MAT2A INHIBITORS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. Application No. 62/804,352, filed February 12, 2019, the disclosure of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0001] The invention relates to compounds that inhibit Methionine Adenosyltransf erase 2 A (MAT2A). In particular, the invention relates to compounds, pharmaceutical compositions comprising the compounds and methods for use therefor.

BACKGROUND OF THE INVENTION

[0002] The transfer of a methyl group from one molecule to another molecule plays a central role in a wide variety of biological processes, including gene expression, cellular proliferation and apoptosis. The main donor of methyl groups in these cellular processes is S- adenosylmethionine (SAM), which is synthesized by the enzyme methionine

adenosyltransferase (MAT).

[0003] Mammals express three MAT genes, matla, mat2a and mat2b. MAT1 A and MAT2A are catalytic subunits that catalyze the production of S-adenosylmethionine from methionine and ATP. MAT1 A and MAT2A each form dimers with MAT2B, which acts as a regulatory subunit.

[0004] Homozygous deletions of pl6/CDKN2a are prevalent in cancer and these mutations commonly involve the co-deletion of adjacent genes, including the gene encoding

methylthioadenosine phosphorylase (MTAP). It is estimated that approximately 15% of all human cancers have a homozygous deletion of the MTAP gene (e.g., see Firestone &

Schramm (2017) J. Am. Chem Soc. 139(39): 13754-13760. doi: 10.1021/jacs.7b05803. Epub 2017 Sep 20).

[0005] Cells lacking MTAP activity have elevated levels of the MTAP substrate,

methylthioadenosine (MTA), which is a potent inhibitor of the arginine methyltransferase PRMT5. Inhibition of PRMT5 activity results in reduced methylation activity and increased sensitivity of cellular proliferation to PRMT5 depletion or loss of activity. Since PRMT5 utilizes SAM as a methyl donor substrate, the inhibition of MAT2A activity reduces intracellular SAM concentrations decreasing PRMT5 methylation activity selectively in MTAP deleted cells below the threshold level required for growth (e.g., see Marjon et al., (2016) Cell Reports 15:574-587).

[0006] Thus, the loss of MTAP activity reduces methylation activity of PRMT5 making the cells selectively dependent on PRMT5 activity. Since MAT2a activity produces the SAM substrate for PRMT5, the inhibition of MAT2A activity results in synthetic lethality in MTAP-deleted cells through inhibition of PRMT5 activity and, thus, may provide therapeutic benefit for a wide range of cancers.

SUMMARY OF THE INVENTION

[0007] There is a need to develop new MAT2A inhibitors that are capable of reducing SAM levels to inhibit PRMT5 activity, particularly in MTAP-deficient cells.

[0008] In one aspect of the invention, compounds are provided represented by Formula (I):

Formula (I)

[0009] and pharmaceutically acceptable salts thereof:

[00010] wherein:

[00011] R ¹ is cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein the cycloalkyl, heterocyclyl, aryl or heteroaryl are each optionally substituted with one or more R ³ ;

[00012] R ² is hydrogen, -L-SO2CI - C3 alkyl, -L-S02NR ^5a R ^5b , aralkyl, heteroarylalkyl or -O-heteroarylalkyl, wherein the aryl or heteroaryl portion of the aralkyl, heteroarylalkyl, or -O-heteroarylalkyl are each optionally substituted with one or more R ⁴ ;

[00013] each R ³ is independently cyano, acyl, halogen, hydroxyl, -Cl - C3 alkyl, -Cl - C3 alkoxy, or haloalkyl; [00014] each R ⁴ is independently oxo, halogen, Cl - C3 alkyl, or -L-NR ^5a R ^5b ; [00015] L is a bond or Cl - C3 alkylene;

[00016] R ^5a and R ^5b are each independently hydrogen or Cl - C3 alkyl;

[00017] each R ⁶ is independently Cl - C3 alkyl or alkoxy; and [00018] m is 0, 1 or 2.

[00019] In another aspect of the invention, compounds are provided represented by Formula (IA):

Formula (I A)

[00020] and pharmaceutically acceptable salts thereof:

[0010] wherein:

[0011] R ¹ is cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein the cycloalkyl, heterocyclyl, aryl or heteroaryl are each optionally substituted with one or more R ³ ;

[0012] R ² is hydrogen, -L-SO2CI - C3 alkyl, -L-S02NR ⁵ R ⁵ , aralkyl, heteroarylalkyl or heteroarylalkyl-O-, wherein the aryl or heteroaryl portion of the aralkyl, heteroarylalkyl, or heteroarylalkyl-O- are each optionally substituted with one or more R ⁴ ;

[0013] each R ³ is independently acyl, halogen, hydroxyl, -Cl - C3 alkyl, -Cl - C3 alkoxy, or haloalkyl;

[0014] each R ⁴ is independently oxo, halogen, Cl - C3 alkyl, or -L-NR ⁵ R ⁵ ;

[0015] L is a bond or Cl - C3 alkylene;

[0016] each R ⁵ is independently hydrogen or Cl - C3 alkyl;

[0017] each R ⁶ is independently Cl - C3 alkyl or alkoxy; and [0018] m is 0, 1 or 2.

[0019] In one aspect of the invention, compounds are provided represented by Formula (II):

Formula (II)

[0020] and pharmaceutically acceptable salts thereof:

[0021] wherein:

[0022] X is a 5-membered partially saturated ring comprising one or two heteroatoms independently selected from O, N and S;

[0023] R ¹ is cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein the cycloalkyl, heterocyclyl, aryl or heteroaryl are each optionally substituted with one or more R ² ;

[0024] each R ² is independently acyl, halogen, Cl - C3 alkyl, or haloalkyl;

[0025] R ^3a and R ^3b are each independently hydrogen or Cl - C3 alkyl;

[0026] each R ⁴ is independently oxo, halogen, Cl - C3 alkyl, or -L-NR ^3a R ^3b ;

[0027] L is a bond or Cl - C3 alkylene; and

[0028] n is 0, 1 or 2

[0029] The compounds of the invention offer therapeutic benefit as inhibitors of MAT2A and areuseful for negatively modulating the activity of MAT2A in a cell, particularly a MTAP-deficient cell, and for treating various forms of MTAP-associated cancer.

[0030] In another aspect of the invention, pharmaceutical compositions are provided comprising a therapeutically effective amount of a compound of the invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient. [0031] In yet another aspect of the invention, methods for inhibiting MAT2A activity in a in a cell, comprising contacting the cell with a compound of Formula (I) or Formula (II). In one embodiment, the contacting is in vitro. In one embodiment, the contacting is in vivo.

[0032] Also provided herein is a method of inhibiting cell proliferation, in vitro or in vivo, the method comprising contacting a cell with an effective amount of a compound of Formula (I) or Formula (II), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof as defined herein.

[0033] Also provided are methods for treating cancer in a patient comprising administering a therapeutically effective amount of a compound or pharmaceutical composition of the invention or a pharmaceutically acceptable salt thereof to a patient in need thereof.

[0034] Also provided herein is a method for treating cancer in a patient in need thereof, the method comprising (a) determining that the cancer is associated with MTAP double deletion (e.g., a MTAP-associated cancer); and (b) administering to the patient a therapeutically effective amount of a compound of Formula (I) or Formula (II), or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof.

[0035] Also provided herein is a use of a compound of Formula (I) or Formula (II), or a pharmaceutically acceptable salt or solvate thereof, as defined herein in the manufacture of a medicament for the inhibition of activity of MAT2A.

[0036] Also provided herein is the use of a compound of Formula (I) or Formula (II), or a pharmaceutically acceptable salt or solvate thereof, as defined herein, in the manufacture of a medicament for the treatment of a MTAP-associated disease or disorder.

DETAILED DESCRIPTION OF THE INVENTION

[0037] The invention relates to MAT2A inhibitors. In particular, the invention relates to compounds that inhibit MAT2A activity, pharmaceutical compositions comprising a therapeutically effective amount of the compounds, and methods of use therefor.

DEFINITIONS

[0038] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. All patents, patent applications, and publications referred to herein are incorporated by reference to the extent they are consistent with the present disclosure. Terms and ranges have their generally defined definition unless expressly defined otherwise.

[0039] For simplicity, chemical moieties are defined and referred to throughout primarily as univalent chemical moieties (e.g., alkyl, aryl, etc.). Nevertheless, such terms may also be used to convey corresponding multivalent moieties under the appropriate structural circumstances clear to those skilled in the art. For example, while an“alkyl” moiety generally refers to a monovalent radical (e.g. CH3-CH2-), in certain circumstances a bivalent linking moiety can be“alkyl,” in which case those skilled in the art will understand the alkyl to be a divalent radical (e.g., -CH2-CH2-), which is equivalent to the term

“alkylene.” (Similarly, in circumstances in which a divalent moiety is required and is stated as being“aryl,” those skilled in the art will understand that the term“aryl” refers to the corresponding divalent moiety, arylene.) All atoms are understood to have their normal number of valences for bond formation (i.e., 4 for carbon, 3 for N, 2 for O, and 2, 4, or 6 for S, depending on the oxidation state of the S).

