The isocitrate dehydrogenase (IDH) protein is an important enzyme in the citric acid (tricarboxylic acid or Krebs) cycle. The citric acid cycle is centrally important to many biochemical pathways and is one of the earliest established components of cellular metabolism.
Isocitrate dehydrogenases catalyze the oxidative decarboxylation of isocitrate to a-ketoglutarate (2-oxoglutarate). These enzymes belong to two distinct subclasses, one of which utilizes nicotinamide adenine dinucleotide (NAD(+)) as the electron acceptor and the other nicotinamide adenine dinucleotide phosphate (NADP(+)). Three mammalian isocitrate dehydrogenases have been reported: one NAD(+)-dependent isocitrate dehydrogenase, a multisubunit enzyme which localizes to the mitochondrial matrix, and two NADP(+)-dependent isocitrate dehydrogenases, one of which is mitochondrial and the other predominantly cytosolic. Each NADP(+)-dependent isozyme is a dimer. The protein encoded by the IDH1 gene is the NADP(+)-dependent isocitrate dehydrogenase found in the cytoplasm and peroxisomes. The cytoplasmic enzyme serves a significant role in cytoplasmic NADPH production. IDH1 is expressed in a wide range of species and in organisms that lack a complete citric acid cycle.

Recently, mutations in IDH1, and the related isoform IDH2, have been found in several types of cancers. Mutations were found to occur at specific amino acids along the protein sequence and to be heterozygously expressed, consistent with a gain of function. These mutations occur at functionally conserved residues and biochemical studies of the mutant forms of IDH1 and IDH2 demonstrated a loss of normal function , the reversible conversion of isocitrate to a-ketoglutarate. The result of these mutations is to allow a new (or neomorphic) conversion of α-ketoglutarate (aKG) to 2-hydroxyglutarate (2HG). As a result, cancers cells that harbor mutant forms of IDH1 or IDH2 form substantially higher concentrations of 2HG. High levels of 2HG result in a block in cell differentiation that can be reversed by mutant IDHlor IDH2 inhibition.

Application PCT/US2016/043264 discloses covalent inhibitors of mutant IDHl . There is a further need for compounds that selectively inhibit mutant IDHl and IDH2 enzyme for the treatment of various cancers. There is a further need for compounds that selectively inhibit mutant IDHl and IDH2 enzyme demonstrating neomorphic activity over wild type IDHl and IDH2 for the treatment of various cancers. The present invention provides compounds of Formula I or la that are inhibitors of mutant IDHland IDH2 . The compounds of Formula I or la are covalent inhibitors that selectively inhibit mutant IDHland IDH2.

One aspect of the invention is to provide mutant IDHland IDH2 enzyme inhibitor compounds of the Formula:

I

wherein:

R1 is -CH2CH(CH3)2, -CH2CH3, -CH2CH2OCH3, or -CH2-cyclopropyl;

R2 is -CH3 or -CH2CH3;

X is N or CH; or

a pharmaceutically acceptable salt thereof.

A further aspect of the invention is to provide mutant IDHland IDH2 enzyme inhibitor compounds of the Formula:

la

wherein

R1 is -CH2CH(CH3)2, -CH2CH3, -CH2CH2OCH3, or -CH2-cyclopropyl;

R2 is -CH3 or -CH2CH3: or

a pharmaceutically acceptable salt thereof.

A further aspect of the present invention provides a compound of Formula I or la which is:

7-[[(l S)-l -[4-[(lR)-2-Cyclopropyl- 1 -(4-prop-2-enoylpiperazin- 1 -yl)ethyl]phenyl]ethyl]amino]-l-ethyl-4H-pyrimido[4,5-d][l,3]oxazin-2-one;

7-[[( 1 S)- 1 -[4-[( 1 S)-2-cyclopropyl- 1 -(4-prop-2-enoylpiperazin- 1 -yl)ethyl]phenyl]ethyl]amino]-l-ethyl-4H-pyrimido[4,5-d][l,3]oxazin-2-one;

1 -Ethyl-7-[[( 1 S)- 1 -[4-[ 1 -(4-prop-2-enoylpiperazin- 1 -yl)propyl]phenyl]ethyl]amino]-4H-pyrimido[4,5-d][l,3]oxazin-2-one, isomer 1;

1 -Ethyl-7-[[( 1 S)- 1 -[4-[ 1 -(4-prop-2-enoylpiperazin- 1 -yl)propyl]phenyl]ethyl]amino]-4H-pyrimido[4,5-d][l,3]oxazin-2-one, isomer 2;

or a pharmaceutically acceptable salt of any of them.

Another aspect of the present invention is a compound of Formula I or la which is

7-[[( 1 S)- 1 -[4-[( 1 S)-2-cyclopropyl- 1 -(4-prop-2-enoylpiperazin- 1 -yl)ethyl]phenyl]ethyl]amino]-l-ethyl-4H-pyrimido[4,5-d][l,3]oxazin-2-one or a pharmaceutically acceptable salt thereof.

Another aspect of the present invention provides a pharmaceutical composition comprising a mutant IDH1 inhibitor compound of Formula I or la, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

A further aspect of the present invention provides a method of treating a cancer expressing mutant IDH1 or mutant IDH2 which is glioma, glioblastoma, glioblastoma multiforme, astrocytomas, oligodendrogliomas, paraganglioma, fibrosarcoma, angioimmunoblastic T-cell lymphoma (AITL), myelodysplastic syndrome (MDS), B cell acute lymphoblastic leukemia (B-ALL), thyroid cancer, colorectal cancer, acute myeloid leukemia (AML), melanoma, prostate cancer, chondrosarcoma or cholangiocarcinoma in a patient comprising administering to a patient in need thereof a therapeutically effective amount of a compound of Formula I or la, or a pharmaceutically acceptable salt thereof.

Another aspect of the present invention provides a method of treating a cancer expressing mutant IDHlor mutant IDH2 which is fibrosarcoma, acute myeloid leukemia, glioma, or glioblastoma in a patient comprising administering to a patient in need thereof a therapeutically effective amount of a compound of Formula I or la, or a pharmaceutically acceptable salt thereof.

A further aspect of the present invention provides a compound of Formula I or la, or a pharmaceutically acceptable salt thereof, for use in therapy.

Another aspect of the present invention provides a compound of Formula I or la, or a pharmaceutically acceptable salt thereof, for use in the treatment of a cancer expressing mutant IDHlor mutant IDH2 which is glioma, glioblastoma, glioblastoma multiforme, astrocytomas, oligodendrogliomas, paraganglioma, fibrosarcoma, angioimmunoblastic T-cell lymphoma (AITL), myelodysplastic syndrome (MDS), B cell acute lymphoblastic leukemia (B-ALL), thyroid cancer, colorectal cancer, acute myeloid leukemia (AML), melanoma, prostate cancer, chondrosarcoma or cholangiocarcinoma .

A further aspect of the present invention provides a compound of Formula I or la, or a pharmaceutically acceptable salt thereof, for use in the treatment of a cancer expressing mutant IDHlor mutant IDH2 which is fibrosarcoma, acute myeloid leukemia, glioma, or glioblastoma.

Another aspect of the present invention provides use of a compound of Formula I or la, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a cancer expressing mutant IDHlor mutant IDH2 which is glioma, glioblastoma, glioblastoma multiforme, astrocytomas, oligodendrogliomas,

paraganglioma, fibrosarcoma, angioimmunoblastic T-cell lymphoma (AITL),

myelodysplastic syndrome (MDS), B cell acute lymphoblastic leukemia (B-ALL), thyroid cancer, colorectal cancer, acute myeloid leukemia (AML), melanoma, prostate cancer, chondrosarcoma or cholangiocarcinoma.

A further aspect of the present invention provides use of a compound of Formula I or la, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a cancer expressing mutant IDHlor mutant IDH2 which is fibrosarcoma, acute myeloid leukemia, glioma, or glioblastoma.

The term "patient" means mammal and "mammal" includes, but is not limited to, a human.

"Therapeutically effective amount" means the dosage of the compound of

Formula I or la, or pharmaceutically acceptable salt thereof, or pharmaceutical composition containing the compound, or pharmaceutically acceptable salt thereof,

necessary to inhibit mutant IDH1 or mutant IDH2 in a cancer patient, leading to the release of the block in differentiation with resulting inhibition of tumor cell growth and eliminate or slow or arrest the progression of the cancer in a patient. Anticipated dosages of a compound of Formula I or la, or a pharmaceutically acceptable salt thereof are in the range of 1 mg/patient/day to 2000 mg/patient/day. Preferred dosages are anticipated to be in the range of 5 mg/patient/day to 1800 mg/patient/day. Most preferred dosages are anticipated to be in the range of 40 mg/patient/day to 1600 mg/patient/day. The exact dosage required to treat a patient and the length of treatment time will be determined by a physician in view of the stage and severity of the disease as well as the specific needs and response of the individual patient. Although expressed as dosage on a per day basis, the dosing administration may be adjusted to provide a more optimal therapeutic benefit to a patient and to manage or ameliorate any drug related toxicities. In addition to daily dosing, twice a day (B.I.D.) dosing; three times a day (T.I.D.) dosing; dosing every other day (Q2D); every other day over a five day period followed by two days without dosing (T.I.W.); or every third day (Q3D) may be appropriate.

