regulation of various biological processes, methyl-modifying enzymes, in particular EZH2 and its mutants, are attractive targets for modulation.

The essence of the invention

The compounds described herein, and their pharmaceutically acceptable compositions, have been found to modulate EZH2 activity (see, for example, Table 5). Such compounds include compounds of the structural formulas

or their pharmaceutically acceptable salts, where each of R ¹ , R ⁶ , R ^a , R ^b and X is defined in this document.

In one aspect, it has also been found that certain compounds described herein have increased retention time (eg, >100 h) and increased permeability. It has been found that an increase in retention time (ie slower withdrawal rate) and/or permeability correlates with improved cellular activity.

The disclosed compounds, pharmaceutically acceptable salts, and pharmaceutically acceptable compositions are useful in the treatment of a variety of conditions associated with methyl-modifying enzymes. These conditions include, for example, one or more types of cancer.

- 1 042914

Detailed Description of the Invention

1. General description of connections

Compounds of formula I are provided

About OR ³

^r2 (i);

or their pharmaceutically acceptable salts, where

R ¹ is halogen, -S(C ₁ -C ₄ )alkyl, -S(C ₃ -C ₇ )cycloalkyl or -S[halo(C ₁ -C ₄ )alkyl];

X is CH or N;

R ² is hydrogen, halogen, (C1-C ₄ )alkyl or halo(C1-C ₄ )alkyl;

R ³ is halogen, (C1-C ₄ )alkyl or halo(C1-C ₄ )alkyl;

R ⁴ is (C ₃ -C ₇ )cycloalkyl or 4-7 membered heterocyclyl, each of which is optionally substituted with 1-3 groups selected from halo, (O ₁ -C ₄ )alkyl, halo(C1-C ₄ ) alkyl, (C1C ₄ )alkoxy, halo(C1-C ₄ )alkoxy and -NR ^a R ^b ;

R ² represents hydrogen, (C ₁ -C ₄ )alkyl or halo(C1-C ₄ )alkyl;

R ^b is (C ₁ -C ₄ )alkyl, halo(C1-C ₄ )alkyl or 4-7 membered heterocyclyl, wherein said heterocyclyl is optionally substituted with 1-3 groups selected from halogen, (C ₁ -C ₄ ) alkyl, halo(C ₁ -C ₄ )alkyl, (C ₁ -C ₄ )alkoxy and halo(C ₁ -C ₄ )αlkoxy; or

R ^a and R ^b together with the nitrogen atom to which they are attached form a 4-7 membered heterocyclyl optionally substituted with 1-3 groups selected from halo, (C ₁ -C ₄ )alkyl, halo(C1-C ₄ ) alkyl and -OR ^c ;

R ^c is (C ₁ -C ₄ )alkyl, halo(C 1 -C ₄ )alkyl or (C ₃ -C ₇ )cycloalkyl and

R ⁵ is halogen, (C ₁ -C ₄ )alkyl or halo(C ₁ -C ₄ )αkyl.

2. Definitions

When used in connection with describing a chemical group that may have multiple attachment points, a hyphen (-) denotes the attachment point of that group to the variable for which it is defined. For example, -S(C ₃ -C ₇ )cycloalkyl and -S[halo(C ₁ -C ₄ )alkyl] mean that the point of attachment of this group is on the sulfur atom.

As used herein, the terms halogen and halogen atom refer to an atom selected from fluorine (fluorine, -F), chlorine (chlorine, -Cl), bromine (bromine, -Br), and iodine (iodine, -I).

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

The term haloalkyl includes mono, poly and perhaloalkyl groups, where the halogens are independently selected from fluorine, chlorine, bromine and iodine.

Alkoxy is an alkyl group that is attached to another moiety via an oxygen linker (-O(alkyl)). Its non-limiting examples include methoxy, ethoxy, propoxy and butoxy.

A haloalkoxy is a haloalkyl group that is attached to another moiety via an oxygen atom, such as, but not limited to, -OCCHCF2 or -OCF ₃ .

The term cycloalkyl refers to a 3-12 membered (eg 3-7 membered) monocyclic, bicyclic (eg bridged or spiro bicyclic ring) or polycyclic (eg tricyclic) hydrocarbon ring system that is fully saturated. Monocyclic cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Bridged bicyclic cycloalkyl groups include, without limitation, bicyclo[3.2.1]octane, bicyclo[2.2.1]heptane, bicyclo[3.1.0]hexane, bicyclo[1.1.1]pentane, and the like. Spirobicyclic cycloalkyl groups include, for example, spiro[3.6]decane, spiro[4.5]decane and the like. Fused cycloalkyl rings include, for example, decahydronaphthalene, octahydropentalene, and the like. When indicated as optionally substituted or substituted, substituents on the cycloalkyl (eg, in the case of optionally substituted cycloalkyl) may be present at any substitutable position and include, for example, the position to which the cycloalkyl group is attached.

The term heterocyclyl means a 3-12 membered (e.g. 4-, 5-, 6- and 7-membered) saturated or partially unsaturated heterocyclic ring containing from 1 to 4 heteroatoms independently selected from N, O and S. It may be monocyclic, bicyclic (eg bridged, fused or spiro bicyclic ring) or tricyclic. The terms heterocycle, heterocyclyl, heterocyclyl ring, heterocyclic group, heterocyclic moiety, and heterocyclic radical are used interchangeably herein. A heterocyclyl ring may be attached to its pendant group at any heteroatom or carbon atom such that

- 2 042914 leads to a stable structure. Examples of such saturated or partially unsaturated heterocyclic radicals include, without limitation, tetrahydrofuranyl, tetrahydrothienyl, therahydropyranyl, pyrrolidinyl, pyridinonyl, pyrrolidonyl, piperidinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, morpholinyl, dihydrofuranyl, dihydropyranyl, dihydropyridinyl, tetrahydropyridine silt, dihydropyrimidinyl, oxetanil, azetidinyl and tetrahydropyrimidinyl. The heterocyclyl group may be mono- or bicyclic. The term heterocyclyl also includes, for example, unsaturated heterocyclic radicals fused to another unsaturated heterocyclic radical or an aryl or heteroaryl ring, such as, for example, tetrahydronaphthyridine, indolinone, dihydropyrrolotriazole, imidazopyrimidine, quinolinon, dioxaspirodecane. When indicated as being optionally substituted or substituted, 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 to which the heterocyclyl group is attached.

The term spiro refers to two rings that share a single ring atom (such as carbon).

The term fused refers to two rings that have two ring atoms adjacent to each other.

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

As used herein, the term geometric isomer refers to isomers that differ in the orientation of the substituent atoms with respect to the cycloalkyl ring, i. cis or trans isomers. When a described compound is named or depicted by a structure without specifying a specific cis or transgeometric isomeric form, it is to be understood that the name or structure encompasses one geometric isomer free from other geometric isomers, mixtures of geometric isomers, or mixtures enriched in one geometric isomer relative to the other corresponding geometric isomer. . Unless otherwise indicated, when the stereochemistry of a described compound is named or depicted by structure, the named or depicted stereoisomer is at least 60%, 70%, 80%, 90%, 99%, or 99.9% pure by weight relative to all other stereoisomers. When one enantiomer is named or depicted by a structure, the depicted or named enantiomer is at least 60%, 70%, 80%, 90%, 99%, or 99.9% optically pure by weight. The percentage of optical purity by weight is the ratio of the mass of an enantiomer to the sum of the mass of a given enantiomer and the mass of its optical isomer. Enantiomers can be separated by methods known to those skilled in the art, for example by the formation of diastereomeric salts, which can be separated, for example, by crystallization; formation of diastereomeric derivatives or complexes which can be separated, for example, by crystallization, supercritical fluid chromatography or liquid chromatography; selective reaction of one enantiomer with an enantiomer-specific reagent, such as enzymatic esterification; or supercritical fluid or liquid chromatography in a chiral medium, for example, on a chiral carrier, such as silicon dioxide with an associated chiral ligand, or in the presence of a chiral solvent. Particular enantiomers can also be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents, or by converting one enantiomer to another by asymmetric conversion. Unless otherwise indicated, when a described compound is named or depicted by a structure without indicating 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 not containing the corresponding an optical isomer or geometric isomer, a racemic mixture of a compound, and mixtures enriched in one enantiomer or geometric isomer relative to the other corresponding optical isomer or geometric isomer.

Unless otherwise indicated, when only some of the stereochemical centers in a described compound are named or depicted by a structure, the named or depicted configuration is enriched relative to the remaining configurations, for example, a molar excess of at least 60%, 70%, 80%, 90%, 99%, or 99.9%. For example, the substructure \

Cl means that the configuration of the chiral carbon is stereochemically enriched as R (for example, by a molar excess of at least 60%, 70%, 80%, 90%, 99% or 99.9%) and that the cyclohexyl geometry can be cis or trans - or a mixture of them. In some instances, a compound described has a structure with no indication of stereochemistry but is identified as stereochemically enriched (eg, single enantiomer; single geometric isomer, or single enantiomer in cases where geometric isomers are not possible). For example, unless otherwise noted,

Example 1 (single enantiomer, single geometric isomer) means that the configuration of the chiral dioxolanyl carbon is stereochemically enriched as R (for example, by a molar excess of at least 60%, 70%, 80%, 90%, 99%, or 99.9%) or is stereochemically enriched as S (for example, by a molar excess of at least 60%, 70%, 80%, 90%, 99%, or 99.9%) and that the geometric configuration of the cyclohexyl ring is enriched as cis (for example, by a molar excess of at least 60%, 70%, 80%, 90%, 99%, or 99.9%) or enriched as trans (e.g., by a molar excess of at least 60%, 70%, 80%, 90%, 99%, or 99.9%.

As used herein, the term patient means an animal such as a mammal and such as a human. The terms subject and patient may be used interchangeably.

The term pharmaceutically acceptable carrier refers to a non-toxic carrier, adjuvant or vehicle that does not interfere with the pharmacological activity of the compound with which it is formulated. Pharmaceutically acceptable carriers, adjuvants, or carrier vehicles that may 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, mixtures of partial glycerides 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, polyvinylpyrrolidone , cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene polyoxypropylene block polymers, polyethylene glycol and wool grease. The terms treatment, treat, and treating refer to the elimination, alleviation, or suppression of the progression of a disease or disorder, or one or more symptoms thereof, as described herein. Treatment can also be continued after the symptoms have disappeared, for example, to delay a relapse. Disease, disorder, and condition are used interchangeably herein.

The term effective amount or therapeutically effective amount refers to the amount of a compound described herein that will elicit a biological or medical response in a subject, such as a dosage of 0.01 to 100 mg/kg body weight/day. The terms subject and patient may be used interchangeably and refer to a mammal in need of treatment, such as pets (eg, dogs, cats, and the like), farm animals (eg, cows, pigs, horses, sheep, goats, and the like), and laboratory animals (eg rats, mice, guinea pigs, etc.). Typically, the subject is a human in need of treatment. The term inhibit, inhibition or inhibitory includes the reduction of the underlying activity of a biological activity or process. The compounds described herein may be present in the form of pharmaceutically acceptable salts. For use in medicines, the salts of the compounds described herein are referred to as non-toxic, pharmaceutically acceptable salts. Forms of pharmaceutically acceptable salts include pharmaceutically acceptable acid/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, phosphoric, nitric and sulfuric acids) and organic acids (such as acetic acid, benzenesulfonic, benzoic, methanesulfonic and para-toluenesulfonic acid).

3. Description of the Example Compounds

In a first embodiment, this specification provides compounds of formula I

or their pharmaceutically acceptable salts, where the variables are the same as indicated above for formula I.

In the second embodiment of the invention in the compounds of formula I

R ¹ is halogen or -S(C ₁ -C ₄ )alkyl;

R ² is halogen;

R ³ is (C ₁ -C ₄ )alkyl;

R ⁴ is (C ₃ -C ₇ )cycloalkyl or (C ₄ -C ₇ )heterocyclyl, each of which is optional

- 4 042914 heavily substituted with 1-2 groups selected from halo(C1- _C4 )alkyl and -NR ^a R ^b ;

R ^a is hydrogen or (C ₁ -C ₄ )alkyl;

R ^b is (C ₁ -C ₄ )alkyl or (C ₄ -C ₇ )heterocyclyl, wherein said heterocyclyl is optionally substituted with halo(C ₁ -C ₄ )alkyl; or

R ^a and R ^b together with the nitrogen atom to which they are attached form a 4-7 membered nitrogen-containing heterocyclyl optionally substituted with 1-3 groups selected from halogen and -OR ^c ;

R ^c is (C ₁ -C ₄ )alkyl, halo(C ₁ -C ₄ )alkyl or (C ₃ -C ₇ )cycloalkyl and

R ⁵ is halo or (C ₁ -C ₄ )alkyl.

In a third embodiment of the invention, the compound of formula I has the formula II

O O I lxv ⁴ _r s>x^r' d (P);

or a pharmaceutically acceptable salt thereof, wherein the other variables are as described above for formula I or the second embodiment of the invention. In a fourth embodiment of the invention, R1 in the compound of formula I or formula II is chlorine, where the remaining variables are as described above for formula I or the second embodiment of the invention.