[0040] As used herein,“MAT2A” refers to a mammalian methionine adenosyltransferase 2A (MAT2A) enzyme.

[0041] As used herein, an“MAT2A inhibitor” refers to compounds of the invention that are represented by Formula (I) and Formula (II) as described herein. These compounds are capable of negatively modulating or inhibiting all or a portion of the enzymatic activity of the MAT2A.

[0042] As used herein,“MTAP” refers to a mammalian methylthioadenosine phosphorylase (MTAP) enzyme.

[0043] An“MTAP-associated disease or disorder" as used herein refers to diseases or disorders associated with or mediated by or having a loss of MTAP activity resulting in sensitizing the disorder to selective inhibition of PRMT5 activity through the reduction in SAM levels by inhibition of MAT2A activity. A non-limiting example of an MTAP- associated disease or disorder is a MTAP-associated cancer.

[0044] The term“amino” refers to -NFh.

[0045] The term“acetyl” refers to“-C(0)CH3. [0046] As herein employed, the term "acyl" refers to an alkylcarbonyl or arylcarbonyl substituent wherein the alkyl and aryl portions are as defined herein.

[0047] The term "alkyl" as employed herein refers to straight and branched chain aliphatic groups having from 1 to 12 carbon atoms. As such,“alkyl” encompasses Ci, C2, C3, C4, C5, C ₆ , C7, Cs, C ₉ , C10, C11 and C12 groups. Examples of alkyl groups include, without limitation, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, and hexyl.

[0048] The term "alkenyl" as used herein means an unsaturated straight or branched chain aliphatic group with one or more carbon-carbon double bonds, having from 2 to 12 carbon atoms. As such,“alkenyl” encompasses C2, C ₃ , C4, C5, C ₆ , C7, Cs, C ₉ , C10, C11 and C12 groups. Examples of alkenyl groups include, without limitation, ethenyl, propenyl, butenyl, pentenyl, and hexenyl.

[0049] The term "alkynyl" as used herein means an unsaturated straight or branched chain aliphatic group with one or more carbon-carbon triple bonds, having from 2 to 12 carbon atoms. As such,“alkynyl” encompasses C2, C ₃ , C4, C5, C ₆ , C7, Cs, C ₉ , C10, C11 and C12 groups. Examples of alkynyl groups include, without limitation, ethynyl, propynyl, butynyl, pentynyl, and hexynyl.

[0050] An "alkylene," "alkenylene," or "alkynylene" group is an alkyl, alkenyl, or alkynyl group, as defined hereinabove, that is positioned between and serves to connect two other chemical groups. Examples of alkylene groups include, without limitation, methylene, ethylene, propylene, and butylene. Exemplary alkenylene groups include, without limitation, ethenylene, propenylene, and butenylene. Exemplary alkynylene groups include, without limitation, ethynylene, propynylene, and butynylene.

[0051] The term“alkoxy” refers to -OC1 - C6 alkyl.

[0052] The term "cycloalkyl" as employed herein is a saturated and partially unsaturated cyclic hydrocarbon group having 3 to 12 carbons. As such,“cycloalkyl” includes C ₃ , C4, C5, C ₆ , C7, Cs, C ₉ , C10, C11 and C12 cyclic hydrocarbon groups. Examples of cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl. [0053] The term "heteroalkyl" refers to an alkyl group, as defined hereinabove, wherein one or more carbon atoms in the chain are independently replaced O, S, or NR ^X , wherein R ^x is hydrogen or Cl - C3 alkyl. Examples of heteroalkyl groups include methoxymethyl, methoxy ethyl and methoxypropyl.

[0054] An "aryl" group is a C6-C14 aromatic moiety comprising one to three aromatic rings. As such,“aryl” includes C ₆ , Cio, C13, and Ci4 cyclic hydrocarbon groups. An exemplary aryl group is a C6-C10 aryl group. Particular aryl groups include, without limitation, phenyl, naphthyl, anthracenyl, and fluorenyl. An“aryl” group also includes fused multicyclic (e.g, bicyclic) ring systems in which one or more of the fused rings is non-aromatic, provided that at least one ring is aromatic, such as indenyl.

[0055] An "aralkyl" or "arylalkyl" group comprises an aryl group covalently linked to an alkyl group wherein the moiety is linked to another group via the alkyl moiety. An exemplary aralkyl group is -(Cl - C6)alkyl(C6 - C10)aryl, including, without limitation, benzyl, phenethyl, and naphthylmethyl.

[0056] A "heterocyclyl" or "heterocyclic" group is a mono- or bicyclic (fused or spiro) ring structure having from 3 to 12 atoms, (3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 atoms), for example 4 to 8 atoms, wherein one or more ring atoms are independently -C(O)-, N, NR ⁴ , O, or S, and the remainder of the ring atoms are quaternary or carbonyl carbons. Examples of heterocyclic groups include, without limitation, epoxy, oxiranyl, oxetanyl, azetidinyl, aziridinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiophenyl, pyrrolidinyl, piperidinyl, piperazinyl, imidazolidinyl, thiazolidinyl, thiatanyl, dithianyl, trithianyl, azathianyl, oxathianyl, dioxolanyl, oxazolidinyl, oxazolidinonyl, decahydroquinolinyl, piperidonyl, 4- piperidonyl, thiomorpholinyl, dimethyl-morpholinyl, and morpholinyl. Specifically excluded from the scope of this term are compounds having adjacent ring O and/or S atoms.

[0057] As used herein,“L-heterocyclyl” refers to a heterocyclyl group covalently linked to another group via an alkylene linker

[0058] As used herein, the term "heteroaryl" refers to a group having 5 to 14 ring atoms, preferably 5, 6, 10, 13 or 14 ring atoms; having 6, 10, or 14 p electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to three heteroatoms that are each independently N, O, or S.“Heteroaryl” also includes fused multicyclic (e.g, bicyclic) ring systems in which one or more of the fused rings is non-aromatic, provided that at least one ring is aromatic and at least one ring contains an N, O, or S ring atom.

[0059] Examples of heteroaryl groups include acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzo[d]oxazol-2(3H)-one, 2H-benzo[b][l,4]oxazin-3(4H)-one,

benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzthiazolyl, benztriazolyl,

benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH- carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, furanyl, furazanyl, imidazolinyl, imidazolyl, lH-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl,

isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3- oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxazolidinyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole,

pyridoimidazole, pyridothi azole, pyridinyl, pyridyl, pyrimidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl,

tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-l,2,5-thiadiazinyl, 1,2,3- thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl, triazinyl, 1,2,3- triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl, and xanthenyl.

[0060] A“L-heteroaryl”, "heteroaralkyl" or "heteroarylalkyl" group comprises a heteroaryl group covalently linked to another group via an alkylene linker. Examples of heteroalkyl groups comprise a Ci- Cr _> alkyl group and a heteroaryl group having 5, 6, 9, or 10 ring atoms. Examples of heteroaralkyl groups include pyridylmethyl, pyridylethyl, pyrrolylmethyl, pyrrolylethyl, imidazolylmethyl, imidazolylethyl, thiazolylmethyl, thiazolyl ethyl, benzimidazolylmethyl, benzimidazolylethyl quinazolinylmethyl, quinolinylmethyl, quinolinylethyl, benzofuranylmethyl, indolinylethyl isoquinolinylmethyl, isoinodylmethyl, cinnolinylmethyl, and benzothiophenyl ethyl. Specifically excluded from the scope of this term are compounds having adjacent ring O and/or S atoms.

[0061] An "arylene," "heteroarylene," or "heterocyclylene" group is an bivalent aryl, heteroaryl, or heterocyclyl group, respectively, as defined hereinabove, that is positioned between and serves to connect two other chemical groups. [0062] As employed herein, when a moiety (e.g., cycloalkyl, aryl, heteroaryl, heterocyclyl, urea, etc.) is described as“optionally substituted” without expressly stating the substituents it is meant that the group optionally has from one to four, preferably from one to three, more preferably one or two, non-hydrogen substituents.

[0063] The term "halogen" or "halo" as employed herein refers to chlorine, bromine, fluorine, or iodine.

[0064] The term“haloalkyl” refers to an alkyl chain in which one or more hydrogens have been replaced by a halogen. Exemplary haloalkyls are trifluoromethyl, difluoromethyl, flurochloromethyl, chi orom ethyl, and fluorom ethyl.