The terms "treatment," "treat," and "treating," are meant to include the full spectrum of intervention for the cancer from which the patient is suffering, such as administration of the active compound to alleviate, slow, or reverse one or more of the symptoms and to delay progression of the cancer even if the cancer is not actually eliminated.

The term -CH2CH(CH3)2 means 2-methylpropyl, the term -CH2CH2OCH3 means 2-methoxy ethyl, and the term -CIHb-cyclopropyl means cyclopropylmethyl.

A compound of Formula I or la, or a pharmaceutically acceptable salt thereof, is preferably formulated as a pharmaceutical composition using a pharmaceutically acceptable carrier and administered by a variety of routes. Preferably, such compositions are for oral administration. Such pharmaceutical compositions and processes for preparing them are well known in the art. See, for example, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY, L.V. Allen, Editor, 22nd Edition, Pharmaceutical Press, 2012.

In a particular embodiment, the pharmaceutical composition comprises 7-{[(l S)- 1 - {4-[( 1 S)- 1 -(4-acryloylpiperazin- 1 -yl)-2-cyclopropylethyl]phenyl } ethyljamino} - 1 -ethyl -l,4-dihydro-2H-pyrimido[4,5-d][l,3]oxazin-2-one or a pharmaceutically acceptable salt

thereof, together with a pharmaceutically acceptable carrier and optionally other therapeutic ingredients particularly for treatment of cancer generally or a specific cancer type.

A compound of Formula I or la, or a pharmaceutically acceptable salt, may be administered either simultaneously with, or before, or after, one or more other therapeutic agents. The compound of Formula I or la, or a pharmaceutically acceptable salt, when administered with one or more other therapeutic agents, may be administered separately, by the same or different route of administration, or together in the same pharmaceutical composition as the other therapeutic agent(s). Where one or more additional therapeutic agents are administered, the administration of each therapeutic agent may be

simultaneous, separate, or sequential.

A compound of Formula I or la is capable of reaction with a number of inorganic and organic acids to form pharmaceutically acceptable acid addition salt. Such pharmaceutically acceptable salts and common methodology for preparing them are well known in the art. See, for example, P. Stahl, et al, HANDBOOK OF

PHARMACEUTICAL SALTS: PROPERTIES, SELECTION AND USE,

(VCHA/Wiley-VCH, 2002); S.M. Berge, et al., "Pharmaceutical Salts, "Journal of Pharmaceutical Sciences, Vol. 66, No. 1, January 1977.

A compound of Formula I ot la, or a pharmaceutically acceptable salt thereof, may be prepared by a variety of procedures known in the art, as well as those described below. The specific synthetic steps may be combined in a different order to prepare a compound of Formula I or la, or a pharmaceutically acceptable salt thereof.

Additionally, certain intermediates described in the following preparations may contain one or more nitrogen protecting groups. It is understood that protecting groups may be varied as appreciated by one of skill in the art depending on the particular reaction conditions and the particular transformations to be performed. The protection and deprotection conditions are well known to the skilled artisan and are described in the literature (See for example "Greene 's Protective Groups in Organic Synthesis", Fifth Edition, by Peter G.M. Wuts and Theodora W. Greene, John Wiley and Sons, Inc. 2014).

Compounds of Formula I or la are named according to IUPAC, and may also be named according to CAS, and other names may be used to unambiguously identify a compound of Formula I or la, or a pharmaceutically acceptable salt thereof.

It will be understood a compound of Formula I or la may be depicted as a single stereoisomer. There are two chiral centers giving rise to four diastereomers. As used herein, references to a single stereoisomer are meant to also include stereoisomeric mixtures including the named or depicted compound of Formula I or la. Herein, the Cahn-Ingold-Prelog designations of (R)- and (S)- may be used to refer to specific stereoisomers. Specific stereoisomers can be prepared by stereospecific synthesis using enantiomerically pure or enriched starting materials. The specific stereoisomers of either starting materials, intermediates, or racemic mixtures including compounds of Formula I or la can be resolved by techniques well known in the art, such as those found in

Stereochemistry of Organic Compounds, E. I. Eliel and S. H. Wilen (Wiley 1994) and Enantiomers, Racemates, and Resolutions, J., Jacques, A. Collet, and S. H. Wilen (Wiley 1991), including chromatography on chiral stationary phases, enzymatic resolutions, or fractional crystallization or chromatography of diastereomers formed for that purpose, such as diastereomeric salts. For compounds of Formula I or la having a configuration with all stereocenters shown, "substantially enantiomerically pure" means the isomeric purity is greater than 90% enantiomeric excess. In another embodiment a compound of Formula I or la isomeric purity is greater than 95% enantiomeric excess. In still another embodiment a compound of Formula I or la isomeric purity is greater than 98% enantiomeric excess. In yet another embodiment a compound of Formula I or la isomeric purity is greater than 99% enantiomeric excess. All stereoisomers, individually and including diastereomeric mixtures of the compounds of Formula I or la are contemplated within the scope of the present invention. The designations "isomer 1" and "isomer 2" and "diastereomer 1" and "diastereomer 2" refer to the compounds that elute from chiral chromatography first and second, respectively, and if chiral chromatography is initiated early in the synthesis, the same designation is applied to subsequent intermediates and examples.

The compounds employed as initial starting materials in the synthesis of the compounds of Formula I or la are well known and, to the extent not commercially available, are readily synthesized using specific references provided, by standard procedures commonly employed by those of ordinary skill in the art or are found in general reference texts.

Examples of known procedures and methods include those described in general reference texts such as Comprehensive Organic Transformations, VCH Publishers Inc, 1989; Compendium of Organic Synthetic Methods, Volumes 1-10, 1974-2002, Wiley Interscience; Advanced Organic Chemistry, Reactions Mechanisms, and Structure, 5th Edition, Michael B. Smith and Jerry March, Wiley Interscience, 2001; Advanced Organic Chemistry, 4th Edition, Part B, Reactions and Synthesis, Francis A. Carey and Richard J. Sundberg, Kluwer Academic / Plenum Publishers, 2000, etc., and references cited therein.

Certain abbreviations are defined as follows: "ACN" means acetonitrile; "aKG" means Alpha-ketoglutarate or 2-ketoglutarate; "alloc" means allyloxycarbonyl; "ATCC" means American Type Culture collection; "BCA" means bicinchoninic acid; "BSA" means Bovine Serum Albumin; "CDF' means Ι, Γ-carbonyldiimidazole; "DCC" means 1,3-dicyclohexylcarbodiimide; "DCM" means dichloromethane; "DEAD" means diethyl azodicarboxylate; "DIAD" means diisopropyl azodicarboxylate; "DIC" means

diisopropylcarbodiimide; "DIPEA" means diisopropylethylamine or N-ethyl-N-isopropyl-propan-2-amine; "DMAP" means dimethylaminopyridine; "DMF" means dimethylformamide; "DMSO" means dimethyl sulfoxide; "DFF" means dithiothreitol; "EDC" means EDAC, EDCI, or l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; "EDFA" means ethylenediaminetetraacetic acid; "EGFA" means ethylene glycol tetraacetic acid; "EtOAc" means ethyl acetate; "EtOH" means ethanol or ethyl alcohol; "Ex" means example; "HAFU" means (dimethylamino)-N,N-dimethyl(3H-[l,2,3]triazolo[4,5-^]pyridin-3-yloxy)methaniminium hexafluorophosphate; "HBFU" means 2-(lH-benzotriazole- 1 -yl)- 1,1,3,3 -tetram ethyl uronium hexafluorophosphate;

"2HG" means 2-hydroxyglutarate; "d5-3HG" means 3 -hydroxy- 1,5 -pentanedioi c-2,2,3, 4,4-d5 acid; "HILIC" means hydrophilic interaction liquid chromatography; "HO At" means l-hydroxy-7-azobenzotriazole; "HOBt" means 1-hydroxylbenzotriazole hydrate; "HPLC means high-performance liquid chromatography; "IC50" means the concentration of an agent that produces 50% of the maximal inhibitory response possible for that agent; "wCPBA" means weto-chloroperbenzoic acid; "MeOH" means methanol or methyl alcohol; "NADP+ and NADPH" means the oxidized and reduced forms of nicotinamide adenine dinucleotide phosphate respectively; "NMP" means N-methyl-2-pyrrolidone;

"PG" means protecting group; "Prep" means preparation; "PyBOP" means benzotriazol-1-yloxytripyrrolidino-phosphonium hexafluorophosphate; "PyBrop" means bromo-tris-

pyrrolidino phosphoniumhexafluoro phosphate; "rpm" means revolutions per minute; "(R)-RUCY®-XylBINAP" means RuCl[(R)-daipena][(R)-xylbinap; "SNAr" means nucleophilic aromatic substitution; "TEA" means triethylamine; "TFA" means trifluoroacetic acid; "THF" means tetrahydrofuran; and "Tris" means

tris(hydroxymethyl)aminomethane .