In a fifth embodiment, R ¹ in a compound of formula I or formula II is -SCH3, with the remaining variables as described above for formula I or the second embodiment of the invention. In a sixth embodiment, R ⁴ in a compound of formula I or formula II is cyclohexyl or piperidinyl, each of which is optionally substituted with 1-2 groups selected from halo(C1-C ₄ )alkyl and -NR ^a R ^b , wherein the remaining variables are as described above for formula I or the second, fourth or fifth embodiments of the invention. In the seventh embodiment of the invention, the compound of formula I or formula II has the formula III ,R ^{6 0 0} । f/^ri ^{x Cl} (III);

or a pharmaceutically acceptable salt thereof, wherein R ⁶ is halo(C ₁ -C ₄ )alkyl and where the remaining variables are as described above for formula I or the second, fourth or fifth embodiments of the invention.

In an eighth embodiment of the invention, the compound of formula I, formula II or formula III has the formula IV

or a pharmaceutically acceptable salt thereof, wherein the other variables are as described above for formula I or the second, fourth, fifth or seventh embodiments of the invention.

In a ninth embodiment, the compound of formula I or formula II has the formula V

or a pharmaceutically acceptable salt thereof, where the remaining variables are as described above for formula I or the second, fourth or fifth embodiments of the invention.

In a tenth embodiment of the invention, the compound of formula I, formula II or formula V has formula VI or VII

or a pharmaceutically acceptable salt thereof, where the remaining variables and features are the same

- 5 042914 mi, as described above for formula I or the second, fourth or fifth embodiments of the invention.

In an eleventh embodiment of the invention, R ^b in the compounds of formula I, formula II, formula V, formula VI and formula VII is (C ₁ -C ₄ )alkyl or oxetanyl, where the specified oxetanyl is optionally substituted with halo(C ₁ -C ₄ )alkyl ; or R ^a and R ^b together with the nitrogen atom to which they are attached form azetidinyl, optionally substituted with halogen or -OR ^c , where the remaining variables and features are as described above for formula I or the second, fourth or fifth embodiments of the invention .

In the twelfth embodiment of the invention, R ^a in the compounds of formula I, formula II, formula V, formula VI and formula VII is hydrogen or methyl; and R ^b is methyl or oxetanyl, wherein said oxetanyl is optionally substituted with -CH2F or -CF ₃ , where the remaining variables and features are as described above for formula I or the second, fourth, fifth or eleventh embodiments of the invention.

In a thirteenth embodiment, R ^a and R ^b in the compounds of formula I, formula II, formula V, formula VI and formula VII, together with the nitrogen atom to which they are attached, form azetidinyl optionally substituted with 1-2 fluorines or -OR ^c ; and R ^c represents -CH ₃ , -CHF2 or cyclopropyl, where the remaining variables and features are as described above for formula I or the second, fourth, fifth or eleventh embodiments of the invention. In the fourteenth embodiment of the invention, the configuration of 1,3-dioxolanyl and the NR ^a R ^b group in any of formulas I, II, V, VI and VII is trans oriented relative to cyclohexyl, while the remaining features are as described above for formula I or the second , fourth, fifth, sixth, eleventh, twelfth or thirteenth embodiments of the invention. Alternatively, the configuration of 1,3-dioxolanyl and the NR ^a R ^b group of formulas I, II, V, VI, and VII is cis-oriented relative to cyclohexyl, with the remaining features as described above for formula I or the second, fourth, fifth, sixth, eleventh, twelfth or thirteenth embodiments of the invention. In the fifteenth embodiment of the invention, the stereochemical configuration of the chiral center of 1,3-dioxolanyl in any of formulas I, II, III, IV, V, VI and VII is R, while the remaining features are as described above for formula I or the second , fourth, fifth, sixth, eleventh, twelfth, thirteenth or fourteenth embodiments of the invention. Alternatively, the stereochemical configuration of the chiral center of 1,3-dioxolanyl in any of formulas I, II, III, IV, V, VI and VII is S, with the remaining features being as described above for formula I or the second, fourth , fifth, sixth, eleventh, twelfth, thirteenth or fourteenth embodiments of the invention.

Specific examples of compounds are presented in the EXAMPLES section and are included herein as part of the fourteenth embodiment of the invention. Also included are pharmaceutically acceptable salts as well as neutral forms of these compounds. In one aspect, this description includes racemic forms of any compound described herein.

4. Use, compilation and introduction

In some embodiments of the invention, this description refers to a composition containing a compound described herein, or a pharmaceutically acceptable derivative thereof, and a pharmaceutically acceptable carrier. The amount of the compound in the inventive composition is such that it is effective to measurably modulate a histone methyl-modifying enzyme or mutant thereof in a biological sample or patient.

In some embodiments, the composition described herein is formulated for administration to a patient in need of such a composition. The compositions described herein may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir. As used herein, the term parenteral includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. In some embodiments of the invention, these compositions are administered orally, intraperitoneally or intravenously. Sterile injectable forms of the compositions described herein may be an aqueous or oily suspension. These suspensions may be formulated according to methods known in the art using suitable dispersing or wetting agents and suspending agents.

In some embodiments of the invention, these compositions are administered orally.

The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the potency of the particular compound used, age, body weight, general health, sex, diet, timing of administration, rate of elimination, combination of drugs, and physician judgment and severity. specific disease being treated. The amount of a compound described herein in a composition will also depend on the particular compound in the composition.

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The compounds and compositions described herein are generally useful for modulating the activity of one or more enzymes involved in epigenetic regulation, and in particular EZH1 and EZH2, and even more specifically EZH2 and its mutant forms. In some embodiments, the compounds described herein negatively regulate or downregulate EZH2 activity. In some embodiments, the compounds described herein are antagonists of EZH2 activity. In some embodiments of the invention, the compounds described herein negatively regulate or suppress the activity of EZH1. In some embodiments, the compounds described herein are antagonists of EZH1 activity.

In some embodiments, the compounds and compositions described herein are useful in the treatment of diseases and/or disorders associated with overexpression of EZH1 or EZH2 and/or expression of a mutant form of EZH2, such as those mutants that alter the activity of an EZH2 substrate. Studies of EZH2 deletions, missense mutations, and frameshift mutations suggest that EZH2 acts as a tumor suppressor in blood disorders such as myelodysplastic syndromes (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 are useful in the treatment of diseases and/or disorders associated with the presence of EZH2 having the Y641N, Y641C, Y641F, Y641H, Y641S, A677G, or A687 mutation. In a specific aspect of this embodiment, EZH2 has the Y641N mutation.

In some embodiments, provided herein is a method of treating a subject suffering from a disease and/or disorder associated with overexpression of EZH1 or EZH2 and/or expression of a mutant form of EZH2, comprising the step of administering a compound described herein, or a pharmaceutically acceptable salt thereof, or compositions. In some embodiments of the invention, the above method further includes the preliminary step of determining whether the subject overexpresses EZH2 or a mutant form of EZH2. In some embodiments of the invention, said disease or disorder associated with the presence of a mutant form of EZH2 is human B-cell lymphoma. In some embodiments, said disease and/or disorder associated with the presence of Y641N EZH2 is follicular lymphoma or diffuse large B cell lymphoma. In some embodiments of the invention, these compounds or compositions described herein are useful in the treatment of blood disorders such as myelodysplastic syndromes, 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. Dec 2010; 109(48):20980-20985. In some embodiments of the invention, these compounds and compositions described herein are suitable for the treatment of diseases and/or disorders associated with cell proliferation. In some embodiments, the compounds and compositions described herein are useful in the treatment of diseases and/or disorders associated with cell cycle misregulation or DNA repair. In some embodiments of the invention, these compounds and compositions described herein are suitable for the treatment of cancer.

In one aspect, cancers that may be treated with the compounds, compositions, and methods described herein include, but are not limited to:

Heart: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma;

Lungs: bronchogenic carcinoma (squamous, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma;

Gastrointestinal tract: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small intestine (adenocarcinoma, lymphoma , carcinoid tumors, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large intestine (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma);

Genitourinary tract: kidneys (adenocarcinoma, Wilm's tumor (nephroblastoma), lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testicles (seminoma, teratoma, embryonic carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma);

Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma;

Bones: osteosarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytosis

- 7 042914 volumes, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticular cell sarcoma), multiple myeloma, malignant giant cell chordoma, osteochronfrom (bone cartilage extosis), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors;

Nervous system: skull (osteoma, hemangioma, granuloma, xanthoma, osteitis deformans), meninges (meningioma, meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma, glioma, ependymoma, germinoma (pinealoma), glioblastoma multiforme, oligodendroglioma, schwanoma, retin oblastoma , congenital tumors), spinal cord (neurofibroma, meningioma, glioma, sarcoma);

Gynecology: uterus (endometrial carcinoma), cervix (cervical carcinoma, premalignant cervical dysplasia), ovaries (ovarian carcinoma (serous cystadenocarcinoma, mucinous cystadenocarcinoma, clear cell carcinoma, unclassified carcinoma), serous cell tumor), granulosa cell tumors, tumors from Sertoli-Leydig cells, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonic rhabdomyosarcoma)), fallopian tubes (carcinoma);

Hematology: blood (myeloid leukemia (acute and chronic), acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma (malignant lymphoma);

Skin: malignant melanoma, basalioma, squamous cell carcinoma, Kaposi's sarcoma, moles, dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis and

Adrenal glands: neuroblastoma.

In one aspect, said cancer being treated with the compounds, compositions and methods described herein is selected from the following: adrenal cancer, acinar cell tumor, acoustic neuroma, acral lentiginous melanoma, acrospiroma, acute eosinophilic leukemia, acute erythroid leukemia , острый лимфобластный лейкоз, острый мегакариобластный лейкоз, острый моноцитарный лейкоз, острый промиелоцитарный лейкоз, аденокарцинома, аденокистозная карцинома, аденома, аденоматоидная одонтогенная опухоль, аденосквамозная карцинома, новообразование подкожно-жировой клетчатки, адренокортикальный рак, Т-клеточный лейкоз/лимфома взрослых, агрессивный лейкоз NK cell, AIDS-associated 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 prolymphocytic leukemia, B-cell lymphoma, basalioma, bile duct cancer, bladder cancer, blastoma, bone cancer, Brenner's tumor, brown tumor, Burkitt's lymphoma, breast cancer, brain cancer, carcinoma, preinvasive cancer, carcinosarcoma, cartilage tumor, cementoma, myeloid sarcoma, chondroma, chordoma, choriocarcinoma, choroid papilloma, renal clear cell sarcoma, craniopharyngioma, cutaneous T-cell lymphoma, cervical cancer, colorectal cancer, Degos disease, desmoplastic small round cell tumor, diffuse Large cell lymphoma, dysem bryoplastic neuroepithelial tumor, dysgerminoma, embryonic carcinoma, endocrine gland neoplasm, endodermal sinus tumor, enteropathy-associated T-cell lymphoma, esophageal cancer, fetal fetus in fetus, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, ganglioneuroma, gastrointestinal cancer, gonocytoma, uterine choriocarcinoma, giant cell fibroblastoma, giant cell bone tumor, glial tumor, glioblastoma multiforme, glioma, cerebral gliomatosis, glucagonoma, gonadoblastoma, granulosa cell tumor, gynandroblastoma, gallbladder cancer, gastrointestinal cancer, hairy cell leukemia, hemangioblastoma, head tumors and neck, hemangiopericytoma, hemoblastosis, hepatoblastoma, hepatolienal T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, invasive lobular carcinoma, colon cancer, kidney cancer, larynx cancer, lentigial melanoma, lethal median carcinoma, leukemia, Leydig cell tumor, liposarcoma, lung cancer, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphoma, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, liver cancer, small cell lung cancer, non-small cell lung cancer, MALT lymphoma, malignant fibrous histiocytoma, malignant peripheral nerve sheath tumor, malignant tritonitis tumor, mantle cell lymphoma, marginal zone B-cell lymphoma, mast cell leukemia, mediastinal germ cell tumor, medullary breast cancer, medullary thyroid cancer, medulloblastoma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, metastatic urothelial carcinoma, mixed müllerian tumor, mucosal tumor, multiple myeloma, neoplasms of muscle tissue, mycosis fungoides, myxoid liposarcoma, myxoma of the heart, myxosarcoma, nasopharyngeal carcinoma, neurinoma, neuroblastoma, neurofibroma, neuroma, nodular melanoma, eye cancer, oligoastrocytoma, oligodendroglioma, oncocytoma, meningioma of the membranes viewer leg nerve, tumor optic nerve, oral cancer, osteosarcoma,

- 8 042914 ovarian cancer, Pancoast tumor, papillary thyroid cancer, paraganglioma, pineoblastoma, pineocytoma, pituicytoma, pituitary adenoma, pituitary tumor, plasmacytoma, polyembryoma, T-lymphoblastic progenitor cell lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, primary peritoneal cancer, prostate cancer, pancreatic cancer, pharyngeal cancer, abdominal pseudomyxoma, renal cell carcinoma, renal medullary carcinoma, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter's transformation, rectal cancer, sarcoma, Schwannomatosis, seminoma, Sertoli cell tumors, sex cord stromal tumor, cricoid cell carcinoma, skin cancer, small blue round cell tumors, small cell carcinoma, soft tissue sarcoma, somatostatinoma, scrotum cancer, spinal cord tumor, splenic marginal zone lymphoma, squamous cell carcinoma, synovial sarcoma, Cesari's disease, small intestine cancer, squamous cell carcinoma , gastric cancer, T-cell lymphoma, testicular cancer, thecoma, thyroid cancer, transitional cell carcinoma, throat cancer, urachus cancer, genitourinary system cancer, urothelial carcinoma, uveal melanoma, uterine cancer, verrucous carcinoma, optic tract glioma, vulvar cancer, vaginal cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, and Wilms' tumor. In one aspect, said cancer being treated with the compounds, compositions and methods described herein is selected from the following: adenocarcinoma, adult T-cell leukemia/lymphoma, bladder cancer, blastoma, bone cancer, breast cancer, brain cancer brain, carcinoma, myeloid sarcoma, cervical cancer, colorectal cancer, esophageal cancer, gastrointestinal cancer, glioblastoma multiforme, glioma, gallbladder cancer, gastrointestinal cancer, head and neck tumors, Hodgkin's lymphoma, non-Hodgkin's lymphoma, colon 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 tumor nerve, oral cancer, ovarian cancer, pituitary tumor, primary central nervous system lymphoma, prostate cancer, pancreatic cancer, pharyngeal cancer, renal cell carcinoma, rectal cancer, sarcoma, skin cancer, spinal cord tumor, small intestine cancer, gastric cancer, T-cell lymphoma, testicular cancer, thyroid cancer, throat cancer, genitourinary cancer, urothelial carcinoma, uterine cancer, vaginal cancer, and Wilms tumor.