[0065] The term“hydroxyalkyl” refers to -alkylene-OH.

[0066] As used herein,“an effective amount” of a compound is an amount that is sufficient to negatively modulate or inhibit the activity of MAT2A enzyme.

[0067] As used herein, a“therapeutically effective amount” of a compound is an amount that is sufficient to ameliorate or in some manner reduce a symptom or stop or reverse progression of a condition, or negatively modulate or inhibit the activity of MAT2A. Such amount may be administered as a single dosage or may be administered according to a regimen, whereby it is effective.

[0068] As used herein,“treatment” means any manner in which the symptoms or pathology of a condition, disorder or disease in a patient are ameliorated or otherwise beneficially altered.

[0069] As used herein,“amelioration of the symptoms of a particular disorder by administration of a particular compound or pharmaceutical composition” refers to any lessening, whether permanent or temporary, lasting or transient, that can be attributed to or associated with administration of the composition.

COMPOUNDS

[0070] In one aspect of the invention, compounds are provided represented by Formula (I):

Formula (I)

[0071] and pharmaceutically acceptable salts thereof:

[0072] wherein:

[0073] R ¹ is cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein the cycloalkyl, heterocyclyl, aryl or heteroaryl are each optionally substituted with one or more R ³ ;

[0074] R ² is hydrogen, -L-SO2CI - C3 alkyl, -L-S02NR ^5a R ^5b , aralkyl, heteroarylalkyl or - O-heteroarylalkyl, wherein the aryl or heteroaryl portion of the aralkyl, heteroarylalkyl, or - O-heteroarylalkyl are each optionally substituted with one or more R ⁴ ;

[0075] each R ³ is independently cyano, acyl, halogen, hydroxyl, -Cl - C3 alkyl, -Cl - C3 alkoxy, or haloalkyl;

[0076] each R ⁴ is independently oxo, halogen, Cl - C3 alkyl, or -L-NR ^5a R ^5b ;

[0077] L is a bond or Cl - C3 alkylene;

[0078] R ^5a and R ^5b are each independently hydrogen or Cl - C3 alkyl;

[0079] each R ⁶ is independently Cl - C3 alkyl or alkoxy; and [0080] m is 0, 1 or 2.

[0081] In one embodiment for the compounds of Formula (I), R ¹ is cycloalkyl optionally substituted with one or more R ³ . In certain embodiments, the cycloalkyl is cyclohexyl. In one embodiment, cycloalkyl is substituted with one or two R ³ , wherein R ³ is halogen. In certain embodiments, the cycloalkyl is 4,4-difluoro-cyclohexyl.

[0082] In one embodiment for the compounds of Formula (I), R ¹ is aryl optionally substituted with one or more R ³ . In certain embodiments, the aryl is indenyl or phenyl. In one embodiment, the aryl is 5-methyl-2,3-dihydro-lH-indenyl. In certain embodiments, the R ^5a attached to the imidazole is C1-C3 alkyl and the R ^5b attached to the amide nitrogen is hydrogen. In certain embodiments, the R ^5a is methyl. In certain embodiments, both the R ^5a and the R ^5b are methyl.

[0083] In one embodiment for the compounds of Formula (I), the aryl is phenyl substituted with one, two or three R ³ . In one embodiment, the one, two or three R ³ are each

independently halogen, Cl - C3 alkyl or haloalkyl. In certain embodiments, the halogen is chloro, fluoro or bromo. In other embodiments, the Cl - C3 alkyl is methyl or ethyl. In certain embodiments, the haloalkyl is trifluorom ethyl. In certain embodiments, the R ^5a attached to the imidazole is C1-C3 alkyl and the R ^5b attached to the amide nitrogen is hydrogen. In certain embodiments, the R ^5a is methyl. In certain embodiments, both the R ^5a and the R ^5b are methyl.

[0084] In one embodiment for the compounds of Formula (I), R ¹ is phenyl substituted with two or three Cl - C3 alkyl. In other embodiments, the Cl - C3 alkyl is methyl or ethyl. In one embodiment for the compounds of Formula (I), R ¹ is phenyl substituted with halogen and two Cl - C3 alkyl. In certain embodiments, the halogen is chloro, fluoro or bromo and the two Cl - C3 alkyl are each independently methyl or ethyl. In certain embodiments, the R ^5a attached to the imidazole is C1-C3 alkyl and the R ^5b attached to the amide nitrogen is hydrogen. In certain embodiments, the R ^5a is methyl. In certain embodiments, both the R ^5a and the R ^5b are methyl.

[0085] In one embodiment for the compounds of Formula (I), R ¹ is phenyl substituted with hydroxyl. In one embodiment for the compounds of Formula (I), R ¹ is phenyl substituted with Cl- C3 alkoxy. In one embodiment for the compounds of Formula (I), the alkoxy is methoxy. In certain embodiments, the R ^5a attached to the imidazole is C1-C3 alkyl and the R ^5b attached to the amide nitrogen is hydrogen. In certain embodiments, the R ^5a is methyl.

In certain embodiments, both the R ^5a and the R ^5b are methyl.

[0086] In one embodiment for the compounds of Formula (I), R ² is heterocyclylalkyl, wherein the heterocyclyl portion is optionally substituted with one or more R ⁴ . In one embodiment, the heterocyclyl portion of the heterocyclylalkyl is azetidinyl, pyrrolidinyl, imidazolidinyl, thiazolidinyl, piperdinyl or morpholinyl. In one embodiment, the

heterocyclyl portion of the heterocyclylalkyl is substituted with one R ⁴ , wherein the R ⁴ is oxo. In certain embodiments, the oxo-substituted heterocyclyl portion of heterocyclylalkyl is azetidin-2-one, pyrrolidin-2-one, or imidazolidin-2-one. In one embodiment, the

heterocyclyl portion is pyrrolidin-2-one. In certain embodiments, the R ^5a attached to the imidazole is C1-C3 alkyl and the R ^5b attached to the amide nitrogen is hydrogen. In certain embodiments, the R ^5a is methyl. In certain embodiments, both the R ^5a and the R ^5b are methyl.

[0087] In one embodiment for the compounds of Formula (I), R ² is heterocyclylalkyl, wherein the heterocyclyl portion is substituted with two R ⁴ groups wherein the two R ⁴ groups are each oxo. In certain embodiments, the dioxo-substituted heterocyclyl portion of heterocyclylalkyl is imidazolidin-2,5-dione, thiazolidin-2,5-dione or piperdin-2,5-dione. In certain embodiments, the R ^5a attached to the imidazole is C1-C3 alkyl and the R ^5b attached to the amide nitrogen is hydrogen. In certain embodiments, the R ^5a is methyl. In certain embodiments, both the R ^5a and the R ^5b are methyl.

[0088] In one embodiment, the heterocyclyl portion of the heterocyclylalkyl is substituted with one R ⁴ , wherein the R ⁴ is -L-S02NR ^5a R ^5b . In one embodiment, L is methylene and R ^5a and R ^5b are each hydrogen. In one embodiment, L is methylene and R ^5a and R ^5b are each Cl - C3 alkyl.

[0089] In one embodiment of the compounds of Formula (I), R ² is L-heteroaryl optionally substituted with one or more R ⁴ . In certain embodiments, L is a bond. In other

embodiments, L is Cl - C3 alkylene. In one embodiment, the Cl - C3 alkylene is methylene. In one embodiment, the heteroaryl is pyrolyl, pyrazolyl, triazolyl, imidazolyl, pyridyl, pyrimidinyl, pyridine-2-one or benzothiophenyl. In one embodiment, the heteroaryl is substituted with one R ³ group selected from the group consisting of oxo, Cl - C3 alkyl, halogen, haloalkyl and -L-NR5 ^a R5 ^b .

[0090] In one embodiment of the compounds of Formula (I), R ² is -L-SO2CI - C3 alkyl. In one embodiment, L is methylene and the Cl - C3 alkyl is methyl or ethyl.

[0091] In one embodiment of the compounds of Formula (I), R ² is -L-S02NR ^5a R ^5b . L is methylene and R ^5a and R ^5b are each hydrogen. In one embodiment, L is methylene and R ^5a and R ^5b are each Cl - C3 alkyl.

[0092] In one embodiment of the compounds of Formula (I), R ² is aralkyl optionally substituted with one or more R ⁴ . In one embodiment, the aralkyl is benzyl.

[0093] In certain embodiments of the compounds of Formula (I), R ² is hydrogen. [0094] In one embodiment of the compounds of Formula (I), n is zero. In one embodiment, n equals one and R ⁴ is Cl - C3 alkyl or alkoxy. In certain embodiments, the Cl - C3 alkyl is methyl. In other embodiments, the alkoxy is methoxy.