The compounds of Formula I or la, or pharmaceutically acceptable salts thereof, may be prepared by a variety of procedures known in the art, some of which are illustrated in the Schemes, Preparations, and Examples below. The specific synthetic steps for each of the routes described may be combined in different ways, or in conjunction with steps from different schemes, to prepare compounds of Formula I or la, or pharmaceutically acceptable salts thereof. The products of each step in the schemes below can be recovered by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, trituration, and crystallization. In the schemes below, all substituents unless otherwise indicated, are as previously defined. The reagents and starting materials are readily available to one of ordinary skill in the art.

Scheme 1

Oxidation Step C

In Scheme 1, a series of reactions leads to a l-substituted-7-(methylsulfonyl)-l,4-dihydro-2H-pyrimido[4,5-d][l,3]oxazin-2-one, (4), the product of Step C where R2 is as previously defined. "PG" is a protecting group developed for the amino group or oxygen group such as for carbamates, amides or esters. For example, 5-hydroxy methyl 4-amino-2-methylsulfanyl-pyrimidine can be cyclized under standard carbamoylation conditions to the oxazine-2-one using triphosgene and an organic base such as DIPEA or TEA at a temperature of about -30 to -35 °C to give Compound (2), the product of Step A.

Alternatively a dihalide carbonyl or a di-pseudohalide carbonyl such as CDI, phosgene, or diphosgene can be used instead of triphosgene to complete the carbamoylation. The amine of the oxazine can be alkylated with the appropriate substituted alkyl halide such as an iodo reagent in a solvent such as MP and an inorganic base such as K2CO3 at a temperature of about 50-65 °C to give Compound (3), the product of Step B.

Alternatively, a Mitsunobu reaction can be accomplished to alkylate the amine of the oxazine using an appropriate alcohol such as MeOH. Mitsunobu reactions are well known in the art and can convert a hydroxyl group into a leaving group that is displaced by a wide variety of nucleophiles such as a carbamate using triphenylphosphine and an azodicarboxylate such as DIAD or DEAD in a solvent such as THF to give Compound (3). The sulfide can be oxidized to the sulfone under conditions well known in the art such as wCPBA or potassium peroxymonosulfate at a temperature of about 10 to 25 °C in a solvent such as ACN or DCM to give Compound (4), the product of Step C.

Scheme 2

Step G Grignard Reaction Step I Chlorination

(11)

In Scheme 2, a (4-(l-aminoethyl)phenyl)methanol (7) can be generated over several steps from an aryl halide such as a bromide (5) using procedures well known to the skilled artisan. The amine can be protected in substep 1, Step D, where "PG" is a protecting group developed for the amino group such as an amide. The protected aryl bromide (5) can be converted to a ketone under lithium carbonylation conditions to give the aryl ketone (6) in substep 2, Step D. The ketone can then be reduced using a reducing agent such as sodium borohydride in a solvent such as MeOH in substep 1, Step E. The amine can be deprotected at this step (substep 2, Step E) or at a later point in the synthesis to give Compound (7). Alternatively, Compound (6) can be directly converted to Compound (12) in a reductive amination using titanium(IV) isopropoxide in a solvent such as THF and with heating to about 60 °C followed by cooling and the addition of MeOH and a reducing agent such as sodium cyanoborohydride to give Compound (12). Compound (7) can be converted to a benzyl halide such as a chloride under standard halogenation conditions using a halogenating agent such as thionyl chloride or POCh in a solvent such as DCMto give compound (10), Step I. Compound (10) can be protected if needed and alkylated in Step J. For example, the amine, (10) can be protected in substep 1 of Step J using a protecting group such as a trifluoroacetyl or CBZ. Such protecting groups are well known and appreciated in the art. Alternatively, Compound 10 can be prepared from an aldehyde, Compound (8). Compound (8) can be prepared by oxidation of the corresponding benzyl alcohol under such conditions as Dess-Martin periodinane in a solvent such as DCM to give an aldehyde (8). A Grignard reaction can be

accomplished in Step G on the aldehyde to give Compound (7). Compound (7) from Step G can be chlorinated in Step H to give Compound (10) as discussed above for Step I. The chloride of Compound (10) can be displaced with a mono-protected piperazine in a two-step, one pot procedure. It is not always necessary to protect the 1 -phenylethylamine but if protection is chosen, it is advantageous to use a different protecting group on the 1-phenylethylamine than the piperazine amine product (11), Step J, substep 1, to selectively deprotect one or the other PG at the desired step. For example, the 1 -phenylethylamine can be reacted with trifluoroacetic anhydride using an organic base such as TEA in a solvent such as DCM at a temperature of about 0-5 °C to give the protected amine product of substep 1, Step J. One skilled in the art would understand other protecting groups can be utilized on the amine such as CBZ. Displacement of the chloride can then be accomplished under conditions well known by one skilled in the art. For example, the halide can be displaced by the protected or unprotected piperazine using an inorganic base such as K2CO3 and utilizing KI, or Nal as a nucleophilic catalyst to accelerate the reaction. The mixture can be heated to about 60-80 °C in a solvent such as ACN to give the protected Compound (11) of substep 2, Step J. The protecting group on the 1-phenylethylamine can be removed with a base such as aqueous potassium hydroxide to give Compound (11) of substep 3, Step J.

Scheme 3

In Scheme 3, Compound (11) can be reacted with Compound (4), Scheme 1, in a SNAr reaction using an organic base such as DIPEA, CsF to accelerate the reaction, a solvent such as DMSO, and a temperature of about 70-80 °C to give the product of Step L. In Step M, substep 1, a tert-butoxy protected piperazine can be deprotected using an acid such as HC1 in dioxane and MeOH or TFA in DCM whereas an alloc protected piperazine can be deprotected in the presence of a palladium source such as catalytic tetrakis(triphenylphosphine)palladium(0) in a solvent such as THF using a soft nucleophile such as dimedone to give the deprotected piperazine of substep 1, Step M. In substep 2, Step M, the piperazine can be amidated with acryloyl chloride at a temperature of about -50 to -78 °C with or without an organic base such as TEA if the amine is an acid salt in a solvent such as DCM to give compounds of Formula la. Alternatively, an amide coupling can be accomplished with acrylic acid and the appropriate amine in a solvent such as DMF with a coupling reagent such as EDC and an additive such as HOBt. One skilled in the art will recognize that there are a number of methods and reagents for amide formation resulting from the reaction of carboxylic acids and amines. For example, the reaction of the appropriate amine and acrylic acid in the presence of a coupling reagent with or without an organic base such as DIPEA or TEA can provide a compound of Formula la. Other coupling reagents include carbodiimides, such as DCC, DIC, or a carbonyldiimidazole such as CDI. Other amide coupling additives, such as HO At can also be used to enhance the reaction. Additionally, uronium or phosphonium salts of non-nucleophilic anions, such as HBTU, HATU, PyBOP, and PyBrOP could be used in place

of the more traditional coupling reagents. An additive such as DMAP may be used to accelerate the desired amidation reaction.

Scheme 4

Chlorination Step O

1. Deprotect

Step Q 2. Amidation

Alternatively in Scheme 4, the amine of the deprotected product of Compound (7), Scheme 2 can be reacted with Compound (4) as described in Scheme 3, Step L in a SNAr alkylation to give Compound 13. The hydroxyl can be chlorinated as described in Step I, Scheme 2 to give Compound 14, Step O. The chlorine of Compound (14) can be displaced in an alkylation with the piperazine as described in Scheme 2, Step 11, substep 2 to give Compound (5). The protected piperazine can be deprotected and the piperazine then ami dated as described in Scheme 3, Step M to give compounds of Formula la.

Scheme 5

PG = protecting group

1

2

Step I Chlorination

1. Alkylation

Step J (PG-piperazine)

2. Deprotect

(1 1 a)

In scheme 5, a chiral (4-(l-aminoethyl)phenyl)methanol (7a) can be generated over several steps from a chiral aryl halide such as a bromide (5a) using procedures well known to the skilled artisan. The amine can be protected in substep 1, Step D, where "PG" is a protecting group developed for the amino group such as an amide. The aryl bromide (5a) can be converted to a ketone under lithium carbonylation conditions to give the aryl ketone (6a) in sub step 2, Step D. The ketone can then be asymmetrically reduced using a chiral reducing agent such as (R)-RUC Y-XylBINAP in a solvent such as EtOH to provide compound 7a in substep 1, Step E. Compound (7a) can be converted to a chiral benzyl halide such as a chloride under standard halogenation conditions using a halogenating agent such as benzoyl chloride in a solvent such as t-butyl ether to give compound (10a), Step I. Compound (10a) is first reacted with a protected piperazine (PG-piperazine) in the presence of a base such as sodium bicarbonate in a solvent such as acetonitrile to give a protected form in substep 1, Step J. The protected form is then deprotected with a base such as aqueous potassium hydroxide in a solvent such as EtOH to provide compound (11a), substep 2, Step J.