In one aspect, said cancer being treated with the compounds, compositions and methods described herein is selected from the following: breast cancer, prostate cancer, colon cancer, renal cell carcinoma, glioblastoma multiforme, bladder cancer, melanoma, bronchial cancer, lymphoma and liver cancer.

Also provided is the use of a compound described herein, or a pharmaceutically acceptable salt thereof, or a composition containing the described compound or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of the condition described herein. Also provided is a compound described herein, or a pharmaceutically acceptable salt thereof, or a composition containing the described compound, or a pharmaceutically acceptable salt thereof, for use in the treatment of the condition described herein.

Examples

The representative examples that follow are intended to help illustrate the invention and are not intended to and should not be construed as limiting the scope of the invention.

Obtaining Intermediates

Intermediate 1: 3-(aminomethyl)-6-methyl-4-(methylthio)pyridin-2(1H)-one (hydrochloride salt) o

O tBuONa, CS ₂ O SNa Mel, MeOH O

Toluene ^^^SNa tBuONa, tBuOH, 80 °C

Stage 1: Synthesis of sodium 3-oxobut-1-ene-1,1-bis(thiolate)

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 slow addition of carbon disulfide (6.6 g, 5.24 ml, 86 mmol). The resulting mixture was stirred at 0°C for 4 h, then filtered. The filter cake was dried under vacuum to give the title compound (15.4 g, crude) as a yellow solid, which was used in the next step.

- 9 042914 blowing stage without further purification.

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

To a solution of sodium 3-oxobut-1-ene-1,1-bis(thiolate) (15.4 g, 86.4 mmol) in methanol (90 ml) was added slowly 173 mmol). The mixture was stirred at 70°C for 1 h, then evaporated to dryness. Water (30 ml) was added and the desired product was extracted with ethyl acetate (60 ml x 3). The combined organic layers were dried over sodium sulfate and evaporated 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. LC-MS [M+H]+ m/z: calc. 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).

Stage 3: Synthesis of 6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridine-3-carbonitrile

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 reaction were performed and combined at this point). Water (20 ml) was added and the pH was adjusted to 5-6 with 10% hydrochloric acid. The resulting mixture was filtered and the filter cake was washed with petroleum ether (20 ml x 2), then the cake was dried under vacuum to give the title compound (4.8 g, 74% yield) as a greyish solid, which was used in the next step without further purification. . LC-MS [M+H]+ m/z: calc. 181.0; found 181.0.

¹ H NMR (400 MHz, dimethylsulfoxide-d ₆ ) δ 6.27 (s, 1H), 2.56 (s, 3H), 2.25 (s, 3H).

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

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 complex with dimethyl sulfide (10 M, 8.0 ml, 80 mmol) was then slowly added at 0°C, after which the reaction mixture was heated to 70°C and stirred for 2 h. Methanol (15 ml) was added slowly at 0° C to quench the reaction before the mixture was evaporated under reduced pressure to give the title compound (3.8 g, crude) as a light yellow solid, which was used in the next step without further purification. LC-MS [M+H]+ m/z: calc. 185.1; found 185.0.

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

To a solution of 3-(aminomethyl)-6-methyl-4-(methylthio)pyridin-2(1H)-one (3.6 g, 20 mmol) in tetrahydrofuran (80 ml) was added triethylamine (5.9 g, 1 ml, 59 mmol). The mixture was stirred for 30 min before adding di-tert-butyl dicarbonate (6.4 g, 29 mmol) and the reaction mixture was stirred at 25°C for 12 h. The reaction mixture was then evaporated to dryness under reduced pressure, after which water (35 ml) and the desired product was extracted with a mixture of petroleum ether/ethyl acetate 5:1 (30 ml x 3). The combined organic layers were dried over sodium sulfate and evaporated to give the title compound (5.8 g, crude) as a white solid, which was used in the next step without further purification. LC-MS [M+H]+ m/z: calc. 285.12; found 284.9.

1H NMR (400 MHz, dimethylsulfoxide-d6) δ 6.05 (s, 1H), 4.03-4.00 (m, 2H), 2.42 (s, 3H), 2.15 (s, 3H) , 1.39 (s, 9H).

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

To a solution of hydrogen chloride in 1,4-dioxane (4 M, 100 ml, 400 mmol) at 25°C was added tert-butyl ((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridine-3- yl)methyl)carbamate (5.0 g, 17.6 mmol). The reaction mixture was stirred at 25°C for 2 h, then evaporated to dryness under reduced pressure. The residue was washed with dichloromethane (30 ml x 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. LC-MS [M+H]+ m/z: calc. 185.1; found 185.0.

1H NMR (400 MHz, D ₂ O) δ 6.31 (s, 1H), 4.03 (s, 2H), 2.41 (s, 3H), 2.18 (s, 3H).

Intermediate 2: 5-(aminomethyl)-2-methyl-6-(methylthio)pyrimidin-4(3H)-one (hydrochloride salt)

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

A suspension of sodium hydride (60% dispersion in mineral oil) (4.23 g, 106 mmol) in tetrahydrofuran (400 ml) was cooled to 0°C before methyl cyanoacetate (8.90 ml, 101 ml) was added dropwise.

- 10 042914 mmol). The reaction mixture was vigorously stirred for 15 min, after which carbon disulfide (6.1 mL, 101 mmol) was added dropwise while maintaining the reaction temperature below 20° C. (white semi-solid turned yellow). Iodomethane (15.7 ml, 252 mmol) was then added dropwise and the reaction mixture was stirred at room temperature for 15 minutes. Then the reaction mixture was evaporated under reduced pressure and the residue was poured into ice water. The resulting precipitate was filtered and dried under vacuum to give the title compound (11.9 g, 58% yield) as a tan solid, which was used in the 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).

Stage 2: Synthesis of 2-methyl-4-(methylthio)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile

Methyl 2-cyano-3,3-bis(methylthio)acrylate (11.9 g, 58.5 mmol) was dissolved in ethanol (1000 ml). Acetamidine hydrochloride (5.53 g, 58.5 mmol) and triethylamine (36.7 ml, 263 mmol) were added to the mixture and stirred at reflux overnight. Then the reaction mixture was evaporated under reduced pressure and the residue was washed with 10% hydrochloric acid. The precipitate was filtered and the filter cake was washed with water and diethyl ether before drying in vacuo to give the title compound (5 g, 47% yield) as a solid, which was used in the next step without further purification. LC-MS [M+H]+ m/z: calc. 182.03; found 182.10.

1 H NMR (400 MHz, dimethyl sulfoxide-d ₆ ) δ 2.57 (s, 3H), 2.37 (s, 3H).

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

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 in tetrahydrofuran (2 M, 6.2 ml, 12.4 mmol). The reaction mixture was slowly warmed to room temperature and after completion the reaction was quenched with aqueous sodium hydroxide solution (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 succession. The biphasic mixture was vigorously stirred until the amine was completely 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 evaporated to dryness under reduced pressure. The residue was triturated with dichloromethane and the filtrate (containing the desired product) was evaporated to dryness under reduced pressure. The resulting solid was purified by reverse phase flash chromatography (column C18, gradient 5 to 95% acetonitrile in water buffered with 0.1% trifluoroacetic acid) followed by normal phase flash chromatography (silica gel, gradient 30 to 90% ethyl acetate in dichloromethane ) to give the title compound (2.57 g, 26% yield, >99% purity) as a white solid. LC-MS [M-(C ₄ H ₈ )+H]+ m/z: calc. 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).

Stage 4. Synthesis of 5-(aminomethyl)-2-methyl-6-(methylthio)pyrimidin-4(3H)-one (hydrochloride salt)

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 (4 M, 2.10 ml, 8.40 mmol). The reaction mixture was stirred at 23°C for 3 h, then a solution of hydrogen chloride in 1,4-dioxane (4 M, 2.10 ml, 8.40 mmol) was added. The reaction mixture was then evaporated to dryness under reduced pressure after 14 hours to give the title compound (254 mg, 94% yield) as a white solid, which was used in the next step without further purification. LC-MS [M+Na]+ (free base) m/z: calc. 208.1; found 208.1.

Intermediate 3: 3-(aminomethyl)-6-chloro-4-(methylthio)pyridin-2(1H)-one (hydrochloride salt)

Stage 1: Synthesis of 2-(benzyloxy)-6-chloronicotinic acid

To a suspension of sodium hydride (60% dispersion in mineral oil) (11.5 g, 286 mmol) in tetrahydrofuran (500 ml) at 0°C was added benzyl alcohol (14 ml, 143 mmol). The reaction mixture

- 11 042914 was stirred for 20 min, 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 h. Then the reaction quenched with 10% hydrochloric acid (300 ml), treated with solid NaHCO ₃ to pH ~8, acidified with acetic acid (50% in water) and finally extracted with ethyl acetate (200 ml x 3). The combined organic layers were washed with brine (200 ml), dried over magnesium sulfate, filtered and evaporated to dryness. The residue was purified by flash chromatography (silica gel, 5 to 50% ethyl acetate in dichloromethane gradient) to give the title compound (33.68 g, 98% yield).

Stage 2: Synthesis of 2-(benzyloxy)-6-chloronicotinamide

To a solution of 2-(benzyloxy)-6-chloronicotinic acid (33.68 g, 128.2 mmol) in dichloromethane (500 ml) at 0°C was added N,N-dimethylformamide (1 ml) followed by oxalyl chloride (11, 9 ml, 141 mmol). The reaction mixture was stirred at 23°C for 2 h, then evaporated to dryness. The residue was dissolved in dichloromethane (200 ml) and added slowly at 0°C to a concentrated ammonium hydroxide solution (50 ml). After 1 h the organic layer was collected and the aqueous phase was extracted with dichloromethane (50 ml x 3). The combined organic layers were dried over magnesium sulfate, filtered and evaporated to dryness under reduced pressure. The residue was purified by flash chromatography (silica gel, gradient 0 to 50% ethyl acetate in dichloromethane) to give the title compound (quantitative) as a solid.

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

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 heated to 0°C for 30 min, then cooled again to -78°C. Then a solution of 2-(benzyloxy)6-chloronicotinamide (11.5 g, 43.8 mmol) in tetrahydrofuran (100 ml) was added while maintaining the internal temperature below -60°C. The reaction mixture was stirred for 1 h at -78° C., then dimethyl disulfide (15.77 mL, 175.1 mmol) was added. The reaction mixture was further stirred at -78°C for 2 h, then quenched at -78°C with a solution of acetic acid in water (50% v/v). 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 hydrogen carbonate solution (20 ml), brine (20 ml), dried over magnesium sulfate, filtered and evaporated to dryness under reduced pressure . The residue was purified by flash chromatography (silica gel, 0 to 20% ethyl acetate in dichloromethane gradient) to give the title compound (4.86 g, 36% yield).

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

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 h, then quenched by slow addition of methanol at room temperature. The reaction mixture was evaporated to dryness under reduced pressure and the residue was purified by flash chromatography (silica gel, gradient 0 to 30% methanol in dichloromethane) to give the title compound (710 mg, 15% yield) as a white solid.

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

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 h, then evaporated to dryness under reduced pressure. The residue was recrystallized from a mixture of methanol and diethyl ether to give the title compound (400 mg, 69% yield) as a white solid.

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

Di-tert-butyl dicarbonate (2, 85 g, 13.1 mmol) followed by N,N-diisopropylethylamine (3.4 ml, 19.6 mmol) at room temperature. The reaction mixture was stirred at room temperature for 3 h (until complete dissolution), then quenched with water.

The organic layer was separated, dried over magnesium sulfate, filtered and evaporated to dryness under reduced pressure. The residue was purified by flash chromatography (silica gel, gradient 0 to 10% methanol in dichloromethane) to give the title compound (1.5 g, 75% yield) as a white solid. LC-MS [M+H]+ m/z: calc. 305.1; found 305.1.