[0095] In one embodiment of the compounds of Formula (I), R ^5a is hydrogen. In one embodiment, R ^5a is Cl - C3 alkyl. In certain embodiments, the Cl - C3 alkyl is methyl.

[0096] In one embodiment of the compounds of Formula (I), R ^5b is hydrogen. In one embodiment, R ^5a is Cl - C3 alkyl. In certain embodiments, the Cl - C3 alkyl is methyl.

[0097] In one embodiment, the R ^5a attached to the imidazole is C1-C3 alkyl and the R ^5b attached to the amide nitrogen is hydrogen. In certain embodiments, the R ^5a is methyl. In certain embodiments, both the R ^5a and the R ^5b are methyl.

[0098] In one embodiment, the compound of Formula (I) is:

[0099] or a pharmaceutically acceptable salt of any of the foregoing compounds.

[00100] In another aspect of the invention, compounds are provided represented by Formula (II):

Formula (II)

[00101] and pharmaceutically acceptable salts thereof:

[00102] wherein:

[00103] X is a 5-membered partially saturated ring comprising one or two heteroatoms independently selected from O, N and S; [0103] R ¹ is cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein the cycloalkyl, heterocyclyl, aryl or heteroaryl are each optionally substituted with one or more R ² ;

[0104] each R ² is independently cyano, acyl, halogen, Cl - C3 alkyl, or haloalkyl;

[0105] R ^3a and R ^3b are each independently hydrogen or Cl - C3 alkyl;

[0106] each R ⁴ is independently oxo, halogen, Cl - C3 alkyl, or -L-NR ^3a R ^3b ;

[0107] L is a bond or Cl - C3 alkylene; and

[0108] n is 0, 1 or 2.

[0109] In one embodiment for the compounds of Formula (II), R ¹ is aryl optionally substituted with one or more R ² . In certain embodiments, the aryl is indenyl or phenyl. In one embodiment, the aryl is 5-methyl-2,3-dihydro-lH-indenyl.

[0110] In one embodiment for the compounds of Formula (II), the aryl is phenyl substituted with one, two or three R ² . In one embodiment, the one, two or three R ² are each

independently halogen, Cl - C3 alkyl or haloalkyl. In certain embodiments, the halogen is chloro, fluoro or bromo. In other embodiments, the Cl - C3 alkyl is methyl or ethyl. In certain embodiments, the haloalkyl is trifluorom ethyl.

[0111] In one embodiment for the compounds of Formula (II), R ¹ is phenyl substituted with two or three Cl - C3 alkyl. In other embodiments, the Cl - C3 alkyl is methyl or ethyl. In one embodiment for the compounds of Formula (I), R ¹ is phenyl substituted with halogen and two Cl - C3 alkyl. In certain embodiments, the halogen is chloro, fluoro or bromo and the two Cl - C3 alkyl are each independently methyl or ethyl.

In one embodiment for the compounds of Formula (II), R ¹ is phenyl substituted with hydroxyl. In one embodiment for the compounds of Formula (II), R ¹ is phenyl substituted with -Cl- C3 alkoxy. In one embodiment for the compounds of Formula (II), the alkoxy is methoxy. In one embodiment for the compounds of Formula (II), R ¹ is phenyl substituted with one -Cl - C3 alkoxy and one or two -Cl - C3 alkyl.

[0112] In one embodiment for the compounds of Formula (II), X and the phenyl ring to which it is attached is benzimidazolyl, benzthiazolyl, benzo[d]oxazolyl or isoindolyl. [0113] In one embodiment of the compounds of Formula (II), R ^3a is hydrogen. In one embodiment, R ^3a is Cl - C3 alkyl. In certain embodiments, the Cl - C3 alkyl is methyl.

[0114] In one embodiment of the compounds of Formula (II), R ^3b is hydrogen. In one embodiment, R ^3a is Cl - C3 alkyl. In certain embodiments, the Cl - C3 alkyl is methyl.

[0115] In one embodiment, the compound of Formula (II) is:

[0116] or a pharmaceutically acceptable salt of any of the foregoing compounds.

[0117] The compounds of Formula (I) and Formula (II) may be formulated into

pharmaceutical compositions.

PHARMACEUTICAL COMPOSITIONS

[0118] In another aspect, the invention provides pharmaceutical compositions comprising a MAT2A inhibitor according to the invention and a pharmaceutically acceptable carrier, excipient, or diluent. Compounds of the invention may be formulated by any method well known in the art and may be prepared for administration by any route, including, without limitation, parenteral, oral, sublingual, transdermal, topical, intranasal, intratracheal, or intrarectal. In certain embodiments, compounds of the invention are administered intravenously in a hospital setting. In certain other embodiments, administration may preferably be by the oral route.

[0119] The characteristics of the carrier will depend on the route of administration. As used herein, the term "pharmaceutically acceptable" means a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism, and that does not interfere with the effectiveness of the biological activity of the active ingredient(s). Thus, compositions according to the invention may contain, in addition to the inhibitor, diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. The preparation of pharmaceutically acceptable formulations is described in, e.g., Remington's Pharmaceutical Sciences, 18th Edition, ed. A. Gennaro, Mack Publishing Co., Easton, Pa., 1990.

[0120] As used herein, the term“pharmaceutically acceptable salts” refers to salts that retain the desired biological activity of the above-identified compounds and exhibit minimal or no undesired toxicological effects. Examples of such salts include, but are not limited to acid addition salts formed with inorganic acids (for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like), and salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, and polygalacturonic acid. The compounds can also be administered as pharmaceutically acceptable quaternary salts known by those skilled in the art, which specifically include the quaternary ammonium salt of the formula— NR+Z-, wherein R is hydrogen, alkyl, or benzyl, and Z is a counterion, including chloride, bromide, iodide, -O-alkyl, toluenesulfonate, methyl sulfonate, sulfonate, phosphate, or carboxylate (such as benzoate, succinate, acetate, glycolate, maleate, malate, citrate, tartrate, ascorbate, benzoate, cinnamoate, mandeloate, benzyloate, and diphenylacetate).

[0121] The active compound is included in the pharmaceutically acceptable carrier or diluent in an amount sufficient to deliver to a patient a therapeutically effective amount without causing serious toxic effects in the patient treated. A dose of the active compound for all of the above-mentioned conditions is in the range from about 0.01 to 300 mg/kg, preferably 0.1 to 100 mg/kg per day, more generally 0.5 to about 25 mg per kilogram body weight of the recipient per day. A typical topical dosage will range from 0.01-3% wt/wt in a suitable carrier. The effective dosage range of the pharmaceutically acceptable derivatives can be calculated based on the weight of the parent compound to be delivered. If the derivative exhibits activity in itself, the effective dosage can be estimated as above using the weight of the derivative, or by other means known to those skilled in the art.

[0122] The pharmaceutical compositions comprising compounds of the invention may be used in the methods described herein.

METHODS OF USE

[0123] In yet another aspect, the invention provides for methods for inhibiting MAT2A activity in a cell, comprising contacting the cell in which inhibition of MAT2A activity is desired in vitro with an effective amount of a compound of Formula (I) or Formula (II), pharmaceutically acceptable salts thereof or pharmaceutical compositions containing the compound or pharmaceutically acceptable salt thereof.

[0124] The compositions and methods provided herein are particularly deemed useful for inhibiting MAT2A activity in a cell. In one embodiment, a cell in which inhibition of MAT2A activity is desired is contacted in vivo with a therapeutically effective amount of a compound of Formula (I) or Formula (II) to negatively modulate the activity of MAT2A. In other embodiments, a therapeutically effective amount of pharmaceutically acceptable salt or pharmaceutical compositions containing the compound of Formula (I) or Formula (II) may be used. In one embodiment, the cell is a MTAP-deficient cell.

[0125] By negatively modulating the activity of MAT2A, particularly in cases for cells that lack MTAP activity, the methods are designed to reduce intracellular SAM substrate concentrations to further inhibit PRMT5 activity to block cellular proliferation. The cells may be contacted in a single dose or multiple doses in accordance with a particular treatment regimen to affect the desired negative modulation of MAT2A. The degree intracellular SAM depletion may be monitored in the cell using well known methods, including those described in Example B below, to assess the effectiveness of treatment and dosages may be adjusted accordingly by the attending medical practitioner.