Scheme 6

(15) (16) (17) (18)

In Scheme 6, a series of reactions leads to a l-substituted-7-chloro-4H-pyrido[4,3-d][l,3]oxazin-2-one (18), the product of Step C where R2 is as previously defined. For example, ethyl 4,6-dichloropyridine-3-carboxylate (15) can be reacted with an amine under standard conditions to give the 6-chloro-4-(amino)pyridine-3-carboxylate (16) in a solvent such as acetonitrile. Reduction of the ester group in Compound (16) using a hydride reagent such as lithium aluminum hydride in a solvent such as THF affords the (4-amino-6-chloro-3-pyridyl)methanol (17). Compound such as 17 can be cyclized under standard carbamoylation conditions to the oxazine-2-one using triphosgene and an organic base such as DIPEA or TEA at a temperature of about -20 °C to give Compound (18), the product of Step C. Alternatively a dihalide carbonyl or a di-pseudohalide carbonyl such as CDI, phosgene, or diphosgene can be used instead of triphosgene to complete the carbamoylation. Compound 11, prepared as shown above in Schemes 2 or 5, or as described in alternatives to either Scheme, may be reacted with Compound 18 under standard alkylating conditions. The resulting intermediate compound is then deprotected and amidated as described above in Schemes 3 or 4 to afford the compounds of Formula I where X is CH.

In an optional step, a pharmaceutically acceptable salt of a compound of Formula I or la can be formed by reaction of an appropriate free base of Formula I or la with an appropriate pharmaceutically acceptable acid in a suitable solvent under standard conditions. Additionally, the formation of such salts can occur simultaneously upon deprotection of a nitrogen protecting group. The formation of such salts is well known and appreciated in the art. See, for example, Gould, P.L., "Salt selection for basic drugs," International Journal of Pharmaceutics, 33 : 201-217 (1986); Bastin, R.J., et al. "Salt Selection and Optimization Procedures for Pharmaceutical New Chemical Entities," Organic Process Research and Development, 4: 427-435 (2000); and Berge, S.M., et al., "Pharmaceutical Salts," Journal of Pharmaceutical Sciences, 66: 1-19, (1977). One of ordinary skill in the art will appreciate that a compound of Formula I or la is readily converted to and may be isolated as a pharmaceutically acceptable salt.

Preparation 1

7-Methylsulfanyl- 1 ,4-dihydropyrimido[4, 5-d] [ 1 ,3 ]oxazin-2-one

Triphosgene (859 g, 2.9 mol) is added to a solution of (4-amino-2-methylsulfanyl-pyrimidin-5-yl)methanol (900 g, 5.26 mol) in THF (22.5 L) over 15 minutes at -30 °C. DIPEA (2.449 g, 18.92 mol) is added over 1 hour, while maintaining the reaction temperature between -35 and -30 °C. The reaction mixture is then poured over ice water (30 L) and 2-methyltetrahydrofuran (10 L) is added. The organic phase is washed with water and brine. The organic phase is dried over Na2S04 and is concentrated to dryness. The crude product is slurried with petroleum ether / EtOAc (1 : 1), filtered, and

concentrated to give a yellow solid which is carried on without further purification (890.5 g, 1.62 mol, 83% purity, 86% yield). MS (m/z): 198 (M+H).

Preparation 2

l-Ethyl-7-(methylthio)-l,4-dihydro-2H-pyrimido[4,5-d][l,3]oxazin-2-one

To a solution of 7-methylsulfanyl-l,4-dihydropyrimido[4,5-d][l,3]oxazin-2-one (280 g, 1.42 mol) in MP (2.24 L) is added K2C03 (294.2 g, 2.13 mol) and ethyl iodide (336.3 g, 1.99 mol) at room temperature. The mixture is stirred for 16 hours at 50 °C and then diluted with DCM (3 L) and water (6 L). The organic phase is separated and washed with water and brine and concentrated to dryness to give the crude title compound (286 g, 1.27 mol, 83% purity, 91% yield). MS (m/z): 226 (M+H).

Preparation 3

l-Methyl-7-methylsulfanyl-4H-pyrimido[4,5-d][l,3]oxazin-

To a solution of triphenylphosphine (1.61 g, 6.08 mmol) and 7-methylsulfanyl-l,4-dihydropyrimido[4,5-d][l,3]oxazin-2-one (1.00 g, 5.07 mmol) in THF (25 mL) is added MeOH (0.248 mL, 6.08 mmol) followed by dropwise addition of DIAD (1.21 mL, 6.08 mmol) at ambient temperature. After stirring overnight the solvent is removed under vacuum and the resulting yellow oil is purified by silica gel chromatography (40-50%) EtOAc/hexanes) to give the title compound as a white solid (1.08 g, 5.11 mmol, quantitative). MS (m/z): 212 (M+H).

Preparation 4

l-Ethyl-7-(methylsulfonyl)-l,4-dihydro-2H-pyrimido[4,5-d][l,3]oxazin-

To a stirred solution of l-ethyl-7-(methylthio)-l,4-dihydro-2H-pyrimido[4,5-d][l,3]oxazin-2-one (286 g, 1.24 mol) in ACN (2.8 L) and water (1.4 L) is added potassium peroxymonosulfate (1526 g, 2.48 mol) as a solid over 20 minutes, and the

resulting mixture is stirred for 16 hours at 10-20 °C. The reaction mixture is filtered and the obtained filter cake is washed with DCM. The combined filtrate and DCM are washed with 5% Na2S03, water, and brine. The organic phase is dried over Na2S04 and concentrated to provide the title compound (133.8 g, 93% purity, 41% yield). MS (m/z): 258 (M+H).

The following compound is prepared essentially by the method of Preparation 4.

Table 1

Preparation 6

N-[(l S)-l-(4-Bromophenyl)ethyl]-2,2,2-trifluoro-acetamide

Trifluoroacetic anhydride (165 mL, 1.17 mol) is added dropwise to a solution of (l S)-l-(4-bromophenyl)ethanamine (213 g, 1.06 mol) in ACN (1.3 L) at 5 °C followed by the dropwise addition of TEA (326 mL, 2.34 mol) over 1 hour. After 30 minutes, water (3 L) and brine (1 L) are added resulting in the formation of a colorless precipitate. The slurry is stirred for 15 minutes and then the solid is filtered, washed with water and hexanes, and dried by air current followed by drying at 40 °C under vacuum to give the title compound (290 g, 92%). ¾ NMR (de-DMSO) δ 1.44 (d, 3H, J= 7.1 Hz), 4.98 (dddd, 1H, J= 7.6, 7.1, 7.1, 7.1 Hz), 7.30 (d, 2H, J= 8.4 Hz), 7.55 (d, 2H, J= 8.4 Hz), 9.91 (d, lH, J= 7.6 Hz).

Preparation 7

N-[(l S)-l-[4-(2-Cyclopropylacetyl)phenyl]ethyl]-2,2,2-trifluoro-acetamide

«-Butyl lithium (2.5 M in hexanes, 53 mL, 130 mmol) is added dropwise to a solution of N-[(l S)-l-(4-bromophenyl)ethyl]-2,2,2-trifluoro-acetamide (18.00 g, 60.79 mmol) in THF (600 mL) at -78 °C so as to maintain an internal temperature below -70 °C. After the addition is complete, the mixture is stirred for 45 minutes at -78 °C and then 2-cyclopropyl-N-methoxy-N-methyl-acetamide (11.4 g, 79.6 mmol) is added as a solution in THF (10 mL). The mixture is stirred at -78 °C for 45 minutes, saturated aqueous ammonium chloride is added, and the mixture is warmed to room temperature. The layers are separated and the organic layer is dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. To the solid is added a small amount of DCM and the mixture is heated briefly to dissolve the solids. The mixture is concentrated until just before precipitation and then hexanes (150 mL) is added dropwise with vigorous stirring to give a colorless solid. The solid is collected via filtration, washed with a small amount of hexanes, and dried under reduced pressure to give the title compound (13.82 g, 76%) as a colorless solid. MS (m/z): 298.3 (M-H).

The following compound is prepared essentially by the method of Preparation 7.

Table 2

Preparation 9

l-[4-[(l S)-l-Aminoethyl]phenyl]-2-cyclopropyl-ethanol

Sodium borohydride (0.1411 g, 2 equiv., 3.729 mmol,) is added to a solution of N-[(l S)-l-[4-(2-cyclopropylacetyl)phenyl]ethyl]-2,2,2-trifluoro-acetamide (558 mg, 1.86 mmol) in MeOH (15 mL) cooled in an ice water bath. The mixture is stirred for about 2.5 hours, and then potassium hydroxide (800 mg, 14.2 mmol) in water (3 mL) is added. The mixture is stirred at room temperature for about 20 hours. The mixture is concentrated and partitioned between DCM and water. The organic layer is washed with saturated aqueous sodium chloride, dried over sodium sulfate, filtered, and concentrated to give the title compound (354 mg, 1.38 mmol, 74%) as a white solid. The material is used without further purification. ES/MS (m/z): 189.0 (M-OH).