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

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 (4 M, 1.96 ml, 7.87 mmol) at room temperature. The reaction mixture was stirred at room temperature for 3 h, then a solution of hydrogen chloride in 1,4-dioxane (4 M, 1.96 ml, 7.87 mmol) was added. After 14 hours, the reaction mixture was evaporated to dryness under reduced pressure to give the title compound (253 mg, quant.) as a white solid, which

- 12 042914 was used in the next step without further purification. LC-MS [M+Na]+ (free base) m/z: calcd. 227.0; found 227.0.

Intermediate 4: Methyl 7-chloro-2,4-dimethyl-2-(4-oxocyclohexyl)benzo[d][1,3]dioxol-5-carboxylate

Intermediate 4

Intermediate 4 Intermediate 4 (R enantiomer) (S enantiomer)

Stage 1: Synthesis of methyl 5-chloro-3,4-dihydro-2-methylbenzoate

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

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

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

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

The racemic mixture of methyl 7-chloro-2,4-dimethyl-2-(4-oxocyclohexyl)benzo[d][1,3]dioxol-5carboxylate (4.4 g, 13 mmol) was separated by preparative SFC [Column: ChiralPak AY from Daicel chemical industries (250 mm x 50 mm inner diameter, 10 µm). Mobile phase A: CO ₂ /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) (undesired enantiomer/distomer): Retention time = 6.2 min. Recovery = 1.4 g, 4.05 mmol, 31% yield, 90% ee, 98% purity (yellow solid).

1H NMR (400 MHz, chloroform-d) δ 7.48 (s, 1H), 3.78 (s, 3H), 2.44-2.36 (m, 2H), 2.35-2.25 ( m, 6H), 2.19 (tdd, J=2.8, 5.6, 13.1 Hz, 2H), 1.70-1.57 (m, 5H).

Intermediate 4 (Peak 2) (desired enantiomer/eutomer): Retention time = 7.0 min. Isolation = 1.1 g, 3.08 mmol, Yield 23.75%, 99% ee, 95% pure (yellow solid).

1H NMR (400 MHz, chloroform-d) δ 7.49 (s, 1H), 3.78 (s, 3H), 2.44-2.36 (m, 2H), 2.36-2.25 ( m, 6H), 2.20 (tdd, J=2.8, 5.6, 13.1 Hz, 2H), 1.72-1.59 (m, 5H).

SFC Analytical Method: [Column: ChiralPak AY-3 (150 x 4.6 mm ID, 3 µm). Mobile phase A: CO ₂ /Mobile phase B: 0.05% Et ₂ NH in iPrOH. 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 - undesired enantiomer/distomer): Retention time = 2.853 min. Intermediate 4 (Peak 2 - desired enantiomer/eutomer): Retention time = 2.979 min.

- 13 042914

Intermediate 5: Methyl 7-chloro-2,4-dimethyl-2-(trans-4-((3-(trifluoromethyl)oxetan-3yl)amino)cyclohexyl)benzo[b][1,3]dioxol-5- carboxylate

PI (single enantiomer) Intermediate 5 (single enantiomer, with cis or trans geometric isomers)

Mixture of 1-chloro-N-[3-(trifluoromethyl)oxetan-3-yl]hydrogenamine (678 mg, 3.82 mmol) and N,N-diisopropylethylamine (698 µl, 4.01 mmol) in dichloroethane (9.54 ml) stirred at room temperature for 30 min before adding acetic acid (218 μl, 3.82 mmol) followed by methyl 7-chloro-2,4-dimethyl-2-(4-oxocyclohexyl)benzo[b][1.3 ]dioxol-5-carboxylate (650 mg, 1.91 mmol) to this reaction mixture. After 5 minutes at room temperature the mixture became clear (yellow brown) then sodium triacetoxyborohydride (404 mg, 1.91 mmol) was added. The reaction mixture was stirred at room temperature for 3 hours, then quenched with saturated aqueous sodium hydrogen carbonate (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 evaporated to dryness under reduced pressure. The residue was purified by reverse phase flash chromatography (C18 column, acetonitrile/water 1:1 with 0.1% trifluoroacetic acid) to give two geometric isomers (cis and trans). The first elution peak corresponds to geometric isomer 1.

Analytical method LC-MS (for geometric isomers)

[Column: Zorbax SB-C8 (75 x 4.6 mm i.d., 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 min), then 100% mobile phase B (4.5 min). Flow rate: 1.5 ml/min. Column temperature: 20°C] Geometric isomer 1 (desired): Retention time = 4.073 min (278 mg, 31%). Geometric isomer 2 (undesired): Retention time = 4.277 min (253 mg, 29%).

The enantiomers of Geometric Isomer 1 (preferably based on LC-MS) were separated by preparative SFC [Column: Chiralcel OX-H (250 x 21 mm ID). Mobile phase A: CO ₂ /Mobile phase B: 0.25% isopropylamine in isopropanol/hexane 1:1. 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 min. LC-MS [M+H]+ m/z: calc. 464.15; found 464.

Intermediate 5 (Peak 2): Retention time = 2.1 min. LC-MS [M+H]+ m/z: calc. 464.15; found 464.2.

Intermediate 6: 3-(aminomethyl)-4-chloro-6-methylpyridin-2(1H)-one

Stage 1: Synthesis of 2,4-dichloro-6-methylnicotinonitrile

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 h, then quenched with saturated aqueous sodium hydrogen carbonate (to pH = 8). It was partitioned between water (2000 ml) and ethyl acetate (1000 ml), the organic layer was dried over sodium sulfate, filtered and evaporated to dryness under reduced pressure to give the title compound (85 g, 86% yield) as a brown solid which was used on next step without further purification. LC-MS [M+H]+ m/z: calc. 186.98; found 186.6.

Stage 2: Synthesis of 2-chloro-4-hydroxy-6-methylnicotinonitrile

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 reaction was stirred at 80° C. overnight 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 evaporated to dryness under reduced pressure. The title compound (8.8 g, 98% yield) was obtained as a brown solid and used in the next step without further purification. LC-MS [M+H]+ m/z: calc. 169.01; found 168.8.

Stage 3: Synthesis of 4-hydroxy-2-methoxy-6-methylnicotinonitrile

A mixture of 2-chloro-4-hydroxy-6-methylnicotinonitrile (8.8 g, 52.2 mmol) and sodium methoxide (14.1 g,

- 14 042914

261 mmol) in methanol (50 ml) was stirred at 60°C overnight. The mixture was quenched with 1 M hydrochloric acid to pH=5. It was partitioned between water (500 ml) and ethyl acetate (500 ml), the organic layer was dried over sodium sulfate and evaporated to give the title compound (8.0 g, 93% yield) in as a brown solid, which was used in the next step without further purification. LC-MS [M+H]+ m/z: calc. 165.1; found 164.8.

Stage 4: 4-Chloro-2-methoxy-6-methylnicotinonitrile

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 N,N-dimethylformamide (5 ml) in chloroform (100 ml) were stirred at 60°C for 30 min. The reaction was quenched with a saturated aqueous sodium bicarbonate solution (up to pH = 8). This was partitioned between water (1000 ml) and ethyl acetate (1000 ml), the organic layer was dried over sodium sulfate and evaporated 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 additional cleaning. LC-MS [M+H] ⁺ m/z: calc. 183.0; found 182.8.

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

To a solution of 4-chloro-2-methoxy-6-methylnicotinonitrile (8.0 g, 43.8 mmol) in tetrahydrofuran (50 ml) was added a complex of borane with dimethyl sulfide (10 M, 5.3 ml, 53 mmol). The mixture was stirred at 60°C for 2 h, then extinguished with methanol (10 ml) at 0°C. The mixture was evaporated to dryness under reduced pressure to give the title compound (7.0 g, 93% yield) as a brown solid, which was used directly in the next step without further purification. LC-MS [M+H] ⁺ m/z: calc. 187.1; found 187.1.

Stage 6: tert-butyl ((4-chloro-2-methoxy-6-methylpyridin-3-yl)methyl)carbamate

A mixture of (4-chloro-2-methoxy-6-methylpyridin-3-yl)methanamine (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 h, 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 evaporated to dryness under reduced pressure. The residue was purified by a 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. LC-MS [M+H] ⁺ m/z: calc. 287.1; found 286.9.

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

To a solution of hydrogen chloride in water (4 M, 10 ml, 10 mmol) was added tert-butyl ((4-chloro-2methoxy-6-methylpyridin-3-yl)methyl)carbamate (3.0 g, 10.5 mmol) at room temperature. The reaction mixture was heated at 100° C. for 2 h, then evaporated to dryness under reduced pressure to give the title compound (1.7 g, 94% yield) as a yellow solid. LC-MS [M+H] ⁺ m/z: calc. 173.04; found 173.1.

1H NMR (400 MHz, methanol^): δ 6.38 (s, 1H), 4.15 (s, 2H), 2.32 (s, 3H).

Intermediate 7: Methyl 7-chloro-2-(4-(3,3-difluoroazetidin-1-yl)cyclohexyl)-2,4dimethylbenzo[d][1,3]dioxol-5-carboxylate

F

E ,O ^F GP .

_-o AJyO O Separation of SFC^

MeOH, THF

ClCI

Intermediate 4 Intermediate 7 (Racemic mixture) (single geometric isomer)

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

Methyl 7-chloro-2,4-dimethyl-2-(4-oxocyclohexyl)benzo[b][1,3]dioxol-5-carboxylate solution (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) were stirred overnight at room temperature. The next day, the reaction mixture was cooled to -78°C and a solution of lithium borohydride (2 M in tetrahydrofuran, 1.21 ml, 2.43 mmol) was added dropwise. The thick yellow mixture was gradually warmed to room temperature, then quenched with saturated aqueous sodium carbonate at 0°C. The desired product was extracted with dichloromethane (three times) and the combined organic layers were dried over sodium sulfate, filtered and evaporated to dryness under reduced pressure. The residue was purified twice by flash chromatography (KP-NH silica gel column, 0 to 20% ethyl acetate in heptane gradient) to give the title compound.

- 15 042914 (354 mg, 53% yield) as a racemic mixture of one geometric isomer (cis or trans). LCMS [M+H] + m/z: calc. 416.9; found 416.2.

Step 2: Separation of methyl (2R)-7-chloro-2-(4-(3,3-difluoroazetidin-1-yl)cyclohexyl)-2,4dimethylbenzoA[1,3]dioxol-5-carboxylate and methyl (2S) -7-chloro-2-(4-(3,3-difluoroazetidin-1-yl)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxol-5-carboxylate

The racemic mixture of methyl 7-chloro-2-(4-(3,3-difluoroazetidin-1-yl)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxol-5-carboxylate (560 mg) was separated by preparative SFC [Column: Chiralpak AD-H from Daicel chemical industries. Mobile phase A: CO ₂ /Mobile phase B: 0.25% isopropylamine in a mixture of hexane and ethanol (1:1). Isocratic (90% mobile phase A and 10% mobile phase B). Flow rate: 80 g/min. Column temperature: 25°C]. Analytical method SFC: Column: Chiralpak AD-H from Daicel chemical industries (100 mm x 4.6 mm. Mobile phase A: CO ₂ /Mobile phase B: 0.1% isopropylamine in a mixture of hexane and ethanol (3:1). 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 analytical method) Isolation = 173 mg, yield 15%, ee 96%.

¹ H 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 7 (Peak 2): retention time = 1.16 min (SFC analytical method). Isolation = 150 mg, yield 13%, ee 97%. LC-MS [M+H] ⁺ m/z: calc. 416.86; found 416.2.

Intermediate 8: Perfluorophenyl 7-chloro-2,4-dimethyl-2-(1-(2,2,2-trifluoroethyl)piperidin-4-yl)benzo[d][1,3]dioxol-5-carboxylate

ClCI

F

Intermediate 8 (Racemic mixture)

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

In a 100 ml pear-shaped flask fitted with a condenser, a mixture of methyl 5-chloro-3,4-dihydroxy2-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/vacuum cycles (4 cycles), then toluene (12 ml) was added, purged with nitrogen/vacuum cycles, then stirred at 120° C. for 30 min. To the dark mixture was added a mixture of tert-butyl 4-ethynylpiperidine-1-carboxylate (2.32 g, 11.1 mmol) in toluene (10 mL). The resulting orange solution was further stirred for 2 hours at 120°C. The reaction mixture was evaporated under reduced pressure, then purified by flash chromatography (silica gel, 0 to 100% ethyl acetate in heptane gradient) to give the title compound (2.33 g, 98% yield) as a yellow oil. LC-MS [M+Na] ⁺ m/z: calc. 448.2; found 448.2.

Step 2: Synthesis of methyl 7-chloro-2,4-dimethyl-2-(piperidin-4-yl)benzo[d][1,3]dioxol-5carboxylate (trifluoroacetic acid salt)

To a yellow solution of tert-butyl 4-[7-chloro-5-(methoxycarbonyl)-2,4-dimethyl-2H-1,3-benzodioxol2-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 evaporated under reduced pressure to give the title compound (2.40 g, quant) as a gum, which was used in the next step without further purification. LC-MS [M+H] ⁺ m/z: calc. 326.79; found 326.2.