[0126] In another aspect, methods of treating cancer comprising administering to a patient having cancer a therapeutically effective amount of a compound of Formula (I) or Formula (II), pharmaceutically acceptable salts thereof or pharmaceutical compositions comprising the compound or pharmaceutically acceptable salts thereof are provided. In one

embodiment, the cancer is a MTAP-associated cancer. [0127] The compositions and methods provided herein may be used for the treatment of a wide variety of cancer including tumors such as prostate, breast, brain, skin, cervical carcinomas, testicular carcinomas, etc More particularly, cancers that may be treated by the compositions and methods of the invention include, but are not limited to tumor types such as astrocytic, breast, cervical, colorectal, endometrial, esophageal, gastric, head and neck, hepatocellular, laryngeal, lung, oral, ovarian, prostate and thyroid carcinomas and sarcomas. More specifically, these compounds can be used to treat: Cardiac: sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogenic carcinoma (squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma; Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel

(adenocarcinoma, lymphoma, carcinoid tumors, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma); Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor (nephroblastoma), lymphoma, leukemia), bladder and urethra (squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonal carcinoma, teratocarcinoma,

choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver: hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; Biliary tract: gall bladder carcinoma, ampullary carcinoma, cholangiocarcinoma; Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (osteocartilaginous exostoses), 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 multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors), spinal cord neurofibroma, meningioma, glioma, sarcoma); Gynecological: uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumor cervical dysplasia), ovaries (ovarian carcinoma (serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma), granulosa- thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma), fallopian tubes (carcinoma); Hematologic: 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, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis; and Adrenal glands: neuroblastoma. In certain embodiments, the cancer is diffuse large B-cell lymphoma (DLBCL).

[0128] In one embodiment, the cancer is a MTAP-associated cancer selected from hepatocellular carcinoma, breast cancer, skin cancer, bladder cancer, liver cancer, pancreatic cancer, and head and neck cancer.

[0129] The concentration and route of administration to the patient will vary depending on the cancer to be treated. The compounds, pharmaceutically acceptable salts thereof and pharmaceutical compositions comprising such compounds and salts also may be co administered with other anti-neoplastic compounds, e.g., chemotherapy, or used in combination with other treatments, such as radiation or surgical intervention, either as an adjuvant prior to surgery or post-operatively.

GENERAL REACTION SCHEME INTERMEDIATES AND EXAMPLES GENERAL REACTION SCHEMES

[0130] The compounds of the invention may be prepared using commercially available reagents and intermediates in the synthetic methods and reaction schemes described herein, or may be prepared using other reagents and conventional methods well known to those skilled in the art.

[0131] For instance, intermediates for preparing compounds and compounds of Formula (I) and Formula (II) of the invention may be prepared according to General Reaction Schemes I - VI: General Reaction Scheme I

[0132] Compounds of Formula (I) of the invention may be prepared following General Reaction Scheme I. An R'-aryl or heteroaryl substituted methyl ketone is brominated in Step A to the a-bromoketone, by nucleophilic addition to an electrophilic bromine equivalent, such as NBS. The a-bromoketone is cyclized in the presence of methyl-aminopyrimidine in Step B to R^ R ^5a -substituted 1 -methyl -///-i mi dazol -2-amine. M ethyl -///-i mi dazol -2-amine is coupled in Step C to R ² and R ⁶ substituted benzoic acid in the presence of a suitable coupling agent, e.g., HATU, to provide R ¹ , R ² , R ^5a , R ^5b R ⁶ -substituted amide compounds of Formula (I).

General Reaction Scheme II

[0133] Compounds of Formula (II) of the invention may be prepared following General Reaction Scheme IF To a mixture of fused aryl or heteroaryl-carboxylic acid in suitable polar solvent is added an appropriate coupling agent, e.g., HATU, and base, e.g., a Lewis base, and the reaction mixture is stirred. An appropriately substituted 2-aminoimidazole is added to the mixture, and the reaction mixture is stirred until completion to provide the appropriately R ¹ , (^-substituted amide compounds of Formula (II). General Reaction Scheme III

[0134] Compounds of Formula (I) of the invention, wherein R ² is -L-SO2CI - C3 alkyl and R ^5b is hydrogen, may be prepared following General Reaction Scheme III. To a solution of a compound of Formula (I) wherein R ² is -L-SC1 - C3 alkyl in polar protic solvent is added a solution of appropriate oxidant, e.g., Oxone, in polar protic solvent, e.g., methanol. The mixture is stirred to completion to provide the sulfone product of Formula (I), wherein L is a bond or L is methylene.

General Reaction Scheme IV

[0135] Compounds of Formula (I) of the invention, wherein R ² is -L-heterocyclyl, may be prepared following General Reaction Scheme IV. To a solution of a secondary heterocyclic- amine and appropriately substituted compound of Formula (I) wherein R ² initially is -CFhBr in polar aprotic solvent, e.g., DMF, is added K2CO3 and 18-crown-6. The mixture is stirred until completion, to provide benzylamine product of Formula (I), wherein R ² is -L- heterocyclyl and L is methylene.

General Reaction Scheme V [0136] Compounds of Formula (I) of the invention, wherein R ² is -L-heterocyclyl substituted with a single R ⁴ , wherein R ⁴ is an amine, may be prepared following General Reaction Scheme V. To a solution of a compound of Formula (I) wherein R ² contains a nitro group in polar aprotic solvent, e.g., DMF, is added Pd/C under inert atmosphere. The suspension is degassed and purged with Fb and stirred under Fb (15 Psi) until completion, to furnish compounds of Formula (I) wherein R ² is -L-heterocyclyl substituted with an amine.

General Reaction Scheme VI

[0137] Compounds of Formula (I) of the invention, wherein the aryl or heteroaryl group of R ¹ is substituted with a single R ³ , wherein R ³ is a hydroxy group, may be prepared following General Reaction Scheme VI. To a solution of appropriately substituted alkyl phenyl ether in non-polar solvent, e.g., DCM, is added an appropriate Lewis acid. The mixture is stirred at - 50— » 30 °C until completion, to provide the appropriately substituted free alcohol compounds of Formula (I).

[0138] The following intermediates may be used to prepare compounds of the invention.

INTERMEDIATE A pyridinium

tribromide,

[0139] An exemplary intermediate, Intermediate A, may be used to synthesize compounds of Formula (I) and Formula (II). To a solution of NaH (4.77 g, 119 mmol, 60% purity, 1.30 eq) in THF (150 mL) was added pyrrolidin-2-one (10.6 g, 124 mmol, 9.53 mL, 1.36 eq) dropwise at 0 °C. After addition, the mixture was stirred at this temperature for 0.5 hour, and then methyl 3-(bromomethyl) benzoate (21.0 g, 91.7 mmol, 1.0 eq) was added at 0 °C. The resulting mixture was stirred at 60 °C for 11.5 hours. After completion, the organic solvent was quenched with water (50.0 mL). The aqueous phase was extracted with ethyl acetate (2 x 200 mL). Combined extracts were washed with brine (300 mL), dried with Na2SC>4, the solvent was then removed under vacuum. The residue was purified by column chromatography (S1O2, petroleum ether: ethyl acetate = 5: 1 to 2: 1). Compound methyl 3-[(2- oxopyrrolidin-l-yl)methyl]benzoate (18.5 g, 79.3 mmol, 87% yield, 100% purity) was obtained as yellow oil. LCMS [ESI, M+l]: 234.

[0140] ¾ NMR (400 MHz, chloroform-rf) d 7.88 (td, J= 1.6, 7.6 Hz, 1H), 7.82 (s, 1H), 7.45 - 7.33 (m, 2H), 4.47 (s, 2H), 3.88 (s, 3H), 3.24 (t, J= 7.2 Hz, 2H), 2.42 (t, J= 8.2 Hz, 2H), 2.03 - 1.93 (m, 2H).

INTERMEDIATE B

[0141] An exemplary intermediate, Intermediate B, may be used to synthesize compounds of Formula (I) and Formula (II). l-(w-tolyl)ethanone (700 mg, 5.22 mmol, 693 pL, 1.0 eq) was dissolved in THF (15.0 mL) under a nitrogen atmosphere at 20 °C. TFA (595 mg, 5.22 mmol, 386 pL, 1.0 eq) was added followed by pyridinium tribromide (2.0 g, 6.26 mmol, 1.20 eq). The mixture was stirred at 15 °C for 3 hours. After completion, the reaction mixture was concentrated under reduced pressure to remove THF. The residue was added H2O (20.0 mL) and extracted with ethyl acetate (3 ^c 20 mL). The combined organic layers were washed with saturated brine (30.0 mL), dried over NaiSCri, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (S1O2, petroleum ether: ethyl acetate = 1 :0 to 100: 1) to give 2-bromo- 1 -(/??-tolyl)ethanone (650 mg, 2.96 mmol, 57% yield, 97% purity) as colorless oil. LCMS [ESI, M+l]: 213.