Preparation 10

2,2,2-Trifluoro-N-[(l S)-l-(4-formylphenyl)ethyl]acetamide

Dess-Martin periodinane (20.9 g, 49.3 mmol) is added to a 0 °C solution of 2,2,2-trifluoro-N-[(l S)-l-[4-(hydroxymethyl)phenyl]ethyl]acetamide (11.1 g, 44.9 mmol) in DCM (450 mL). The reaction mixture is stirred overnight and allowed to warm to room temperature. The reaction mixture is diluted with additional DCM and washed with saturated aqueous NaHCCb, saturated aqueous Na2S203, and brine. The combined organics are dried (Na2S04), filtered, and concentrated to give a residue which is purified by silica gel chromatography eluting with a gradient of 0-50% EtOAc/hexanes to give the title compound as a white solid (9.5 g, 39 mmol, 86%). ES/MS (m/z): 244 (M-H).

Preparation 10a

2,2,2-Trifluoro-N-[(l S)-l-[4-(hydroxymethyl)phenyl]ethyl]acetamide

Trifluoroacetic anhydride (12 mL, 85.4 mmol) is added to a 0 °C solution of [4-[(l S)-l-aminoethyl]phenyl]methanol (10.8 g, 71.4 mmol) in CH2C12 (150 mL). After 10 minutes, triethylamine (24 mL, 172 mmol) in CH2C12 (8 mL) is added dropwise over 30 minutes, the cooling bath is removed and the reaction is stirred overnight. The reaction mixture is concentrated under vacuum, diluted with additional CH2C12, and washed with 1 N aqueous HC1 and water. The organic phase is dried (Na2S04), filtered, and

concentrated. The crude material is purified by silica gel chromatography eluting with a gradient of 0-50% EtOAc/hexanes to give the title compound as a white solid (11.1 g, 44.9 mmol, 63%). ES/MS (m/z): 246 (M-H).

Preparation 11

2,2,2-Trifluoro-N-[(l S)-l-[4-(l-hydroxy-3-methyl-butyl)phenyl]ethyl]acetamide

To a solution of 2,2,2-trifluoro-N-[(l S)-l-(4-formylphenyl)ethyl]acetamide (1.72 g, 7.01 mmol) in THF (35 mL) cooled in an ice water bath is added isobutylmagnesium bromide (2 M in diethyl ether, 7.0 mL, 14.0 mmol) and stirred for about 30 minutes. The mixture is quenched with saturated aqueous ammonium chloride and partitioned between EtOAc and water. The organic layer is washed with saturated aqueous sodium chloride, dried over sodium sulfate, filtered, and concentrated to give the title compound as an oil (1.40 g, 4.15 mmol, 59%) which is used without further purification. ES/MS (m/z): 302.0 (M-H).

The following compound is prepared essentially by the method of Preparation 11.

Table 3

Preparation 13

fert-Butyl 4-[2-cyclopropyl-l-[4-[(l S)-l-[(2,2,2- trifluoroacetyl)amino]ethyl]phenyl]ethyl]piperazine-l-carboxylate

Titanium(IV) isopropoxide (60 mL, 200 mmol) is added to a solution of N-[(1 S)-l-[4-(2-cyclopropylacetyl)phenyl]ethyl]-2,2,2-trifluoro-acetamide (12.0 g, 40.1 mmol) and tert-butyl piperazine-l-carboxylate (17.9 g, 96.1 mmol) in THF (80 mL) and the mixture is stirred at 60 °C overnight. The mixture is cooled to room temperature and MeOH (80 mL) is added followed by the portion-wise addition of sodium

cyanoborohydride (5.3 g, 80 mmol). The mixture is stirred at room temperature for 8 hours and then water and MeOH are added and the mixture is stirred at room temperature overnight. The mixture is filtered to remove solids and the solids are rinsed with MeOH and water. The filtrate is partially concentrated to remove most of the MeOH and the residue is extracted with EtOAc (2χ). The combined organic extracts are dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude material is purified via column chromatography eluting with a gradient of 0% to 30% EtOAc in solvent B where solvent B is 1 : 1 hexanes:DCM to give the title compound (10.5 g, 56%) as a colorless solid. MS (m/z): 470.3 (M+H).

Preparation 14

tert-Butyl 4-[l-[4-[(l S)-l-aminoethyl]phenyl]-2-cyclopropyl-ethyl]piperazine-l- carboxylate

Aqueous potassium hydroxide (5 M, 69 mL, 350 mmol) is added to a solution of fert-butyl 4-[2-cyclopropyl-l-[4-[(l S)-l-[(2,2,2-trifluoroacetyl)amino]ethyl]phenyl]ethyl] piperazine-l-carboxylate (32.24 g, 68.67 mmol) in EtOH (350 mL) and the resulting mixture is stirred at room temperature for 4 hours. The EtOH is removed under reduced pressure and to the residue is added saturated aqueous sodium bicarbonate and the mixture is extracted with DCM. The combined organic extracts are dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give the title compound (24.33 g, 96.5% purity containing 3.5% residual DCM, 92% yield) as a colorless viscous oil. MS (m/z): 374.3 (M+H).

The following compound is prepared essentially by the method of Preparation 14.

Table 4

Alternate Preparation 14

A solution of potassium hydroxide (1.28 g, 22.9 mmol) in water (4 mL) is added to a solution of fert-butyl 4-[2-cyclopropyl-l-[4-[(ls)-l -[(2,2,2-trifluoroacetyl)amino]ethyl]phenyl]ethyl]piperazine-l-carboxylate (2.15 g, 4.58 mmol) in EtOH (23 mL) and the mixture stirred at room temperature. After about 6 hours, the mixture is concentrated. The residue is partitioned between DCM and saturated sodium bicarbonate solution. The organic layer is washed with water and saturated aqueous sodium chloride, dried over sodium sulfate, filtered, and concentrated to give the title compound as an oil which is used without purification (1.73 g, 4.49 mmol, 98%). ES/MS (m/z): 374.2 (M+H).

Preparation 18

tert-Butyl 4-[(lR)-l-[4-[(l S)-l-aminoethyl]phenyl]-2-cyclopropyl-ethyl]piperazine-l- carboxylate, diastereomer 1

Preparation 19

tert-Butyl 4-[(l S)-l-[4-[(l S)-l-aminoethyl]phenyl]-2-cyclopropyl-ethyl]piperazine-l- carboxylate, diastereomer 2

A 1 : 1 mixture of fert-butyl 4-[(lR)-l-[4-[(l S)-l-aminoethyl]phenyl]-2-cyclopropyl-ethyl]piperazine-l-carboxylate, diastereomer 1 and tert-butyl 4-[(l S)-l-[4-[(l S)-l-aminoethyl]phenyl]-2-cyclopropyl-ethyl]piperazine-l-carboxylate, diastereomer 2 (3.23 g) is dissolved in MeOH (40 mL) and is separated into individual diastereomers by preparative chiral HPLC chromatography using the following conditions: column Chiralpak AD, 20 μιη, (8 x 33 cm); injection volume 10 mL; eluent 100% MeOH with 0.2% DMEA; detection wavelength 220 nm; flow rate 400 mL/min. Preparation 12, tert-butyl 4-[(lR)-l-[4-[(l S)-l-aminoethyl]phenyl]-2-cyclopropyl-ethyl]piperazine-l-carboxylate, diastereomer 1, is obtained from the first eluting peak as a clear viscous oil (1.50 g, 46%, >99% de). MS (m/z): 374.3 (M+H). Preparation 13, tert-butyl 4-[(l S)-l-[4-[(l S)-l-aminoethyl]phenyl]-2-cyclopropyl-ethyl]piperazine-l-carboxylate, diastereomer 2, is obtained from the second eluting peak as a clear viscous oil (1.46 g, 45%, >98.2% de). MS (m/z): 374.3 (M+H).

Preparation 20

N,3-Dimethoxy-N-methyl-propanamide

A solution of 3-methoxypropanoic acid (62 g, 577.7 mmol) in DCM (1200 mL) is treated portion-wise slowly with Ι,Γ-carbonyldiimidazole (103 g, 635.2 mmol) and stirred at room temperature for 2 hours. N,0-dimethylhydroxylamine hydrochloride (62 g, 635.6 mmol) is added and the mixture is stirred at room temperature overnight. The mixture is washed with water (2x), 0.1M aq HCl (2x), and with saturated aqueous sodium bicarbonate (2x), dried over magnesium sulfate, filtered and concentrated to dryness to give the crude material. The crude material is chromatographed over silica gel eluting with a gradient of 20-40% acetone in hexanes. The resulting oil is dried overnight under vacuum to give the title compound (69.5g, 81.7%). ¾ MR (CDCb) δ 2.72 (t, 2H), 3.2 (s, 3H), 3.38 (s, 3H), 3.7 (m, 5H).