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

To a solution of the trifluoroacetic acid salt of methyl 7-chloro-2,4-dimethyl-2-(piperidin-4-yl)-2H-1,3-benzodioxol-5-carboxylate (2.40 g, 5.45 mmol) in toluene (20 ml) and acetonitrile (10 ml) were 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 evaporated under reduced pressure. Water (50 ml) was added and the mixture was extracted with EtOAc (30 ml x 3). The combined organic layers were washed with brine (50 ml x 2),

- 16 042914 was dried over sodium sulfate, filtered and evaporated under reduced pressure, then purified by flash chromatography (silica gel, gradient from 0 to 50% ethyl acetate in heptane) to obtain the title compound (2.04 g, 92%) as a yellowish oil . LC-MS [M+H]+ m/z: calc. 408.81; found 408.2.

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

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

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

To a stirred solution of 7-chloro-2,4-dimethyl-2-[1-(2,2,2-trifluoroethyl)piperidin-4-yl]-2H1,3-benzodioxole-5-carboxylic acid (665 mg, 1 68 mmol) in dichloromethane (5 ml) was added pyridine (1 ml, 12.4 mmol) followed by the addition of 2,3,4,5,6-pentafluorophenyl 2,2,2-trifluoroacetate (500 μl, 2.90 mmol) . After 20 minutes, the reaction mixture was evaporated under reduced pressure to give the title compound, which was used in the next step without further purification. LCMS [M+H] ⁺ m/z: calc. 560.84; found 560.2.

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

ClCI

Intermediate 4 (single geometric isomer) (Peak 2)

CI

Intermediate 9 (single enantiomer, single geometric isomer)

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

A solution of 3-methoxyazetidine hydrochloride (8 g, 64.75 mmol) and Ν,N-diisopropylethylamine (12 mL, 68.9 mmol) in methanol (30 mL) was stirred at room temperature for 30 min before another solution of methyl 7- chloro-2,4-dimethyl-2-(4-oxocyclohexyl)-1,3-benzodioxole-5-carboxylate (Intermediate 4 - Peak 2) (4.1 g, 12.10 mmol) in tetrahydrofuran (30 ml). The reaction mixture was stirred at room temperature for 1 h, then cooled to -70°C. Lithium borohydride (500 mg, 22.96 mmol) was added and the reaction mixture was stirred at -70°C for 30 min [or until complete consumption of the starting material, observed by TLC, ethyl acetate/methanol 5:1]. The two reaction batches were then combined and quenched with saturated aqueous ammonium chloride (120 ml) at 0° C. and the desired product was extracted with dichloromethane (200 ml x 3). The combined organic layers were dried over sodium sulfate, filtered and evaporated to dryness under reduced pressure. The residue was purified by flash chromatography (silica gel, gradient 0 to 14% methanol in dichloromethane) to give the title compound (8.05 g, 67% yield, 83% purity) as a light yellow oil. A sample (50 mg) was further purified by preparative thin layer chromatography (silica gel, ethyl acetate:methanol 15:1). LC-MS [M+H] ⁺ m/z: calc. 410.2; found 410.1. ¹ H NMR (400 MHz, Methanol-bd) 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 (w 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).

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

To a solution of methyl 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4dimethylbenzo[b][1,3]dioxol-5-carboxylate (4 g, 9.75 mmol) in methanol (48 ml) was added a solution of lithium hydroxide hydrate (4.03 g, 96.06 mmol) in water (12 ml). The reaction mixture was stirred at

- 17 042914

70°C for 2 h, then the two batches were combined and evaporated under reduced pressure. Water (50 ml) was added and the pH was adjusted to 6 with saturated aqueous citric acid at 0°C. The desired product was extracted with a mixture of dichloromethane and isopropanol (3:1) (300 ml x 5). The combined organic layers were dried over sodium sulfate, filtered and evaporated to dryness under reduced pressure to give the title compound (6.1 g, crude) as a greyish solid which was used in the next step without further purification. LC-MS [M+H]+ m/z: calc. 396.2; found 396.1.

¹ H NMR (400 MHz, methanol-d ₄ ) δ 7.07 (s, 1H), 4.05-4.10 (m, 2H), 3.76-3.88 (m, 1H), 3, 67 (w dd, J=10, 3.6 Hz, 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).

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]dioxol-5-carboxamide

Intermediate 4 (single geometric isomer)

Example 1 (single enantiomer, single geometric isomer)

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

Methyl 7-chloro-2,4-dimethyl-2-(4-oxocyclohexyl)benzo[d][1,3]dioxol-5-carboxylate solution (Intermediate 4 - Peak 2) (224 mg, 0.661 mmol), 3 -(difluoromethoxy)azetidine (336 mg, 2.72 mmol) and triethylamine hydrochloride (373 mg, 2.71 mmol) in a mixture of methanol (2 ml) and tetrahydrofuran (2 ml) were stirred at room temperature for 1.5 h, then cooled to -78°C. A solution of lithium borohydride in tetrahydrofuran (2 M, 500 µl, 1 mmol) was added dropwise at -78°C, after which the reaction mixture was allowed to warm to room temperature over 15 min. The mixture was then cooled to 0° C., then quenched with saturated aqueous sodium hydrogen carbonate solution, 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 using a hydrophobic filter and evaporated to dryness under reduced pressure. The residue was purified by flash chromatography (silica gel, gradient 10 to 100% ethyl acetate in heptane then 0 to 100% ethanol in ethyl acetate) to give one geometric isomer (cis or trans) of the title compound (205 mg, 70% yield). LC-MS [M+H] ⁺ m/z: calc. 445.9; found 446.2.

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

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

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

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 pyri

- 18 042914 dyne (1.0 ml, 12.4 mmol) followed by the addition of 2,3,4,5,6-pentafluorophenyl 2,2,2-trifluoroacetate (150 μl, 0.871 mmol) at room temperature. After 20 minutes, the reaction mixture was evaporated to dryness under reduced pressure to give a mixture of the title compound (100% theoretical yield = 243 mg) and side products. The crude mixture was used in the next step without further purification. LC-MS [M+H]+ m/z: calc. 597.91; found 598.2.

Step 4: Synthesis of 7-chloro-2-(4-(3-(difluoromethoxy)azetidin-1-yl)cyclohexyl)-2,4-dimethyl-N-((6methyl-4-(methylthio)-2-oxo- 1,2-dihydropyridin-3 -yl)methyl)benzo[d][1,3]dioxol-5-carboxamide

To a solution of perfluorophenyl 7-chloro-2-(4-(3-(difluoromethoxy)azetidin-1-yl)cyclohexyl)-2,4dimethylbenzo[d][1,3]dioxol-5-carboxylate (<243 mg, <0.406 mmol) in dimethylsulfoxide (1 ml) was added 3-(aminomethyl)-6-methyl-4-(methylthio)pyridin-2(1H)-one (hydrochloride salt) (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 h, then evaporated to dryness in a strong stream of nitrogen overnight.

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 hydrogen carbonate solution. The aqueous layer was washed with dichloromethane. The combined organic layers were dried over sodium sulfate and evaporated under reduced pressure to give the title compound (166 mg, 68% yield over two steps) as a single geometric isomer (cis or trans). LC-MS [M+H]+ m/z: calc. 598.1; found 598.3.

¹ H NMR (400 MHz, methanol-d ₄ ) δ 6.88 (s, 1H), 6.26 (s, 1H), 6.38 (t, J=74.3 Hz, 2H), 4.71 (quin, J=6.0 Hz, 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, 11.2 Hz, 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).

Table 1. The following compound was prepared analogously to example 1 using the appropriate starting material

Example Structure

(single enantiomer, single geometric isomer) Derived from Intermediate 3 and Intermediate 4 (Peak 2)

Analytical data LC-MS [M+H] ⁺ m/z: calc. 618.1; found 618.1. ^{X H} NMR (400 MHz, Chloroform-7) δ 6.91 - 6.88 (m, 1H), 6.58 (t, 7 = 5.4 Hz, 1H), 6.53 (s, 1H), 6.39 - 5.97 (m, 2H), 3.80 (d, 7=7.3 Hz, 2H), 3.14 (d, 7=6.4 Hz, 2H), 2.53 - 2 .49 (m, ZN), 2.26 (s, ZN), 2.13 (br. s., 1H), 1.99-1.78 (m, 5H), 1.60 (s, ZN) , 1.45 (d,7 = 3.4 Hz, 1H), 1.28-1.18 (m, 2H), 1.15 - 1.01 (m, 2H).

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]dioxol-5-carboxamide

Example 3

To a solution of (R)-7-chloro-2-(trans-4-(dimethylamino)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxol-5 carboxylic acid (obtained by the method described in the patent application US 2017/0073335 A1) (48 mg, 0.1356 mmol) in dimethyl sulfoxide (0.5 ml), triethylamine (56.6 μl, 0.407 mmol) and O(7-azabenzotriazol-1-yl)-N,N were added ,N',N'-tetramethyluronium hexafluorophosphate (41.2 mg, 0.108 mmol) at room temperature. Since the formation of the activated ester was incomplete according to LC-MS ([M+H]+ m/z: 368.2) after 5 min, additional O-(7azabenzotriazol-1-yl)-N,N,N was added twice. ',N'-tetramethyluronium hexafluorophosphate (16 mg, 0.042 mmol) in dimethyl sulfoxide (1 ml). After completion of acid activation, a suspension of 3-(aminomethyl)-6-chloro4-(methylsulfanyl)-1,2-dihydropyridin-2-one hydrochloride (65.3 mg, 0.271 mmol) and triethylamine (56.6 µl, 0.407 mmol) was added in dimethyl sulfoxide (0.5 ml) at room temperature. The reaction mixture for

- 19 042914 heated at 60°C for 45 min, then diluted with dichloromethane and water. The organic layer was dried using a hydrophobic filter and evaporated to dryness under reduced pressure. The residue was purified by flash chromatography [KP-NH silica gel column, gradient 0 to 100% ethyl acetate in heptane, then 0 to 100% ethanol in dichloromethane, then 0 to 100% methanol (with 20% ammonium hydroxide) in ethyl acetate] to give the title compound (30 mg, 41% yield) as a white solid. LC-MS [M+H]+ m/z: calc. 540.5; found 540.2.

¹ H NMR (400 MHz, methanol-J4) δ 6.89 (s, 1H), 6.68 (s, 1H), 4.50 (s, 2H), 3.20 (tt, J=3.2 , 11.9 Hz, 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 (dc, J=3.4, 12.4 Hz, 2H), 1.47-1.37 (m , 2H).

Table 2. The following compounds were obtained analogously to example 3 using the appropriate starting material

Example Structure Analytical data 4 \ O O I Ji Ji 1 nX \ Η N Ν Ύνθχ L II and N J Y; ¹ Cl (R enantiomer; trans geometric isomer) Derived from Intermediate 1 LC-MS [M+H] ⁺ m/z: calc. 520.2; found 520.3. Ή NMR (400 MHz, DMSO-7b) δ 11.52 (br C, 1H), 8.00 (t, 7 = 4.4 Hz, 1H), 6.85 (s, 1H), 6, 08 (s, 1H), 4.27 (d,7=4.4 Hz, 2H), 3.57 (s, 1H), 2.45 (s, 3N), 2.17 (s, 3N), 2.14 (s, 9H), 1.92 - 1.77 (m, 5H), 1.60 (s, ZN), 1.15 (t, 7= 9.0 Hz, 4H) 5 \ ^{0 0} 1 / \ n ^{N Ν} αΛ ^{η 1} Cl (R enantiomer; trans geometric isomer) Derived from Intermediate 2 LC-MS [M+H] ⁺ m/z: calc. 521.2; found 521.2. NMR (400 MHz, DMSO-7b) δ 12.60-12.19 (m, 2H), 8.05 (t, 7 = 4.4 Hz, 1H), 6.87 (s, 1H), 4, 21 (d, 7=4.4 Hz, 2H), 2.47 (s, 3H), 2.29 (s, 2H), 2.19-2.12 (m, 9H), 2.09 (w .s., 1H), 1.94 - 1.76 (m, 5H), 1.60 (s, ZN), 1.15 (t, 7 = 9.5 Hz, 4H)

Example 6 (Enantiomer 1). 7-Chloro-N-((6-chloro-4-(methylthio)-2-oxo-1,2-dihydropyridin-3yl)methyl)-2-(4-(dimethylamino)cyclohexyl)-2,4-dimethylbenzo[ b][1,3]dioxol-5-carboxamide

Example 6 (Enantiomer 1) (single enantiomer, single geometric isomer)

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

To a solution of methyl 7-chloro-2,4-dimethyl-2-[4-{[3-(trifluoromethyl)oxetan-3-yl]amino}cyclohexyl]

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2H-1,3-benzodioxol-5-carboxylate (Intermediate 5 - Peak 1) (81 mg, 0.174 mmol) in methanol (1 mL) sodium hydroxide (72 mg, 1.79 mmol) was added. The reaction mixture was heated at 60°C for 20 min, cooled to 0°C and then acidified to pH=2 with 1 M hydrochloric acid. The desired product was then extracted with dichloromethane (three times) and the combined organic layers were dried using a hydrophobic filter. The filtrate was evaporated 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. LC-MS [M+H]+ m/z: calc. 450.1; found 450.2.