[0142] ¾ NMR (400 MHz, chloroform-rf) d 7.82 - 7.75 (m, 2H), 7.45 - 7.34 (m, 2H), 4.45 (s, 2H), 2.43 (s, 3H).

[0143] The following Examples are intended to illustrate further certain embodiments of the invention and are not intended to limit the scope of the invention.

EXAMPLE 1

N-(l-methyl-5-(2,4,5-trimethylphenyl)-lH-imidazol-2-yl)-3 -((2-oxopyrrolidin-l- yl)methyl)benzamide

[0144] Intermedate B, 2-bromo-l-(2,4,5-trimethylphenyl)ethanone (500 mg, 2.07 mmol,

1.2 eq), and /V-methylpyrimidin-2-amine (189 mg, 1.73 mmol, 1.0 eq) were taken up into a microwave tube in MeCN (2.0 mL). The reaction mixture was stirred at 130 °C for 15 mins under microwave. After cooling to 10 °C, INkH^LLO (721 mg, 8.64 mmol, 700 //L, 60% purity, 5.0 eq) was taken up into the microwave tube. The reaction mixture was heated at 100 °C for 5 mins under microwave. After completion, the reaction mixture was diluted with H2O (5.0 mL) and extracted with ethyl acetate (3 x 10 mL). The combined organic layers were washed with saturated brine (30.0 mL), dried over NaiSCri, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column

chromatography (S1O2, petroleum ether: ethyl acetate = 3: 1 to ethyl acetate: methanol = 20:1). Compound l-methyl-5-(2,4,5- trimethylphenyl)imidazol-2-amine (330 mg, 1.49 mmol, 86% yield, 97% purity) was obtained as yellow solid.

[0145] To a solution of l-methyl-5-(2,4,5-trimethylphenyl)imidazol -2-amine (120 mg, 557 umol, 1.0 eq ), 3-[(2-oxopyrrolidin-l-yl)methyl]benzoic acid (159 mg, 725 pmol, 1.3 eq ), DIEA (288 mg, 2.23 mmol, 388 pL, 4.0 eq) and HATU (276 mg, 725 pmol, 1.3 eq) in DMF (3.0 mL), the mixture was stirred at 80 °C for 2 hours. After completion, the reaction mixture was diluted with H2O (5.0 mL) and extracted with ethyl acetate (3 ^ 10 mL). The combined organic layers were washed with saturated brine (30.0 mL), dried over Na2S04, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (S1O2, petroleum ether: ethyl acetate = 50: 1 to 20:1) and then purified by prep-HPLC (column: Phenomenex Synergi C18 150 x 25 x l0 pm; mobile phase: [water (0.1% TFA)-ACN]; B%: 30%-60%, 9 min) and lyophilized. Title compound /V-[l-methyl-5- (2,4,5-trimethylphenyl)imidazol-2-yl]-3-[(2-oxopyrrolidin-l- yl)methyl]benzamide (28.9 mg, 68.0 umol, 12% yield, 98% purity) was obtained as yellow solid. LCMS [ESI, M+l]: 417.

[0146] ¹ H NMR (400 MHz, chloroform-d) d 8.04 (d, J= 7.2 Hz, 1H), 7.95 (s, 1H), 7.57 - 7.50 (m, 2H), 7.15 (s, 1H), 7.06 - 7.01 (m, 2H), 4.57 (s, 2H), 3.47 - 3.41 (m, 5H), 2.56 (t, J = 8.0 Hz, 2H), 2.32 (s, 3H), 2.28 (s, 3H), 2.19 (s, 3H), 2.09 - 2.05 (m, 2H).

[0147] Following the teachings of General Reaction Schemes I, & III- VI and the procedure described for the preparation of Example 1, the following compounds of Formula (I), Examples 2-78, shown in Table 1 were prepared:

Table 1

-

-

-

[0148] Following the teachings of General Reaction Scheme II and the procedure described for the preparation of Example 1, the following compounds of Formula (II), Examples 79-83, shown in Table 2 were prepared:

Table 2

EXAMPLE 84 N-(l-methyl-5-(2,4,5-trimethylphenyl)-lH-imidazol-2-yl)-3-(m ethylsulfonyl)benzamide

[0149] To a solution of 3 -methyl sulfanyl-A-[l -methyl-5-(2,4,5- trimethylphenyl)imidazol- 2-yl]benzamide (90 mg, 246 //mol, 1.0 eq) in dichloromethane (1.0 mL), methanol (1.0 mL) and H2O (1.0 mL) was added Oxone (454 mg, 739 //mol, 3.0 eq). The mixture was stirred at 25 °C for 1 hour. After completion, the reaction mixture was diluted with H2O (10 mL) and extracted with ethyl acetate (3 x 10 mL). The combined organic layers were washed with saturated brine (30 mL), dried over Na2S04, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge 150 ^c 255//; mobile phase: [water (0.05% ammonia hydroxide v/v)-ACN]; B%: 37% - 67%, 10 min). 3 -methyl sul fony 1 -A-Ti tl e compound [l-methyl-5-(2,4,5-trimethylphenyl)imidazol-2- yljbenzamide (25.5 mg, 63.5 //mol, 26% yield, 99.1% purity) was obtained as white solid. LCMS [ESI, M+l]: 398.

[0150] ¹ H NMR (400 MHz, Chloroform-if) d = 12.28 (br s, 1H), 8.90 (t, J= 1.6 Hz, 1H), 8.58 (dt, J= 1.4, 8.0 Hz, 1H), 8.04 - 8.01 (m, 1H), 7.63 (t, J= 7.8 Hz, 1H), 7.12 (s, 1H), 7.02 (s, 1H), 6.64 (s, 1H), 3.41 (s, 3H), 3.10 (s, 3H), 2.31 (s, 3H), 2.28 (s, 3H), 2.17 (s, 3H).

EXAMPLE 85

N-(l-methyl-5-(2,4,5-trimethylphenyl)-lH-imidazol-2-yl)-3 -

((methylsulfonyl)methyl)benzamide

[0151] To a solution of methyl 3-(bromomethyl)benzoate (5.0 g, 21.8 mmol, 1.0 eq) in DMF (30 mL) was added MeSNa (7.17 g, 43.7 mmol, 2.0 eq) at 25 °C. The mixture was stirred at 25 °C for 4 hours. After completion, the reaction mixture was diluted with FLO (90 mL) and extracted with ethyl acetate (3 ^c 50 mL). The combined organic layers were washed with saturated brine (300 mL), dried over Na2S04, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (S1O2, petroleum ether/ethyl acetate=l/0 to 1 : 1). 3-(methylsulfanylmethyl)benzoic acid (1.70 g,

9.33 mmol, 43% yield) was obtained as white solid.

[0152] ¾ NMR (400 MHz, Chloroform-i7) d = 9.43 (br s, 1H), 8.09 - 8.04 (m, 1H), 8.00 (dt, J = 1.4, 7.6 Hz, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.47 - 7.40 (m, 1H), 3.73 (s, 2H), 2.00 (s, 3H).

EXAMPLE 86

N-(5-(3,4-dimethylphenyl)-l-methyl-lH-imidazol-2-yl)-3-(( 2,5-dioxopyrrolidin-l- yl)methyl)benzamide

[0153] To a solution of pyrrolidine-2, 5-dione (44.8 mg, 452 pmol, 2.00 eq), 3- (bromomethyl)-N-[5-(3,4-dimethylphenyl)-l-methyl-imidazol-2- yl] benzamide (90.0 mg, 226 pmol, 1.00 eq) in DMF (0.4 mL) and THF (2.00 mL) was added K2CO3 (937 mg, 678 pmol, 3.00 eq) and 18-crown-6 (5.97 mg, 22.6 pmol, 0.10 eq). The mixture was stirred at 25 °C for 2 hours. After completion, the reaction mixture was diluted with H2O (10 mL) and extracted with ethyl acetate (3 x 10 mL). The combined organic layers were washed with brine (30 mL), dried over Na2S04, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge 150*25 5p;

mobile phase: [water (0.05% ammonia hydroxide v/v)-ACN]; B%: 32%-62%, 10 min). /V-[5- (3,4-dimethylphenyl)-l-methyl-imidazol-2-yl]-3-[(2,5-dioxopy rrolidin-l- yl)methyl]benzamide (14.6 mg, 35.2 umol, three steps yield 12%, 100% purity) was obtained as white solid. LCMS [ESI, M+l]: 417.

[0154] ¾ NMR (400 MHz, chloroform-if) d vg 12.28 (s, 1H), 8.32 (s, 1H), 8.21 (d, J = 7.6 Hz, 1H), 7.48 (d, J = 7.6 Hz, 1H), 7.40 - 7.35 (m, 1H), 7.25 (d, J = 8.0 Hz, 1H), 7.19 (s, 1H), 7.18 - 7.13 (m, 1H), 6.68 (s, 1H), 4.75 (s, 2H), 3.64 (s, 3H), 2.73 (s, 4H), 2.34 (s, 6H).