Preparation 21

2,2,2-Trifluoro-N-[(l S)-l-[4-(l-hydroxy-3-methoxy-propyl)phenyl]ethyl]acetamide

2,2,2-Trifluoro-N-[(l S)-l-[4-(3-methoxypropanoyl)phenyl]ethyl]acetamide (23.62 g, 73.98 mmol, 95 mass%) is dissolved in MeOH (700 mL) and treated with sodium borohydride (5.6 g, 150 mmol). After stirring at room temperature for 2 hours the mixture is treated with saturated aqueous ammonium chloride and the MeOH is evaporated. The resulting material is partitioned between water and EtOAc, separated and the combined organics are dried over Na2S04, filtered, and concentrated to dryness. The crude material is chromatographed on silica gel eluting with 40% EtOAc in hexanes to give the title compound as a white solid (17.82g, 79%). MS (m/z): 306 (M+H).

2,2,2-trifluoro-N-[(l S)-l-[4-(l-hydroxy-3-methoxy-propyl)phenyl]ethyl]acetamide (21.76g, 71.27 mmol) is dissolved in DCM (350mL) and cooled to -10 °C. Thionyl chloride (26.12 g, 16 mL, 219.6 mmol) is added dropwise and the reaction is stirred for 2 hours. The mixture is concentrated to dryness, re-dissolved in DCM, and re-concentrated. The crude material is dissolved in ACN (300 mL) and t-butyl piperazine-l-carboxylate (26.55 g, 142.6 mmol), potassium carbonate (39.5 g, 286 mmol) and potassium iodide (12.0 g, 72.3 mmol) are added. The mixture is heated to 80 °C for 72 hours. The resulting white solid is filtered and washed with EtOAc. The combined filtrates are washed with aqueous ammonium chloride, dried over magnesium sulfate, filtered, and concentrated. The crude material is chromatographed on silica gel eluting with a gradient of 40-80% EtOAc in hexanes to give the title compound as a white foam (29.63g, 88%). MS (m/z): 474 (M+H).

Preparation 23

tert-Butyl 4-[l-[4-(l S)-l-aminoethyl]phenyl]-3-methoxy-propyl]piperazine-l- carboxylate, diastereomer 1

Preparation 24

tert-Butyl 4-[l-[4-(l S)-l-aminoethyl]phenyl]-3-methoxy-propyl]piperazine-l- carboxylate, diastereomer 2

O

O^

To a solution of fert-butyl 4-[3-methoxy-l-[4-[(l S)-l-[(2,2,2-trifluoroacetyl)amino]-ethyl]propyl]piperazine-l-carboxylate (29.60 g, 62.5 mmol) in EtOH (310 mL) is added aq potassium hydroxide (63 mL, 5 M). The solution is stirred for 4 hours at room temperature and then the mixture is concentrated to dryness. The crude residue is treated with water and saturated aqueous sodium bicarbonate and extracted with DCM (3 χ). The combined organic extracts are dried over sodium sulfate, filtered, and concentrated to give the title compound (23.6g) which is dissolved in MeOH (236 mL) and separated into individual diastereomers by chiral SFC chromatography using the following conditions: column: Lux Cellulose- 1, (5 x 25 cm); injection volume: 1 mL every 2.5 minutes, eluent 15% MeOH/CC , detection wavelength 230 nm; flow rate 300 g/min; column temperature: 40 °C; BPR Setpoint: 100 bar; BPR temperature: 40 °C. The title compound of Preparation 28 is obtained from the first eluting peak as a clear viscous yellow oil (10.1 g, 42.8%, 96.6% de). MS (m/z): 378 (M+H). The compound of Preparation 29 is isolated as the second eluting peak as a clear viscous yellow oil (10.3 g, 43.6%, 95.2% de). MS (m/z): 378 (M+H).

Preparation 25

7-[[(l S)-l-[4-(l-Chloro-2-cyclopropyl-ethyl)phenyl]ethyl]amino]-l-ethyl-4H- pyrimido[4,5-d][l,3]oxazin-2-one

Thionyl chloride (0.23 mL, 3.219 mmol) is added to a mixture of 7-[[(l S)-l-[4-(2-cyclopropyl-l-hydroxy-ethyl)phenyl]ethyl]amino]-l-ethyl-4H-pyrimido[4,5-d][l,3]oxazin-2-one (432 mg, 1.03 mmol) and potassium carbonate (741 mg, 5.37 mmol) in DCM (20 mL) and the mixture is stirred at room temperature for 20 minutes. The mixture is filtered through diatomaceous earth and concentrated to give the title compound (518 mg, 1.07 mmol, 100%) as a white foam, which is used without further purification. ES/MS (m/z): 401.2/403.2 (M+H).

The following compounds are prepared essentially by the method of Preparation 25.

Table 5

Preparation 29

tert-Butyl 4-[(lR)-2-cyclopropyl-l-[4-[(l S)-l-[(l-ethyl-2-oxo-4H-pyrimido[4,5- d][l,3]oxazin-7-yl)amino]ethyl]phenyl]ethyl]piperazine-l-carboxylate

To a solution of fert-butyl 4-[(lR)-l-[4-[(l S)-l-aminoethyl]phenyl]-2-cyclopropyl-ethyl]piperazine-l-carboxylate (864 mg, 3.35 mmol), and l-ethyl-7-(methylsulfonyl)-l,4-dihydro-2H-pyrimido[4,5-d][l,3]oxazin-2-one (1.14 g, 3.05 mmol), in DMSO (15 mL) is added CsF (1.39 g, 9.15 mmol) and DIPEA (0.80 mL, 4.6 mmol). The mixture is stirred at 60 °C for 1.5 hours. The mixture is cooled to room temperature, diluted with EtOAc, and washed with water (2χ). The combined aqueous washes are extracted with EtOAc and the combined organic extracts are dried (Na2S04), filtered, and concentrated to dryness. The resulting crude product is purified by silica gel

chromatography eluting with a gradient of 55% to 95% EtOAc in hexanes to give the title compound as a colorless solid (1.47 g, 88%). MS (m/z): 551.3 (M+H).

The following compounds are prepared essentially by the method of Preparation 29.

Table 6

razine-1 -carboxylate, isomer 1

Preparation 39

7-[[(l S)-l-[4-[(lR)-2-Cyclopropyl-l-piperazin-l-yl-ethyl]phenyl]ethyl]amino]-l-ethyl- 4H-pyrimido[4,5-d][l,3]oxazin-2-one

Hydrochloric acid (95 mL, 5.5 M in isopropanol, 520 mmol) is added dropwise to a solution of tert-butyl 4-[(lR)-2-cyclopropyl-l-[4-[(l S)-l-[(l-ethyl-2-oxo-4H-pyrimido[4,5-d][l,3]oxazin-7-yl)amino]ethyl]phenyl]ethyl]piperazine-l-carboxylate (29.45 g, 53.48 mmol) in EtOAc (570 mL) at 40 °C. The mixture is allowed to stir 3 hours, at room temperature, and then water (300 mL) is added. The layers are separated and the organic layer is extracted with water (2 χ 150 mL). The pH of the combined aqueous extracts are adjusted to pH 10 by the addition of 5 N NaOH resulting in the formation of a colorless solid. The solid is collected by filtration, washed with water, and air-dried to give the title compound (25.18 g, 99%) as a colorless solid. MS (m/z): 451.2 (M+H).

The following compound is prepared essentially by the method of Preparation 39.

Table 7

Preparation 41

7-[[(l S)-l-[4-(2-Cyclopropyl-l-piperazin-l-yl-ethyl)phenyl]ethyl]amino]-l-methyl-4H- pyrimido[4,5-d][l,3]oxazin-2-one, diastereomer 1

fert-Butyl 4-[2-cyclopropyl-l-[4-[(l S)-l-[(l-methyl-2-oxo-4H-pyrimido[4,5-d][l,3]oxazin-7-yl)amino]ethyl]phenyl]ethyl]piperazine-l-carboxylate, diastereomer 1 (245 mg, 0.46 mmol) is dissolved in DCM (2.5 mL). TFA (0.7 mL, 9 mmol) is added and the reaction is stirred at room temperature for 90 minutes. The mixture is quenched with 20% aq K^CO^ and extracted with DCM (3 χ). The combined organic extracts are dried over Na2S04, filtered, and concentrated to dryness on high vacuum overnight to give the title compound as a white foam, (196 mg, 88.5%). MS (m/z): 437 (M+H).

The following compounds are prepared essentially by the method of Preparation 41.