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

To a solution of 7-chloro-2,4-dimethyl-2-(4-((3-(trifluoromethyl)oxetan-3-yl)amino)cyclohexyl)benzo[d][1,3]dioxole-5-carboxylic acid ( 70 mg, 155 µmol) in dichloromethane (0.5 ml) were added pyridine (24.9 µl, 310 µmol) and 2,3,4,5,6-pentafluorophenyl 2,2,2-trifluoroacetate (39.8 µl, 232 µmol) at room temperature. After 15 minutes, the reaction mixture was evaporated under reduced pressure to give a mixture of the title compound and by-products (68 mg, crude). The crude mixture was used in the next step without further purification. LC-MS [M+H] ⁺ m/z: calc. 616.1; found 616.2.

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

To a solution of 2,3,4,5,6-pentafluorophenyl 7-chloro-2,4-dimethyl-2-[4-{[3-(trifluoromethyl)oxetan-3yl]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-(methylsulfanyl)-1,2-dihydropyridin-2-one (free base) (30.4 mg, 0.165 mmol) and N,N-diisopropylethylamine (0.1 ml, 574 µmol) at room temperature. The reaction mixture was stirred at 50°C for 30 min, then directly added to a C18 column for purification by reverse phase flash chromatography (C18 column, gradient from 5 to 50% acetonitrile in water with 0.1% trifluoroacetic acid) to give the salt trifluoroacetic acid of said compound. The salt was dissolved in dichloromethane and the organic layer was washed with saturated aqueous sodium hydrogen carbonate. Finally, the organic layer was evaporated to dryness under reduced pressure to give the title compound (free base) (42 mg, 62% yield) as a white solid. LC-MS [M+H] ⁺ m/z: calc. 616.19; found 616.3.

¹ H NMR (400 MHz, chloroform-d) δ 12.62-12.44 (m, 1H), 7.16 (t, J=5.1 Hz, 1H), 6.91 (s, 1H), 6.04 (s, 1H), 4.76 (s, 3H), 4.62-4.46 (m, 4H), 2.72-2.63 (m, 1H), 2.49 (s, 3H), 2.32 (s, 3H), 2.27 (s, 3H), 1.92 (d, J=5.9 Hz, 4H), 1.80 (br C, 1H), 1, 60 (s, 3H), 1.36-1.10 (m, 6H).

Table 3. The following compounds were prepared analogously to example 6 using the appropriate starting material

Example Structure Analytical data 6 (Enantiomer 2) cr NH ^{0 0} 1 Η Ν N °\ 1 II and H JX ¹ Cl (single enantiomer, single geometric isomer) Derived from Intermediate LC-MS [M+H] ⁺ m/z: calc. 616.19; found 616.3. 'H NMR (400 MHz, Chloroform-7) δ 12.45 12.30 (m, 1H), 7.14 (t, 7=5.4 Hz, 1H), 6.91 (s, 1H), 6 .04 (s, 1H), 4.75 (d, J= Ί.3 Hz, 2H), 4.61 (s, 2H), 4.55-4.51 (m, 2H), 2.68 ( t, J= 9.5 Hz, 1H),

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Compound 1 and Intermediate 5 (Peak 2) 2.49 (s, ZN), 2.32 (s, ZN), 2.27 (s, ZN), 1.92 (d, 7=6.4 Hz, 4H), 1.83 (t, J \u003d 11.7 Hz, 1H), 1.61 (s, ZN), 1.35 - 1.12 (m, 6H). 7 (Enantiomer 1) F N— ^{0 0} 1 ajO ¹ CI (single enantiomer, single geometric isomer) Derived from Intermediate 1 and Intermediate 7 (Peak 1) LC-MS [M+H] ⁺ m/z: calc. 568.2; found 568.3. 'H NMR (400 MHz, Methanol-7^) δ 6.93-6.84 (m, 1H), 6.27 (s, 1H), 4.48 (s, 2H), 3.67 (br. T, J= 12.0 Hz, 4H), 2.52 (s, ZN), 2.29 (s, ZN), 2.26-2.19 (m, 1H), 2.18 (s, ZN ), 2.02-1.81 (m, 5H), 1.61 (s, 3H), 1.34-1.21 (m, 2H), 1.13 1.00 (m, 2H). 7 (Enantiomer 2) F .N— ^{0 0} 1 ¹ CI (single enantiomer, single geometric isomer) Label: Derived from Intermediate 1 and Intermediate 7 (Peak 2) LC-MS [M+H] ⁺ m/z: calc. 568.2; found 568.3. 'H NMR (400 MHz, Methanol-7^) δ 6.89 (s, 1H), 6.27 (s, 1H), 4.49 (s, 2H), 3.61 (t, J = 12, 0 Hz, 4H), 2.53 (s, ZN), 2.29 (s, ZN), 2.23 - 2.08 (m, 4H), 2.03 - 1.78 (m, 5H), 1.61 (s, 3H), 1.35-1.22 (m, 2H), 1.120.99 (m, 2H).

Example 8 7-Chloro-N-((4-chloro-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-(4-(3-cyclopropoxyazetidin-1-yl) cyclohexyl)-2,4-dimethylbenzo^][1,3]dioxol-5-carboxamide

ClCI

Intermediate 4 (single geometric isomer) (Peak 2)

Example 8 (single enantiomer, single geometric isomer)

Step 1: Synthesis of methyl 7-chloro-2-(4-(3-cyclopropoxyazetidin-1-yl)cyclohexyl)-2,4dimethylbenzo[1,3]dioxol-5-carboxylate

A solution of 3-(cyclopropoxy)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 h before adding another solution of methyl 7-chloro-2,4-dimethyl-2-(4-oxocyclohexyl)-1,3-benzodioxole-5-carboxylate (Intermediate 4 - Peak 2) (860 mg, 2.54 mmol) in tetrahydrofuran ( 30 ml). The reaction mixture was stirred at room temperature for 1.5 h, then cooled to -70°C. Lithium borohydride (120 mg, 5.51 mmol) was added and the reaction mixture was stirred at -70°C for 30 min [or until complete consumption of the starting material, observed by TLC, ethyl acetate/methanol 5:1]. The reaction was then quenched with saturated aqueous ammonium chloride (100 ml) and the desired product was extracted with ethyl acetate (50 ml x 2). The combined organic layers were washed with brine (30 ml), dried over sodium sulfate, filtered and evaporated to dryness under reduced pressure. The residue was purified by flash chromatography (silica gel, 50% to 100% ethyl acetate in petroleum ether gradient) to give the title compound (760 mg, 65% yield, 95% purity) as a yellow oil. LC-MS [M+H]+ m/z: calc. 436.1; found 436.0.

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

To a solution of methyl 7-chloro-2-(4-(3-cyclopropoxyazetidin-1-yl)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxol-5-carboxylate (760 mg, 1.74 mmol ) in methanol (15 ml) and water (3 ml) was added lithium hydroxide hydrate (962 mg, 22.93 mmol). The reaction mixture was stirred at 70°C for 15 h,

- 22 042914 was then evaporated under reduced pressure.

Water (15 ml) was added and the pH was adjusted to 6 with saturated aqueous citric acid at 0°C. The desired product was extracted with a mixture of dichloromethane and isopropanol (10:1) (30 ml x 3). The combined organic layers were washed with brine (150 ml x 2), dried over sodium sulfate, filtered and evaporated 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. LC-MS [M+H]+ m/z: calc. 422.1; found 422.0.

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

To a solution of 7-chloro-2-[4-[3-(cyclopropoxy)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-(7azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (500 mg, 1.31 mmol) and N,Ndiisopropylethylamine (1 ml, 5 .74 mmol). The mixture was stirred at 15°C for 30 min before adding 3-(aminomethyl)-4-chloro-6-methyl-1H-pyridin-2-one hydrochloride (Intermediate 6) (250 mg, 1.2 mmol). The reaction mixture was further stirred at 15° C. for 4 h, then filtered. The filtrate was purified by preparative HPLC [Column: Waters Xbridge (150 mm x 25 mm, 5 µm). Mobile phase A: water (0.05% v/v ammonium hydroxide/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 LC-MS [M+H]+ m/z: calc 576.2, found 576.2.

¹ H NMR (400 MHz, methanol^) δ 6.90 (s, 1H), 6.30 (s, 1H), 4.52 (s, 2H), 4.22 (quin, J=6.0 Hz , 1H), 3.61 (dd, J=6.4, 8.6 Hz, 2H), 3.31-3.25 (m, 1H), 2.99 (dd, J=6.3, 8 .4 Hz, 2H), 2.28 (s, 3H), 2.20 (s, 3H), 2.12-2.04 (m, 1H), 1.99-1.85 (m, 5H) , 1.62 (s, 3H), 1.33-1.24 (m, 2H), 1.06-0.95 (m, 2H), 0.56-0.51 (m, 2H), 0 .51-0.44 (m, 2H).

Table 4. The following compounds were prepared analogously to example 8 using the appropriate starting material

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(single enantiomer, single geometric isomer) Derived from Intermediate 1 and Intermediate 4 (Peak 2) 1H), 4.53 (s, 2H), 4.26-4.17 (m, 1H), 3.69 - 3.59 (m, 2H), 3.30 - 3.24 (m, 1H) , 3.06 2.94 (m, 2H), 2.54 (s, ZN), 2.31 (s, ZN), 2.21 (s, ZN), 2.10-2.02 (m, 1H), 1.991.80 (m, 5H), 1.62 (s, 3H), 1.35-1.18 (m, 2H), 1.080.93 (m, 2H), 0.58 - 0.50 (m, 2H), 0.50 - 0.42 (m, 2H). 10 ^{0 0} 1 <C\ ¹ CI (single enantiomer, single geometric isomer) Derived from Intermediate 2 and Intermediate 4 (Peak 2) LC-MS [M+H] ⁺ m/z: calc. 599.2; found 599.1.¾ NMR (400 MHz, Methanol d ₄ ) δ 6.92 (s, 1H), 6.40 (t, J = 76 Hz, 1H), 4.73 (t, 7=8 Hz, 1H ), 4.43 (s, 2H), 3.68 3.64 (m, 2H), 3.18 -3.14 (m, 2H), 2.57 (s, 3H), 2.40 (s , ZN), 2.21 (s, ZN), 2.14-2.09 (m, 1H), 1.97 - 1.83 (m, 5H), 1.62 (s, ZN), 1, 32 - 1.23 (m, 2H), 1.05 - 0.95 (m, 2H). eleven o ° 1 Cy ° ¹ CI (geometric isomer 1) Derived from Intermediate 1 and Intermediate 4 (Peak 2) LC-MS [M+H] ⁺ m/z: calc. 580.2; found 580.2. 'H NMR (400 MHz, Methanol _{d 4} ) δ 6.91 (s, 1H), 6.29 (s, 1H), 4.82-4.79 (m, 1H), 4.68 (s, 1H) , 4.55 (dd, J= 4.0.6.4 Hz, 2H), 4.52 - 4.46 (m, 4H), 2.63 (br. T, J= 10.8 Hz, 1H ), 2.55 (s, ZN), 2.31 (s, ZN), 2.21 (s, ZN), 1.94 (wide d, J = 13.2 Hz, 2H), 1.89 - 1.80 (m, ZN),

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1.62 (s, 3H), 1.31 (q, J = 12.0 Hz, 2H), 1.25-1.15 (m, 2H). eleven H ^{F 0} ° 1 O ⁰ 1 Cl (geometric isomer 2) Derived from Intermediate 1 and Intermediate 4 (Peak 2) LC-MS [M+H] ⁺ m/z: calc. 580.2; found 580.1. ¹ H NMR (400 MHz, Methanol^)5 6.91 (s, 1H), 6.30 (s, 1H), 4.75 (s, 1H), 4.63 (s, 1H), 4.62 - 4.59 (m, 2H), 4.52 - 4.47 (m, 4H), 3.06 (br. C, 1H), 2.55 (s, ZN), 2.31 (s, ZN ), 2.22 (s, 3H), 1.91 (br D, J= 10.4 Hz, 1H), 1.68 (br C, J= 8.4 Hz, 4H), 1.65 (s, 5H), 1.61 (br. C, 1H), 1.57 (br. C, 1H). 12 FN ^{0 0} 1 ¹ Cl (single enantiomer, single geometric isomer) Derived from Intermediate 1 and Intermediate 4 (Peak 2) LC-MS [M+H] ⁺ m/z: calc. 572.2; found 571.9. Ή NMR (400 MHz, Methanol d ₄ ) δ 6.89 (s, 1H), 6.27 (s, 1H), 4.96-5.23 (m, 1H), 4.48 (s, 2H), 3.54 - 3.69 (m, 2H)), 3.18 -3.30 (m, 2H), 2.52 (s, ZN), 2.29 (s, ZN), 2.19 (s , ZN), 2.06-2.15 (m, 1N), 1.80 - 1.98 (m, 5N), 1.61 (s, ZN), 1.19-1.32 (m, 2N ), 0.93 - 1.05 (m, 2H). 13 nO ^{f 0 0} 1 Cl LC-MS [M+H] ⁺ m/z: calc. 586.2; found 586.1. ^Χ Η NMR (400 MHz, Methanold4) δ 6.90 (s, 1H), 6.40 (t, J = 76 Hz, 1H), 6.30 (s, 1H), (single enantiomer, single geometric isomer) Derived from Intermediate 6 and Intermediate 4 (Peak 2) 4.73 (t, ./=8 Hz, 1H), 4.53 (s, 2H), 3.68-3.64 (m, 2H), 3.18-3.14 (m, 2H), 2.28 (s, ZN), 2.20 (s, ZN), 2.15-2.08 (m, 1H), 1.97 - 1.83 (m, 5H), 1.63 (s, 3H), 1.35-1.23 (m, 2H), 1.05-0.90 (m, 2H).