EXAMPLE 87

N-(5-(3 ,4-dimethylphenyl)- 1 -methyl- lH-imidazol-2-yl)-3 -((2-methylpyrrolidin- 1 - yl)methyl)benzamide

[0155] To a solution of 2-methylpyrrolidine (109 mg, 900 umol, 131 pL, 3.0 eq, HC1) and /V-[5-(3,4-dimethylphenyl)-l-methyl-imidazol-2-yl]-3-formyl- benzamide (100 mg, 300 pmol, 1.0 eq) in tetrahydrofuran (1.0 mL) and 1,2-dichloroethane (1.0 mL), the mixture was stirred at 40 °C for 6 hours, and then NaBH(OAc)3 (191 mg, 900 pmol, 3.0 eq) was added at -10 °C. The resulting mixture was stirred at 25 °C for 12 hours. After completion, the reaction mixture was diluted with EhO (10.0 mL) and extracted with ethyl acetate (3 x 10 mL). The combined organic layers were washed with saturated brine (50.0 mL), dried over Na2SC>4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (S1O2, petroleum ether: ethyl acetate = 3 : 1 to ethyl acetate: methanol = 50: 1) and then purified by prep-HPLC (column: Phenomenex Gemini 150 x 25 mm x 10 um; mobile phase: [water (lOmM NH4HC03)-ACN]; B%: 50% - 80%, 10 mins). Compound /V-[5-(3,4-dimethylphenyl)-l-methyl-imidazol-2-yl]-3-[(2- methylpyrrolidin-l-yl)methyl]benzamide (40 mg, 97.0 umol, 32% yield, 98% purity) was obtained as yellow solid. LCMS [ESI, M+l]: 403.

[0156] 1H NMR (400 MHz, chloroform-d) d 8.20 (s, 2H), 7.53 - 7.45 (m, 1H), 7.43 - 7.36 (m, 1H), 7.25 (d, J = 7.6 Hz, 1H), 7.21 - 7.13 (m, 2H), 6.70 (s, 1H), 4.11 (br d, J = 12.8 Hz, 1H), 3.63 (s, 3H), 3.32 - 3.25 (m, 1H), 3.15 - 2.85 (m, 1H), 2.60 - 2.39 (m, 1H), 2.34 (s, 6H), 2.23 - 2.21 (m, 1H), 2.05 - 1.85 (m, 1H), 1.80 - 1.70 (m, 2H), 1.55 - 1.47 (m, 2H), 1.30 - 1.20 (m, 3H).

[0157] Following the teachings of General Reaction Scheme IV and the procedure described for the preparation of Example 88, the following compounds of Formula (I), Examples 88-95, shown in Table 3 were prepared:

Table 3

EXAMPLE 96

3-((3-amino-lH-pyrazol-l-yl)methyl)-N-(l-methyl-5-(2,4,5- trimethylphenyl)-lH-imidazol-

2-yl)benzamide

[0158] To a solution of /V-[l-methyl-5-(2,4,5- trimethylphenyl)imidazol-2-yl]-3-[(3- nitropyrazol-l-yl)methyl]benzamide (140 mg, 315 pmol, 1.0 eq) in ethyl acetate (10 mL) was added Pd/C (25.0 mg, 10% purity) under N2 atmosphere. The suspension was degassed and purged with Th for 3 times. The mixture was stirred under Th (15 Psi) at 25 °C for 1 hour. After completion, the reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge 150*25 5m; mobile phase: [water (0.05% ammonia hydroxide v/v)-ACN]; B%: 35%-65%, 10 min) to give the title compound 3-[(3-aminopyrazol-l-yl)methyl]-N-[l-methyl-5-(2,4,5- trimethylphenyl)imidazol-2-yl]benzamide (101 mg, 242 pmol, 77% yield, 99.7% purity) as white solid. LCMS [ESI, M+l]: 415.

[0159] ¾ NMR (400 MHz, Chloroform-rf) d = 12.26 (br s, 1H), 8.24 (d, J=7.6 Hz, 1H),

8.19 (s, 1H), 7.39 (t, J=7.8 Hz, 1H), 7.30 - 7.27 (m, 1H), 7.14 (d, J=2.4 Hz, 1H), 7.11 (s, 1H), 7.01 (s, 1H), 6.60 (s, 1H), 5.62 (d, J=2.0 Hz, 1H), 5.17 (s, 2H), 3.62 (br s, 2H), 3.38 (s, 3H), 2.31 (s, 3H), 2.27 (s, 3H), 2.16 (s, 3H).

[0160] Following the teachings of General Reaction Scheme V and the procedure described for the preparation of Example 97, the following compounds of Formula (I), Examples 98- 100, shown in Table 4 were prepared:

Table 4

EXAMPLE 100

N-(5-(3 -hydroxy-4-methylphenyl)- 1 -methyl- lH-imidazol-2-yl)-3 -((2-oxopyrrolidin- 1 - yl)methyl)benzamide

[0161] To a solution of /V-[5-(3-methoxy-4-methyl-phenyl)-l- methyl-imidazol-2-yl]-3-[(2- oxopyrrolidin-l-yl)methyl]benzamide (45.0 mg, 108 pmol, 1.0 eq) in dichloromethane (2.0 mL) was added BBn (323 mg, 1.29 mmol, 124 pL, 12.0 eq). The mixture was stirred at -50 °C for 10 minutes, and then stirred at 30 °C for 20 minutes. After completion, the reaction mixture was added water (2 mL) and adjusted with saturated NaHCCb aqueous to pH ~ 7, then extracted with dichloromethane (3 x 10 mL). The organic layer was dried over Na2S04, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge 150*25 5m; mobile phase: [water (10 mM

NH4HCCb)-ACN]; B%: 30%-55%, 7 min) to give the compound A-[5-(3-hydroxy-4-methyl- phenyl)-l-methyl-imidazol-2-yl]-3-[(2-oxopyrrolidin-l-yl)met hyl]benzamide (15.1 mg, 37.2 pmol, 35% yield, 100% purity) as white solid. LCMS [ESI, M+l]: 405.

[0162] 1H NMR (400 MHz, Chloroform-d) d = 8.25 (d, J=7.6 Hz, 1H), 8.13 (s, 1H), 7.39 (t, J=7.6 Hz, 1H), 7.36 - 7.31 (m, 1H), 7.18 (d, J=7.6 Hz, 1H), 6.89 (d, J=1.2 Hz, 1H), 6.82 (dd, J=1.6, 8.0 Hz, 1H), 6.66 (s, 1H), 4.56 (s, 2H), 3.61 (s, 3H), 3.31 (t, J=7.0 Hz, 2H), 2.49 (t, J=8.0 Hz, 2H), 2.31 (s, 3H), 2.06 - 1.96 (m, 2H).

EXAMPLE 101

3-(li7-imidazol-2-ylmethyl)-/V-[l-methyl-5-(2,4,5-trimeth ylphenyl)imidazol-2-yl]benzamide

[0163] A solution of 4-methylbenzenesulfonohydrazide (6.11 g, 32.8 mmol, 1.05 eq) in methanol (30 mL) was stirred at 60 °C for 10 minutes, then 1 T/-i mi dazol e-2-carbal dehy de (3.0 g, 31.2 mmol, 1.0 eq) was added to the mixture in portions. After addition, the mixture was stirred at 60 °C for 1 hour. After completion, the reaction mixture was concentrated. The residue was triturated with ethyl acetate (60 mL), and then filtered. The filter cake was washed with ethyl acetate (2 x 60 mL), then dried under vacuum to give the compound N- [(£)-lif-imidazol-2- ylmethyleneamino]-4-methyl-benzenesulfonamide (6.0 g, 21.3 mmol, 68% yield, 94% purity) as white solid. LCMS [ESI, MTl]: 265.

[0164] ¾ NMR (400 MHz, DMSO-de) d = 12.44 (br s, 1H), 11.47 (br s, 1H), 7.83 - 7.77 (m, 3H), 7.39 (d, J= 8.4 Hz, 2H), 7.10 (s, 2H), 2.36 (s, 3H).