Table 8

Preparation 49

7-[[(l S)-l-[4-(2-Cyclopropyl-l-piperazin-l-yl-ethyl)phenyl]ethyl]amino]-l-ethyl-4H- pyrimido[4,5-d][l,3]oxazin-2-one

A mixture of 7-[[(l S)-l-[4-(l-chloro-2-cyclopropyl-ethyl)phenyl]ethyl]amino]-l-ethyl-4H-pyrimido[4,5-d][l,3]oxazin-2-one (518 mg, 1.07 mmol), potassium carbonate (445 mg, 3.217 mmol), sodium iodide (161 mg, 1.07 mmol) and piperazine (277 mg, 3.22 mmol) in ACN (3 mL) is heated in a sealed vial to 70 °C. After ~8 hours, the mixture is cooled to room temperature, diluted with EtOAc, filtered through diatomaceous earth, and concentrated. The crude material is purified on silica gel eluting with a gradient of 1% to 7% 3 M NH3/MeOH in DCM to give the title compound (306 mg, 0.66 mmol, 62%) as a white solid. ES/MS (m/z): 451.2 (M+H).

The following compounds are prepared essentially by the method of Preparation 49 the appropriate protected piperazine.

Table 9

Preparation 53

tert-Butyl 4-[l-[4-[(l S)-l-[(2,2,2-trifluoroacetyl)amino]ethyl]phenyl]propyl]piperazine- 1 -carboxylate, isomer 1

Preparation 54

tert-Butyl 4-[l-[4-[(l S)-l-[(2,2,2-trifluoroacetyl)amino]ethyl]phenyl]propyl]piperazine- 1 -carboxylate, isomer 2

fert-Butyl 4-[l-[4-[(l S)-l-[(2,2,2-trifluoroacetyl)amino]ethyl]-phenyl]propyl]piperazine-l -carboxylate (29.2 g, 65.8 mmol) is dissolved in MeOH (584 mL) and resolved by chiral SFC chromatography using the following conditions: column: Chiralpak AD-H, 5x25 cm; eluent 85/15 C02/MeOH with 0.5% dimethylethylamine; flow rate 300 g/min; detection wavelength 230 nm; column temperature 40 °C; BPR setpoint 100 bar; 40 °C solvent temperature. Isomer 1 is isolated as the first eluting peak (14.15 g, 31.9 mmol). ES/MS (m/z): 444 (M+H). Isomer 2 is isolated as the second eluting peak (13.87 g, 31.3 mmol). ES/MS (m/z): 444 (M+H).

Preparation 55

fert-Butyl 4-[l-[4-[(l S)-l-[(l-ethyl-2-oxo-4H-pyrimido[4,5-d][l,3]oxazin-7- yl)amino]ethyl]phenyl]-3-methyl-butyl]piperazine-l-carboxylate, isomer 1

Preparation 56

fert-Butyl 4-[l-[4-[(l S)-l-[(l-ethyl-2-oxo-4H-pyrimido[4,5-d][l,3]oxazin-7- yl)amino]ethyl]phenyl]-3-methyl-butyl]piperazine-l-carboxylate, isomer 2

fert-Butyl 4-[l-[4-[(l S)-l-[(l-ethyl-2-oxo-4H-pyrimido[4,5-d][l,3]oxazin-7-yl)amino]ethyl]phenyl]-3-methyl-butyl]piperazine-l-carboxylate (2.6 g, 4.70 mmol) is dissolved in 4: 1 isopropanol: chloroform (50 mL) and resolved by chiral SFC

chromatography using the following conditions: column: Chiralpak AD-H, 5x25 cm; injection volume 1 mL; eluent 75/25 CO2/IPA with 0.5% dimethylethylamine; flow rate 280 g/min; detection wavelength 240 nm; column temperature 40 °C; BPR setpoint 100 bar; 40 °C solvent temperature. Preparation 45 is isolated as the first eluting peak (1.02 g, 1.89 mmol). ES/MS (m/z): 553.4 (M+H). Preparation 46 is isolated as the second eluting peak (1.05 g, 1.90 mmol). ES/MS (m/z): 553.4 (M+H).

Example 1

7-[[(l S)-l-[4-[(lR)-2-Cyclopropyl-l-(4-prop-2-enoylpiperazin-l yl)ethyl]phenyl]ethyl]amino]-l-ethyl-4H-pyrimido[4,5-d][l,3]oxazin-

Acryloyl chloride (305 μΐ., 3.75 mmol, in 2 mL DCM) is added dropwise to a solution of 7-[[(lS)-l-[4-[(l R)-2-cyclopropyl- 1 -piperazin- 1 -yl-ethyl]phenyl]ethyl]amino]-l-ethyl-4H-pyrimido[4,5-d][l,3]oxazin-2-one (1.73 g, 3.26 mmol) in DCM at -78 °C. After 2 minutes at -78 °C a few drops of MeOH are added followed by saturated aqueous sodium bicarbonate and the mixture is allowed to warm to room temperature. DCM is added, the layers are separated, and the aqueous layer is extracted with DCM. The combined organic extracts are dried (Na2S04), filtered, and concentrated to dryness. The resulting crude product is purified by silica gel

chromatography (25% to 40% Solvent A in Solvent B where Solvent A is 10%

MeOH/acetone and Solvent B is hexanes) to give the title compound as a colorless solid (1.16 g, 70%). MS (m/z): 505.3 (M+H).

The following compounds are prepared essentially by the method of Example 1.

Table 11

Determination of the IDHl X-ray crystal structure in complex with 7-[[(l S)-l-[4-[(l S)-2-cyclopropyl- 1 -(4-prop-2-enoylpiperazin- 1 -yl)ethyl]phenyl]ethyl]amino]-l -ethyl -4H-pyrimido[4,5 -d] [ 1 ,3 ]oxazin-2-one.

The crystal structure of IDHl in complex with 7-[[(l S)-l-[4-[(l S)-2-cyclopropyl-l-(4-prop-2-enoylpiperazin-l-yl)ethyl]phenyl]ethyl]amino]-l-ethyl-4H-pyrimido[4,5-d][l,3]oxazin-2-one is determined from X-ray diffraction data collected at the synchrotron beam -line APS 31-ID operated at the Advanced Photon Source at Argonne National Laboratory, Argonne, IL 60439. IDHl protein with a R132H mutation is commercially available from multiple sources. Alternatively, IDHl R132H protein may be isolated from a commercially available cell line harboring the mutation by techniques well known and routinely used by those skilled in the art. Crystals are obtained from sitting drop trays equilibrated at 21 °C with IDHl protein with the mutation R132H at a concentration of 15 mg/ml in a buffer containing 10 mM HEPES pH 7.5, 150 mM sodium chloride, 10% glycerol, 5 mM dithiothreitol and 2 mM 7-[[(l S)-l-[4-[(l S)-2-cyclopropyl-l-(4-prop-2-enoylpiperazin-l-yl)ethyl]phenyl]ethyl]amino]-l-ethyl-4H-pyrimido[4,5-d][l,3]oxazin-2-one, and mixed with an equal volume of reservoir solution containing lOOmM Bis Tris pH 5, 5% DMSO, 22% PEG 3350 and 200mM Ammonium Sulfate. Crystals are soaked overnight in a solution containing 3 mM of 7-[[(l S)-l-[4-[(l S)-2-cyclopropyl-l-(4-prop-2-enoylpiperazin-l-yl)ethyl]phenyl]ethyl]amino]-l-ethyl-4H-pyrimido[4,5-d][l,3]oxazin-2-one, before being transferred to a solution

supplemented with 22% Ethylene Glycol and flash frozen for data collection. Diffraction data to 2.8 A resolution is collected with X-ray radiation of wavelength 0.9793 A. The crystals belong to Space Group P432i2 with cell parameters a=82.74 A, b=82.74 A, c=299.4A, α=β=γ=90°. The structure is determined by Molecular Replacement and contained one dimer molecule of IDHl . The difference electron density maps calculated after modelling the IDHl protein have clear density for two bound molecules of 7-[[(l S)-l-[4-[(l S)-2-cyclopropyl-l-(4-prop-2-enoylpiperazin-l-yl)ethyl]phenyl]ethyl]amino]-l-ethyl-4H-pyrimido[4, 5-d] [ 1 ,3 ]oxazin-2-one.

The stereochemistry of 7-[[(l S)-l-[4-[(l S)-2-cyclopropyl-l-(4-prop-2-enoylpiperazin-l-yl)ethyl]phenyl]ethyl]amino]-l-ethyl-4H-pyrimido[4,5-d][l,3]oxazin-2-one is determined from the electron density, and both molecules of 7-[[(l S)-l-[4-[(l S)-2-cyclopropyl-l-(4-prop-2-enoylpiperazin-l-yl)ethyl]phenyl]ethyl]amino]-l-ethyl-4H-pyrimido[4,5-d][l,3]oxazin-2-one are modeled and the co-complex structure refined to R-factors of Rwork=0.192 and Rfree=0.228.