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

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

To a solution of 2,3,4,5,6-pentafluorophenyl 7-chloro-2,4-dimethyl-2-[1-(2,2,2-trifluoroethyl)piperidin-4-yl]

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2H-1,3-benzodioxol-5-carboxylate (Intermediate 8) (752 mg, 1.34 mmol) in dimethyl sulfoxide (4 ml) was added 3-(aminomethyl)-6-methyl-4-(methylsulfanyl)-1, 2-dihydropyridin-2-one (Intermediate 1) (740 mg, 4.02 mmol) and N,N-diisopropylethylamine (932 μl, 5.36 mmol). The reaction mixture was heated at 60°C and after 1 h was added to a C18 column for purification. The desired product was purified twice by reverse phase flash chromatography (C18 column, gradient from 0 to 100% acetonitrile in water with 0.1% trifluoroacetic acid) to give the title compound (289 mg, 38% yield) as a racemic gum. .

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

Racemic mixture of 7-chloro-2,4-dimethyl-N-((6-methyl-4-(methylthio)-2-oxo-1,2-dihydropyridin-3yl)methyl)-2-(1-(2,2 ,2-trifluoroethyl)piperidin-4-yl)benzo[d][1,3]dioxol-5-carboxamide (289 mg) was separated by preparative SFC [Column: ChromegaChiral CC4 from ES Industries (250 mm x 20 mm ID). Mobile phase A: CO ₂ /Mobile phase B: 0.25% isopropylamine in methanol. Isocratic (55% mobile phase A and 45% mobile phase B). Flow rate: 80 g/min. Column temperature: 25°C]. SFC Analytical Method: Column: Chiralcel OX-H from Chiral Technologies (100 mm x 4.6 mm id). Mobile phase A: CO ₂ /Mobile phase B: 0.1% isopropylamine in methanol. 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/eutomer): Retention time = 3.74 min (SFC analytical method). Isolation = 90 mg, 12% yield, 99% ee (yellow solid). LC-MS [M+H]+ m/z: calc. 560.2; found 560.2.

¹ H NMR (400 MHz, methanol^) 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) (undesired enantiomer/distomer): Retention time = 4.25 min (SFC analytical method). Isolation = 101 mg, 13% yield, 98% ee (yellow solid). LCMS [M+H] + m/z: calc. 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).

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]dioxol-5-carboxamide

To a solution of 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethylbenzo[d][1,3]dioxol-5carboxylic acid (900 mg, 2.27 mmol) in N,N-dimethylformamide (5 ml) was added O-(7azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.04 g, 2.73 mmol) and N,Ndiisopropylethylamine (2, 38 ml, 13.6 mmol). The mixture was stirred at 25°C for 30 min before adding 3-(aminomethyl)-4-chloro-6-methyl-1H-pyridin-2-one hydrochloride (Intermediate 6) (710 mg, 3.4 mmol). The reaction mixture was further stirred at room temperature for 1.5 h, then filtered. The filtrate was purified twice by preparative HPLC [Column: YMCActus Triart C18 (100 mm x 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: 30°C and Column: Xtimate C18 (150 mm x 25 mm, 5 µm). Mobile phase A: water (0.05% v/v ammonium hydroxide)/Mobile phase B: acetonitrile. Gradient (69 to 39% mobile phase A/31 to 61% mobile phase B, over 7 minutes). Column temperature: 30° C.] to give the title compound (533 mg, 43% yield) as a white solid. LC-MS [M+H] + m/z: calc. 550.2; found 550.1.

1H NMR (400 MHz, chloroform-d) δ 11.97 (broad, 1H), 7.06-7.03 (m, 1H), 6.88 (s, 1H), 6.19 (s, 1H) , 4.65 (d, J=6.0 Hz, 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).

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]dioxol-5-carboxamide and (S)-7-chloro-N-((4-chloro-6methyl-2- oxo-1,2-dihydropyridin-3-yl)methyl)-2,4-dimethyl-2-(1-(2,2,2-trifluoroethyl)piperidin-4-yl)benzo[d][1,3] dioxol-5-carboxamide

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Step 1: Synthesis of 7-chloro-N-((4-chloro-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2,4-dimethyl2-(1-(2,2 ,2-trifluoroethyl)piperidin-4-yl)benzo[d][1,3]dioxol-5-carboxamide

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-benzodioxol -5-carboxylate (Intermediate 8) (188 mg, 0.3358 mmol) in dimethyl sulfoxide (1 ml) was added 3-(aminomethyl)-4-chloro-6-methyl-1,2-dihydropyridin-2-one hydrochloride ( Intermediate 6) (70.2 mg, 0.336 mmol) and N,N-diisopropylethylamine (233 μl, 1.34 mmol).

The reaction mixture was heated at 60°C and after 1 h was added to a C18 column for purification. The desired product was purified twice by reverse phase flash chromatography (C18 column, gradient from 0 to 100% acetonitrile in water with 0.1% trifluoroacetic acid) to give the title compound (180 mg, 98% yield) as a racemic gum. .

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

Racemic mixture of 7-chloro-N-((4-chloro-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2,4dimethyl-2-(1-(2,2,2 -trifluoroethyl)piperidin-4-yl)benzo[d][1,3]dioxol-5-carboxamide (180 mg) was separated by preparative SFC [Column: ChromegaChiral CC4 from ES Industries (250 mm x 20 mm inner diameter). Mobile phase A: CO ₂ /Mobile phase B: 0.25% isopropylamine in methanol. Isocratic (65% mobile phase A and 35% mobile phase B). Flow rate: 80 g/min. Column temperature: 25°C].

SFC Analytical Method: Column: Chiralcel OZ-H from Chiral Technologies (100 mm x 4.6 mm i.d., 5 µm). Mobile phase A: CO ₂ /Mobile phase B: 0.1% isopropylamine in methanol. 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/eutomer): Retention time = 0.73 min (SFC analytical method). Recovery = 61 mg, 33% yield, 100% ee (yellow solid). Example 17 (Enantiomer 2) (undesired enantiomer/distomer): Retention time = 0.98 min (SFC analytical method). Recovery = 63 mg, 34% yield, 97% ee (yellow solid). LC-MS [M+H]+ m/z: calc. 548.1; found 548.2.

1H NMR (400 MHz, methanol-d ₄ ) δ 6.89 (s, 1H), 6.29 (s, 1H), 4.52 (s, 2H), 3.04 (q, J=9.8 Hz, 4H), 2.35 (br. T, 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).

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]dioxol-5-carboxamide

To a solution of 7-chloro-2-(4-(3-methoxyazetidin-1-yl)cyclohexyl)-2,4-dimethylbenzo^][1,3]dioxole-5carboxylic 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 min before adding 3-(aminomethyl)-6-methyl-4(methylthio)pyridin-2(1H)-one hydrochloride (Intermediate 1) (4.2 g, 19.03 mmol ). The reaction mixture was further stirred at room temperature for 1.5 h, then filtered. The filtrate was purified by preparative HPLC [Column: Phenomenex Gemini C18 (250 mm x 50 mm, 10 µm). Mobile phase A: water (0.04% v/v ammonium hydroxide and 10 mM ammonium hydrogen carbonate)/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. LC-MS [M+H]+ m/z: calc. 562.2;

- 27 042914 found 562.2.

¹ H NMR (400 MHz, methanol-d ₄ ) δ 6.91 (s, 1H), 6.29 (s, 1H), 4.50 (s, 2H), 4.01 (quin, J=6 Hz , 1H), 3.58 (dd, J=8.8, 6.4 Hz, 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).

EZH2 analyzes

IC ₅₀ measurements for inhibitors using EZH2

Biochemical analysis of EZH2 (IC ₅₀ ): The potency of the compounds was evaluated by incorporating ³ HSAM into the biotinylated H3 peptide. Specifically, 30 pM PRC2 containing wild-type EZH2 (pentameric complex produced by the company) was pre-incubated with 450 nM SAM, 450 nM ³ HSAM, 2 μM H3K27me3 activating peptide (H2N-RKQLATKAAR(Kme3)SAPATGGVKKP-amide) and compound (as a 10-point 2-fold dose response titration in DMSO, final assay 0.8% DMSO (v/v)) for 3-5 h in 50 mM Tris (pH 8.5), 1 mM DTT, 0. 07 mM Brij-35, 0.1% BSA and 0.8% DMSO in a total volume of 12.5 µl. The reaction was initiated with biotinylated H3 substrate peptide (H ₂ N-RKQLATKAAR(Kme1)SAPATGGVKKP-NTPEGBiot) as a 2 μM stock solution in 12.5 μl buffer and allowed to react at room temperature for 18-22 h. Quenching was accomplished by adding 20 μl STOP solution (50 mM Tris (pH 8.5), 200 mM EDTA, 2 mM SAH). 35 µl of the quenched solution was transferred to streptavidin-coated FlashPlates (PerkinElmer), incubated for 1-2 h, washed and read on a TopCount plate reader (PerkinElmer). IC ₅₀ was calculated in the Genedata Screener using non-linear 4-parameter least-squares fitting, where the four parameters were IC ₅₀ , Hill slope, pre-transition baseline (0% INH), and post-transition baseline (100% INH).

EC ₅₀ measurements for inhibitors in HeLa cell assays

H3K27me3 Alpha Hela (AlphaLISA) analysis. Ten different doses of each test compound (in a 3-fold dilution series) were plated in duplicate in 384-well tissue culture plates (catalog # 6007680; Perkin Elmer, Waltham, MA). Hela cells grown in culture were trypsinized and counted with a Countess® cell counter (catalog number C10281; Life Technologies, Grand Island, NY). Cells were diluted to 67,000 cells per ml in 10% DMEM (Part No. 10569-010 Life Technologies, Grand Island, NY) and 15 μl (1000 cells) using a Biotek MicroFlo™ Select dispenser (BioTek Instruments, Inc., Vermont , USA) were applied to each well of a 384-well plate. The plates were incubated at 37° C./5% CO2 for 72 hours. One of the duplicate plates was used for the HeLa assay and the other was used for the viability assay. 5 µl of histone cell lysis buffer (1X) (Part # AL009F1 Perkin Elmer; Waltham, MA) per well was added to the AlphaLISA-treated plate and the plate was incubated at room temperature for 30 min on a low speed plate shaker. (Model No. 4625-Q Thermo Scientific; Waltham, MA). Then 10 μl per well of histone extraction buffer (catalog # AL009F2; Perkin Elmer; Waltham, MA) was added and the plate was further incubated at room temperature for 20 minutes on a low speed plate shaker. Then, 10 μl of a 5x anti-K27me3 acceptor bead mix plus biotinylated anti-histone H3 antibody (C-ter) (diluted to a final concentration of 3 nM) (Catalog No. AL118 Perkin Elmer; Waltham, Massachusetts) was added to each well. Dilution of the acceptor beads and antihistone H3 was performed in 1X histone detection buffer (Perkin Elmer Cat# AL009F3; 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. Then 10 µl of 5x Streptavidin Donor Bead Solution (Part # 6760002 Perkin Elmer; Waltham, Massachusetts) was added (final concentration 20 µg/ml in 1x histone detection buffer) = the plate was sealed with aluminum sealant and incubated at 23°C C for 30 min. The plates were then read using an EnVision-Alpha Reader (Model No. 2104 Perkin Elmer; Waltham, MA). Cell viability was assessed by adding 15 μl of Cell Titer Glo (Promega Cat# G9241, Madison, Wisconsin) to each media cell well. The plates were incubated at room temperature for 15-20 minutes on a low speed plate shaker. The plates were then read using an EnVision-Alpha Reader (Model No. 2104 Perkin Elmer; Waltham, MA).

GI ₅₀ measurements for inhibitors in Karpas-422 viability assays

Karpas-422 cell lines were 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 plates coated with compound. Cells were split and seeded at initial seeding density (based on DMSO well count) every 4 days in plates containing fresh EZH2 inhibitors. Relative cell count was assessed using a fluorescent cell viability assay Cell Titer-Glo (Promega) on day 8 GraphPad Prism 5 was used to fit the curve and GI ₅₀ values were obtained. The data are presented in table. 5.