[0165] To a solution of /V-[(E)-liT-imidazol-2-ylmethyleneamino]- 4-methyl- benzenesulfonamide (3.0 g, 11.4 mmol, 1.0 eq ) and CS2CO3 (11.1 g, 34.1 mmol, 3.0 eq ) in dioxane (60 mL) was added (3 -ethoxy carbonylphenyl)boronic acid (3.30 g, 17.0 mmol, 1.50 eq). The mixture was stirred at 110 °C for 2 hours. After completion, the reaction mixture was added H2O (60 mL) and extracted with ethyl acetate (3 ^c 60 mL). The organic layer was dried over Na2SC>4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (S1O2, petroleum ether : ethyl acetate = 5: 1 - ethyl acetate: methanol = 15: 1) and prep-HPLC (column: Waters Xbridge 150*50 10 m; mobile phase: [water (0.05% ammonia hydroxide v/v)-acetonitrile]; B%: 15%-45%, 11.5 min) to give the compound ethyl 3-(liT-imidazol-2-ylmethyl)benzoate (500 mg, 2.10 mmol, 19% yield, 97% purity) as colorless oil.

[0166] ¾ NMR (400 MHz, Chloroform-d) d = 7.96 - 7.86 (m, 2H), 7.44 - 7.33 (m, 2H), 6.99 - 6.91 (m, 2H), 4.42 - 4.30 (m, 2H), 4.17 - 4.08 (m, 2H), 1.38 (t, J= 3.6 Hz, 3H).

[0167] To a solution of ethyl 3-(liT-imidazol-2-ylmethyl)benzoate (250 mg, 1.09 mmol, 1.0 eq) in THF (2 mL), H2O (0.6 mL) and methanol (0.6 mL) was added LiOH ^» H20 (91.1 mg, 2.17 mmol, 2.0 eq). The mixture was stirred at 20 °C for 1 hour. After completion, the mixture was adjusted with 12 M HC1 aqueous solution to pH~3 and concentrated. Then The residue was added water (5 mL) and lyophilized to give the compound 3-(liT-imidazol-2- ylmethyl)benzoic acid (200 mg, crude) as yellow solid.

[0168] ¾ MR (400 MHz, DMSO-de) d = 15.1 (br s, 1H), 13.07 (br s, 1H), 7.96 (s, 1H), 7.84 (d, J= 8.0 Hz, 1H), 7.75 (br d, J= 8.0 Hz, 1H), 7.56 (s, 2H), 7.47 (t, J= 3.8 Hz, 1H), 4.45 (s, 2H).

[0169] To a solution of 3-(liT-imidazol-2-ylmethyl)benzoic acid (188 mg, 929 pmol, 2.50 eq) and DIEA (144 mg, 1.11 mmol, 194 pL, 3.0 eq) in DMF (3.0 mL) was added HOBt (90.4 mg, 669 umol, 1.80 eq) and EDCI (128 mg, 669 pmol, 1.80 eq). The mixture was stirred at 20 °C for 0.5 hour. Then l-methyl-5-(2,4,5-trimethylphenyl)imidazol-2-amine (80.0 mg, 372 pmol, 1.0 eq ) was added to the mixture, and the mixture was stirred at 80 °C for 1 hour.

After completion, the reaction mixture was added H2O (5 mL) and extracted with ethyl acetate (3 x 5 mL). The combined organic layer was washed with saturated brine (2 ^c 30 mL), dried over NaiSCri, filtered and concentrated under reduced pressure to give a residue. The residue was purified by prep-HPLC (column: Waters Xbridge 150*50 10m; mobile phase: [water (0.05% ammonia hydroxide v/v)-acetonitrile]; B%: 30%-60%, 11.5min) to give the title compound 3-( l//-i mi dazol-2-yl methyl )-/V-[ l -methyl -5-(2, 4,5- trimethylphenyl)imidazol-2-yl]benzamide (56.9 mg, 137 umol, 37% yield, 96.5% purity) as off-white solid. LCMS [ESI, M+l]: 400.

[0170] ¹ H NMR (400 MHz, Chloroform-d) d = 12.29 (br s, 1H), 9.08 (s, 1H), 8.22 - 8.16 (m, 2H), 7.42 - 7.32 (m, 2H), 7.11 (s, 1H), 7.01 (s, 1H), 6.95 (s, 2H), 6.62 (s, 1H), 4.18 (s, 2H), 3.37 (s, 3H), 2.31 (s, 3H), 2.27 (s, 3H), 2.16 (s, 3H).

[0171] Following the teachings of General Reaction Scheme VI and the procedure described for the preparation of Example 101, the following compound of Formula (I), Example 102, shown in Table 5 was prepared:

Table 5

[0172] The compounds in Table 6 are prepared essentially according to the procedures set forth in the above Reaction Schemes and Examples. Table 6

EXAMPLE A

[0173] This Example illustrates that exemplary compounds of the invention inhibit MAT2A enzymatic activity.

[0174] Ten-point dose-response curves for compounds of the invention were determined using a MAT2A Inhibitor Assay Kit (BPS Biosciences Catalog #71402). The assay uses purified human, MAT2A enzyme to covert L-Methionine and ATP to S-adenosylmethionine (SAM). In this assay, the transfer of the adenosyl group from ATP to L-Methionine generates free phosphate, which is quantitated spectrophotometrically to determine the activity of MAT2A enzyme in the presence and absence of compound.

[0175] Briefly, compounds of the invention were solubilized in 100% DMSO at a highest concentration of 30 mM. A series of intermediate compound dilutions were made for each compound in 100% DMSO and then diluted 20-fold in assay buffer (50 MM Tris-HCl, pH 8.0, 15 mM MgCh, 50 mM KC1, 80 mM L-Methionine (Sigma Aldrich Catalog #64319), 100 pM ATP (Sigma Aldrich Catalog A6419) and 0.5 mg/ml BSA) for an intermediate concentration of 5% DMSO. All of the compounds were initially tested at a final concentration of 10 mM and the percent inhibition was calculated as reported below in Table 7.

[0176] For ICso determinations, the initial starting concentration for the serial dilutions of each compound was 300 mM. Control samples lacking compound, MAT2A enzyme or various reaction components also were prepared and processed in parallel with compound test samples. Compound MHC03177 was used as a positive control for assay validation.

[0177] To measure MAT2A inhibitory activity, 100 ng of MAT2A enzyme (BPS

Bioscience Catalog # 71401) was preincubated with test compound in assay buffer lacking Methionine and ATP for 60 minutes at room tempaerture. The enzymatic reaction was initiated by adding Methionine and ATP at the final concentrations above to duplicate samples (25 mΐ total volume, 1% DMSO) and the samples were incubated for 1 hr at room temperature. After 1 hr, a 50 mΐ aliquot of a colorimetric detection reagent (BPS Bioscience Catalog # 74001) was added to each well and the samples were incubated for an additional 20 minutes at room temperature. Absorbance was measured at 630 nM for each sample using a Tecan Infinite M1000 microplate reader. The ICso value for each compound was determined from each 10-point dose-response curve using GraphPad Prism software and the results for exemplary compounds of Formula (I) and Formula (II) are shown in Table 7. Percent Inhibition @ 10 mM Key: A = < 75%; B = > 50% - < 75%; C = > 25% - < 50% and D = < 25%. ICso Key: A = < 500 nM; B = > 500 nM - < 1 mM; and C = > 1 mM.

Table 7

Inhibition of MAT2A-mediated Enzymatic Activity by Exemplary Compounds of Formula

(I)

EXAMPLE B

[0178] This Example illustrates that exemplary compounds of the invention are capable of reducing intracellular SAM levels via the inhibition of MAT2A activity.

[0179] HCT116 cells (ATCC CCL-247) were plated in 10cm dishes. The following day, cells were treated with DMSO vehicle control or MAT2A inhibitor, at 3 mM or 10 pM, for 6 hours. Cell lysates were prepared on ice using non-ionic NP-40 lysis buffer and total protein concentration was determined using a Bradford assay. Total protein concentrations were normalized across samples and stored at -80C.

[0180] A 20 ul aliquot of SAM (2mg/ml) was transferred to an Eppendorf tube containing 980 pi of lOmM NEEOAC in water, pH 10, to make a 40 pg/ml working stock solution of the test articles. Subsequent calibrator stock solutions were prepared by serial dilution in water.

[0181] Calibrator lysates were prepared by transferring 5 pi of each respective sub-stock into Eppendorf tubes containing 95 pi of control cell lysate. Calibration standards and study samples were diluted 10-fold into water (20pl sample into 180pl of water) containing candidate internal standards, including 250 ng/ml oxymorphone-D6 and hydromorphone-D4. Samples were analyzed using a LC-MS-MS instrument according to the manufacturer’s instructions and SAM concentrations in cell lysates were calculated against the standard curve.

[0182] The percent depletion of SAM by a MAT2A inhibitor of the invention, Example 101, is shown in Table 8.

Table 8

[0183] As shown in Table 8, Example 101 is capabale of depleting intracellular SAM concentrations by about 72% and 88% at 10 mM and 30 pM, respectively.

[0184] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.