Table 12

Coordinates of 7-[[(lS)-l-[4-[(lS)-2-cyclopropyl-l-(4-prop-2-enoylpiperazin-l- yl)ethyl]phenyl]ethyl]amino]-l-ethyl-4H-pyrimido[4,5-d][l,3]oxazin-2-one.

ATOM X Y Z

02 48.364 1.353 -2.648

C21 49.136 0.711 -1.964

C22 48.932 -0.779 -1.799

C23 47.694 -1.239 -2.530

N5 50.174 1.294 -1.330

C18 50.422 2.738 -1.428

C17 51.848 3.008 -1.849

C19 51.126 0.603 -0.449

C20 52.568 0.906 -0.834

N4 52.797 2.388 -0.868

C16 54.242 2.810 -1.061

C24 55.094 2.321 0.113

C25 56.411 3.091 0.050

C27 57.062 3.523 1.306

C26 56.432 4.534 0.413

C13 54.761 2.408 -2.428

C12 55.212 1.125 -2.730

Cll 55.701 0.819 -3.988

C14 54.812 3.365 -3.432

C15 55.295 3.057 -4.691

CIO 55.742 1.778 -4.997

C8 56.243 1.464 -6.405

C9 55.107 1.279 -7.400

N3 57.128 0.291 -6.469

CI 58.372 0.310 -5.968

N 59.104 -0.809 -6.086

Nl 58.775 1.474 -5.441

C2 60.041 1.523 -5.009

N2 60.425 2.651 -4.254

C6 59.506 3.796 -4.105

C7 58.569 3.631 -2.901

C5 61.546 2.595 -3.438

01 61.736 3.358 -2.523

0 62.437 1.605 -3.629

C4 62.347 0.684 -4.739

C3 60.923 0.429 -5.132

C 60.369 -0.717 -5.655

Example 13

1 -Ethyl-7-[[( 1 S)- 1 -[4-[3 -methoxy- 1 -(4-prop-2-enoylpiperazin- 1 - yl)propyl]phenyl]ethyl]amino]-4H-pyrimido[4,5-d][l,3]oxazin-2-one, diastereomer 1

fert-Butyl 4-[(l S)-l-[4-[(l S)-l-[(l-ethyl-2-oxo-4H-pyrimido[4,5-d][l,3]oxazin-7-yl)amino]ethyl]phenyl]-3-methoxy-propyl]piperazine-l-carboxylate (1.372 g, 2.473 mmol) is dissolved in DCM (15 mL) and TFA (10 mL, 15.08 g, 132.3 mmol) is added. The reaction is stirred for 1 hour and then concentrated to dryness. The crude material is dissolved in DCM (12 mL) and DIPEA (1.25 mL, 7.17 mmol) and the mixture is cooled to -78 degrees. Acryloyl chloride (0.18 mL, 0.20 g, 2.2 mmol) is added dropwise. After 10 minutes, a few drops of methanol are added to quench the remaining acryloyl chloride and the reaction is concentrated to dryness (cold). The crude material is chromatographed over silica gel eluting with a gradient of 50-70% acetone in hexanes to give the title compound as a white foam (867mg, 73%). MS (m/z): 509 (M+H)

The following compound is prepared essentially by the method of Example 13.

Table 13

Example 15

7-[[( 1 S)- 1 - [4-[(l S)-2-Cyclopropyl- 1 -(4-prop-2-enoylpiperazin- 1 - yl)ethyl]phenyl]ethyl]amino]-l-ethyl-4H-pyrimido[4,5-d][l,3]oxazin-2-one

■2 H2S04

7-[[( 1 S)- 1 - [4-[(l S)-2-Cyclopropyl- 1 -(4-prop-2-enoylpiperazin- 1 -yl)ethyl]phenyl]ethyl]amino]-l-ethyl-4H-pyrimido[4,5-d][l,3]oxazin-2-one (188 mg) is placed in 5 mL of acetone while stirring at 1000 rpm/60°C. The sample is a clear solution. 45 μΙ_, of Sulfuric acid is added dropwise (diluted into 2 mL of acetone). A thick white slurry results after a few drops. After addition of half of the sulfuric acid, the slurry consistency is changed. Addition of the second half of the sulfuric acid is done slowly, dropwise. The slurry gumms slightly before converting to a bright white free-flowing slurry of solid. Heat is shut off to the plate after 30 minutes, and the sample cooled to room temperature, giving a thick slurry of white solid. The white solid is isolated by vacuum filtration. The resulting cake is bright white solid. The sample is dried in place on the filter under air stream for 20 minutes, then in the 70°C vacuum oven overnight. 266 mg recovered (96.9% yield).

We Claim:
1. A compound of the Formula:

wherein:

R1 is -CH2CH(CH3)2, -CH2CH3, -CH2CH2OCH3, or -CH2-cyclopropyl;

R2 is -CH3 or -CH2CH3; or

a pharmaceutically acceptable salt thereof.

A compound of Claim 1 which is:

7-[[(l S)-l -[4-[(lR)-2-Cyclopropyl- 1 -(4-prop-2-enoylpiperazin- 1 - yl)ethyl]phenyl]ethyl]amino]-l-ethyl-4H-pyrimido[4,5-d][l,3]oxazin-2-one; 7-[[( 1 S)- 1 - [4-[( 1 S)-2-cyclopropyl- 1 -(4-prop-2-enoylpiperazin- 1 - yl)ethyl]phenyl]ethyl]amino]-l-ethyl-4H-pyrimido[4,5-d][l,3]oxazin-2-one; 1 -Ethyl-7-[[( 1 S)- 1 - [4-[ 1 -(4-prop-2-enoylpiperazin- 1 - yl)propyl]phenyl]ethyl]amino]-4H-pyrimido[4,5-d][l,3]oxazin-2-one, isomer i ;

1 -Ethyl-7-[[( 1 S)- 1 -[4-[ 1 -(4-prop-2-enoylpiperazin- 1 - yl)propyl]phenyl]ethyl]amino]-4H-pyrimido[4,5-d][l,3]oxazin-2-one, isomer

2;

or a pharmaceutically acceptable salt of any of them.

A compound of Claim 2 which is 7-[[(l S)-l-[4-[(l S)-2-cyclopropyl-l-(4- prop-2-enoylpiperazin- 1 -yl)ethyl]phenyl]ethyl]amino]- 1 -ethyl -4H- pyrimido[4,5-d][l,3]oxazin-2-one or a pharmaceutically acceptable salt thereof.

A pharmaceutical composition comprising a compound of Claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

5. A method of treating a cancer expressing mutant IDH1 or mutant IDH2 which is glioma, glioblastoma, glioblastoma multiforme, astrocytomas,

oligodendrogliomas, paraganglioma, fibrosarcoma, angioimmunoblastic T-cell lymphoma (AITL), myelodysplastic syndrome (MDS), B cell acute lymphoblastic leukemia (B-ALL), thyroid cancer, colorectal cancer, acute myeloid leukemia (AML), melanoma, prostate cancer, chondrosarcoma or cholangiocarcinoma in a patient comprising administering to a patient in need thereof a therapeutically effective amount of a compound of any one of Claims 1 to 3, or a pharmaceutically acceptable salt thereof.

6. The method of claim 5 wherein the cancer expressing mutant IDH1 or mutant IDH2 is fibrosarcoma, acute myeloid leukemia, glioma, or glioblastoma.

7. A compound of Claim 1, or a pharmaceutically acceptable salt thereof, for use in therapy.

8. A compound of any one of Claims 1 to 3, or a pharmaceutically acceptable salt thereof, for use in the treatment of a cancer expressing mutant IDH1 or mutant IDH2 which is glioma, glioblastoma, glioblastoma multiforme, astrocytomas, oligodendrogliomas, paraganglioma, fibrosarcoma,

angioimmunoblastic T-cell lymphoma (AITL), myelodysplastic syndrome (MDS), B cell acute lymphoblastic leukemia (B-ALL), thyroid cancer, colorectal cancer, acute myeloid leukemia (AML), melanoma, prostate cancer, chondrosarcoma or cholangiocarcinoma.

9. The use of Claim 8 wherein the cancer expressing mutant IDH1 or mutant IDH2 is fibrosarcoma, acute myeloid leukemia, glioma, or glioblastoma.

10. Use of a compound of any one of Claims 1 to 3 or a pharmaceutically

acceptable salt thereof, for the manufacture of a medicament for the treatment of a cancer expressing mutant IDH1 or mutant IDH2 which is glioma, glioblastoma, glioblastoma multiforme, astrocytomas, oligodendrogliomas, paraganglioma, fibrosarcoma, angioimmunoblastic T-cell lymphoma (AITL), myelodysplastic syndrome (MDS), B cell acute lymphoblastic leukemia (B- ALL), thyroid cancer, colorectal cancer, acute myeloid leukemia (AML), melanoma, prostate cancer, chondrosarcoma or cholangiocarcinoma.

11. The use of Claim 10 wherein the cancer expressing mutant IDHl or mutant IDH2 is fibrosarcoma, acute myeloid leukemia, glioma, or glioblastoma.