- 28 042914

Table 5

Example EZH2 1C ₅₀ (pM) HeLa ec ₅₀ (nM) Karpas422 GI ₅₀ (nM) N—1 ^{F 0 0} 1 ( \ 1 Cl Example 1 (single enantiomer, single geometric isomer) 91 0.52 3.9 N- ^{1 0 0} 1 6 \ CI^^^S ¹ Cl Example 2 (single enantiomer, single geometric isomer) 1500 15 250 \ ^{0 0} 1 /~L ¹ Cl Example 3 370 2.4 29 \ ^{0 0} 1 \ ¹ CI Example 4 (R enantiomer; trans geometric isomer) 38 0.51 4.0

- 29 042914

\ ο ο I / ( A Al ^H < ) NyAUMCHLA/--' A II ^H ϋ JX ¹ Cl Example 5 (R enantiomer; trans geometric isomer) 54 3.6 22 cxy^ NH ^{0 0} 1 ( \ 1 Cl Example 6 - Enantiomer 1 (single enantiomer, single geometric isomer) 1800 39 720 olu ₃ NH ^{0 0} 1 ( ^/ \ 1 CI Example 6 - Enantiomer 2 (single enantiomer, single geometric isomer) 140 4.2 53 F N - O O I X\ laahl ¹ CI Example 7 - Enantiomer 1 (single enantiomer, single geometric isomer) 8600

- 30 042914

F __/-FN—। ^{0 0} 1 ώ/φΟτ ¹ Cl Example 7 - Enantiomer 2 (single enantiomer, single geometric isomer) 120 12 0°^ ^{0 0} 1 / \ CI Example 8 (single enantiomer, single geometric isomer) 39 0.52 5.0 ⁰ ° 1 cChl ¹ CI Example 9 (single enantiomer, single geometric isomer) 54 0.61 3.7 ^{0 0} 1 -^N^S ¹ Cl Example 10 (single enantiomer, single geometric isomer) 202 1.9 20 H ^{0 0} 1 <2 ςΧ\ ^{0 1} CI 140 1.3 8.6

- 31 042914

Example 11 (single enantiomer; geometric isomer 1) H 1Cl Example 11 (single enantiomer; geometric isomer 2) 180 1.7 ___ F N—। ^{0 0} 1 f \ ¹ CI Example 12 (single enantiomer, single geometric isomer) 92 0.59 4.4 uu o o i tu Cl Example 13 (single enantiomer, single geometric isomer) 84 0.81 9.7 ^{0 0} 1 cG ^{3 1} Cl Example 14 - Enantiomer 1 (single enantiomer, single geometric isomer) 37 1.4

- 32 042914

Hold time measurements

EZH2 retention time analysis: Compound retention time was assessed by monitoring the recovery of enzyme activity after 100-fold dilution of the pre-formed enzyme-inhibitor complex (dilution reaction) and comparing it with the activity of an undiluted control with the same final concentrations of all reagents (control reaction). Enzyme activity was measured by the incorporation of 3H-SAM into the biotinylated H3 peptide. For the dilution reaction, 20 nM PRC2 containing wild-type EZH2 (pentameric complex produced by the company) was pre-incubated with 1 μM activating peptide (H ₂ N-RKQLATKAAR(Kme3)SAPATGGVKKPamide) and compound at 600 times Ki for 2 h in 40 µl of buffer (50 mM Tris pH 8.5, 4 mM DTT, 1 mM MgCl ₂ , 0.07 mM Brij-35 and 0.1 mg/ml BSA), then diluted 100 times and the reaction was initiated by transfer 1, 4 µl to 138.6 µl volume of buffer containing 1 µM activating peptide, 5.05 µM substrate peptide (H2N-RKQLATKAARKSAPATGGVKKP-NTPEGbiot), 1.01 µM SAM and 1.01 µM ³ H-SAM. For the control reaction, 0.202 nM PRC2 was pre-incubated with 1 μM activating peptide, 1.01 μM SAM, 1.01 μM ³ H-SAM and compound at 6.06 times the K _i for 2 h in 138.6 μl of buffer , then the reaction was initiated by adding 1.4 μl of 500 μm substrate peptide in water. Reactions were quenched at various time points up to 10 h by transferring 8 μl aliquots from the reaction vessel to a plate containing 8 μl per well of STOP solution (50 mM Tris pH 8.5, 200 mM EDTA, 2 mM SAH). After the last time point, 12 µl of quenched solutions were transferred to streptavidin-coated FlashPlate (PerkinElmer) containing 40 µl per well of STOP solution, incubated for 1-8 hours, washed and read on a TopCount plate reader (PerkinElmer).

To approximate the data on the progress of the control reaction to a straight line and the data on the progress of the dilution reaction, a special script was used:

Vf-X. y \u003d v _s t + ----- (1 - e + background

Kabs

Where y is the product formed, t is the reaction time, vi is the initial rate, vs is the steady state rate, and ko _bs is the rate constant of the transition from the phase of the initial rate curve to the phase of the steady rate curve. When fitting the progress data of the dilution reaction, vs was limited by the slope of the line fitted to the progress data of the control reaction. The approximate value of ko _bs was then transformed during hold:

[and] kobs CT] where τ is the retention time, Ki is the inhibition constant, [E1] is the calculated equilibrium concentration of enzyme-inhibitor complex, [E] is the calculated equilibrium concentration of free enzyme, and [l] is the

- 33 042914 even equilibrium concentration of the free inhibitor.

Cell Permeability Measurements of Inhibitors in the MDCK Assay

Cell culture: MDCK II cells (obtained from Piet Borst at the Netherlands Cancer Institute) were plated on polyethylene membranes (PET) in 96-well BD insert systems at 2.5 x 10 ⁵ cells/ml for up to 4-7 days to form a monolayer of confluent cells .

Experimental technique:

Test and control compounds (nadolol, metoprolol and digoxin) were diluted with transport buffer (HBSS with 10 mM Hepes, pH 7.4) from stock to 2 μM (<1% DMSO) and applied to the apical (A) or basolateral (B) ) side of the cell monolayer. The permeability of test compounds from A towards B or from B towards A was determined in duplicate with P-gp inhibitor (GF120918, 10 μM). Digoxin was tested at 10 µM from A towards B or from B towards A with/without P-gp inhibitor (GF120918, 10 µM), while nadolol and metoprolol were tested at 2 µM from A towards B to B without P-gp inhibitor (GF120918, 10 μM) in duplicate. The plate was incubated for 2.5 hours in a CO ₂ incubator at 37 ± 1°C with 5% CO 2 at saturated humidity without shaking. In addition, the efflux coefficient of each compound was also determined. Test and control compounds were quantified by LC/MS/MS analysis based on the analyte/BC peak area ratio. After the transfer assay, the Lucifer yellow rejection assay was used to determine the integrity of the cell monolayer. Buffers were removed from both the apical and basolateral chambers, followed by the addition of 75 μl of 100 μM Lucifer yellow in transport buffer and 250 μl of transport buffer to the apical and basolateral chambers, respectively. The plate was incubated for 30 min at 37°C, 5% CO2 and 95% relative humidity without shaking. After a 30-minute incubation, 20 µl samples of Lucifer yellow were taken from the apical sides, followed by the addition of 60 µl of transport buffer. Then, samples of Lucifer yellow were taken from the basolateral sides, 80 µl each. The Lucifer Yellow Relative Fluorescence Unit (RFU) was measured at 425/528 nm (excitation/emission) using a Molecular Device M2e plate reader. Data analysis: The apparent permeability coefficient _Papp (cm/s) was calculated using the formula:

Papp = (dCr/dt) X Vr/(AxCo) where dC _r /dt is the cumulative concentration of the compound in the receiving chamber as a function of time (μM/s);

V _r is the volume of solution in the receiving chamber (0.075 ml on the apical side, 0.25 ml on the basolateral side);

A represents the surface area for transfer, i.e. 0.0804 cm ² for the monolayer area;

Co is the initial concentration in the donor chamber (µM).

The efflux coefficient was calculated by the formula:

Efflux coefficient = P _app (B A) / P _app (AB)

The percentage of extraction was calculated by the formula:

% recovery = 100 x [(V _r x C _r ) + (Vd x Cd)] / (Vd x Co) where V _d is the volume of the donor chambers (0.075 ml on the apical side, 0.25 ml on the basolateral side);

C _d and C _r are the final concentrations of the transferred compound in the donor and receiver chambers, respectively. The percentage of Lucifer Yellow (% Lucifer Yellow) in the basolateral socket was calculated using the formula:

% Lucifer Yellow =

VBasolat^alX RFU _Basolateral

VApical X RFU _Apical + VBasolataal X RFU _{You get} 1 _ta . _a 1 *100 where RFU _Apical and RFUB _asol at _eral are the relative fluorescence units of lucifer yellow in the apical and basolateral wells, respectively;

V _Apical and V _Basolateral represent the volume of the apical and basolateral sockets (0.075 ml and 0.25 ml), respectively. The percentage of yellow lucifer should be less than 2. The data are presented in table. 6.

- 34 042914

Table Example P ^{F 0 0} 1 ( ( ¹ Cl Example 1 (single enantiomer, single 6 EZH2 Holding time (hours) >100 Cell permeability ( ^10'6 cm/s) / Efflux coefficient 5.6/0.79 geometric isomer) \ ⁰ ° 1 Ci^^^S ' ¹ Cl Example 3 3.7/1.2 \ ° ° 1 ¹ Cl Example 4 (R enantiomer; trans geometric isomer) 0.62/2.3 \n^. ^{0 0} 1 AV Vo< ¹ Cl Example 5 (R enantiomer; trans geometric isomer) 1.1/3.6 F .N - O O I / < ¹ Cl Example 7 - Enantiomer 2 (single enantiomer, single geometric isomer) 33 12/0.71

- 35 042914

ϊ and 1 Ci >100 6.1/0.9 Cl Example 8 (single enantiomer, single geometric isomer) o O I / ( ¹ CI Example 9 (single enantiomer, single geometric isomer) >100 3.8/1.4 ^{0 0} 1 ¹ Cl Example 10 (single enantiomer, single geometric isomer) 37 6.5/0.69 ° ¹ CI Example 11 (single enantiomer; geometric isomer 1) 5.3/1.3 X ^{F 0 0} 1 1 \ /0Q yQv ¹ Cl Example 12 (single enantiomer, single geometric isomer) >100 6.3/1.2 O O I /y CI Example 13 (single enantiomer, single geometric isomer) 48 8.9/0.64 ^{0 0} 1 ux ¹ Cl Example 14 - Enantiomer 2 (single enantiomer) 13/0.56 x ⁰ ' O O I XA V'C ¹ Cl Example 17 (single enantiomer, single geometric isomer) >100 2.5/1.8

Claims (15)

1. Compound formula or a pharmaceutically acceptable salt thereof, wherein R ¹ represents -SCH ₃ or chlorine; X is CH; R ^a is hydrogen, (C1-C ₄ )alkyl or halo(C1-C ₄ )alkyl; R ^b is (C1-C ₄ )alkyl, halo(C1-C ₄ )alkyl or 4-7 membered heterocyclyl containing 1 to 4 heteroatoms independently selected from N, O and S, wherein said heterocyclyl may be substituted 1-3 groups selected from halogen, (C ₁ -C ₄ )αalkyl, halo(C1-C ₄ )alkyl, (C1-C ₄ )alkoxy and halo(C ₁ -C ₄ )alkoxy; or R ^a and R ^b together with the nitrogen atom to which they are attached form a 4-7 membered heterocyclyl containing from 1 to 4 heteroatoms independently selected from N, O and S, optionally substituted with 1-3 groups selected from halogen, (C1-C ₄ )alkyl, halo(C1-C ₄ )alkyl and -OR ^c ; R ^c is (C1-C ₄ )alkyl, halo(C1-C ₄ )alkyl or (C ₃ -C ₇ )cycloalkyl and R ⁶ is halo(C1-C ₄ )alkyl. 2. The compound according to claim 1, which has the formula or a pharmaceutically acceptable salt thereof. 3. Connection according to claim 1 or 2, where R ^b represents (C 1 -C ₄ )alkyl or oxetanyl, where said oxetanyl may be substituted with halo(C ₁ -C ₄ )alkyl; or R ^a and R ^b together with the nitrogen atom to which they are attached form azetidinyl, which may be substituted with halogen or -OR ^c . 4. A compound according to any one of claims 1 to 3, wherein R ^a is hydrogen or methyl and R ^b is methyl or oxetanyl, wherein said oxetanyl may be substituted with -CH2F or _-CF3 . 5. Connection according to any one of claims 1-4, where R ^a and R ^b together with the nitrogen atom to which they are attached form azetidinyl which may be substituted with 1-2 fluoro or -OR ^c ; And R ^c represents -CH ₃ , -CHF ₂ or cyclopropyl. 6. Compound having the formula - 37 042914 or a pharmaceutically acceptable salt thereof. 7. The compound according to any one of claims 1-6, where the group and NR ^a R ^b are trans oriented with respect to cyclohexyl. 8. The compound according to any one of claims 1-6, where the group and NR ^a R ^b are cis oriented relative to cyclohexyl. 9. A compound according to any one of claims 1 to 8, wherein the stereochemical configuration of the chiral center of 1,3 dioxolanyl is R. - 38 042914 10. Connection according to any of the pi. 1-8, where the stereochemical configuration of the chiral center of 1,3dioxolanyl is S. 11. A compound having the formula or a pharmaceutically acceptable salt thereof. 12. A compound having the formula or a pharmaceutically acceptable salt thereof. 13. A pharmaceutical composition containing a compound according to any of pi. 1-12 or a pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier. 14. Use of a compound according to any of pi. 1-12 for cancer treatment. 15. Use according to claim 14, characterized in that said cancer is selected from breast cancer, prostate cancer, colon cancer, renal cell carcinoma, glioblastoma multiforme, bladder cancer, melanoma, bronchial cancer, lymphoma, cholangiosarcoma, multiple myeloma, lung cancer, ovarian cancer and liver cancer.