7-phenylethylamino-4h-pyrimido [4,5-d] [1,3] oxazin-2-one compounds as mutant idh1 and idh2 inhibito

专利摘要:
The present invention relates to a compound as defined herein or a pharmaceutical composition containing the compound for use in the treatment of mutant idh1 or idh2 cancer and having the structure:
公开号:BR112019009648A2
申请号:R112019009648
申请日:2017-12-08
公开日:2019-09-10
发明作者:James Hahn Patric；Gilmour Raymond；Alejandro Bauer Renato；Louis Boulet Serge；Paul Burkholder Timothy；Rankovic Zoran
申请人:Lilly Co Eli；
IPC主号:

专利说明:

Invention Patent Descriptive Report for 7-PHENYLETHYLAMINE-4H-PYRIMIDED COMPOUNDS [4,5D] [1,3] OXAZIN-2-ONA AS MUTANT IDH1 AND IDH2 INHIBITORS.
[001] Protein isocitrate dehydrogenase (HDI) is an important enzyme in the citric acid cycle (tricarboxylic acid or Krebs). The citric acid cycle is centrally important to many series of biochemical reactions and is one of the oldest established components of cellular metabolism.
[002] Isocitrate dehydrogenases catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate (2-oxoglutarate). These enzymes belong to two distinct class subdivisions, one of which uses nicotinamide adenine dinucleotide (NAD (+)) as the electron acceptor and the other nicotinamide dinucleotide phosphate (NADP (+)). Three mammalian isocitrate dehydrogenases: one NAD-dependent isocitrate dehydrogenase (+), a multisubunit enzyme that localizes to the mitochondrial matrix, and two NADP-dependent isocitrate dehydrogenases (one) 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 plays a significant role in the production of cytoplasmic NADPH. HDI1 is expressed in a wide range of species and in organisms that do not have a complete citric acid cycle.
[003] Recently, mutations in HDI 1, and the related isoform IDH2, have been found in several types of cancers. Mutations have been found to occur at specific amino acids throughout the protein sequence and to be heterozygous expressed, consistent with a gain in function. These mutations occur in fungi residues
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2/88 internationally conserved and biochemical studies of the mutant forms of IDH1 and IDH2 demonstrated a loss of normal function, the reversible conversion of isocitrate to α-ketoglutarate. The result of these mutations is to allow a new (or neomorphic) conversion of acetoglutarate (aKG) to 2-hydroxyglutarate (2HG). As a result, cancer cells that harbor mutant forms of IDH1 or IDH2 form substantially higher concentrations of 2HG. High levels of 2HG resulted in a block in cell differentiation that can be reversed by inhibiting mutant IDHUor IDH2.
[004] The PCT / US2016 / 043264 application describes covalent inhibitors of mutant HDI1. There is another need for compounds that selectively inhibit mutant IDH1 and IDH2 enzymes for the treatment of various cancers. There is another need for compounds that selectively inhibit mutant IDH1 and IDH2 enzymes demonstrating neomorphic activity on wild-type IDH1 and IDH2 for the treatment of various cancers. The present invention provides compounds of Formula I or Ia that are inhibitors of IDH1 and mutant IDH2. The compounds of Formula I or Ia are covalent inhibitors that selectively inhibit mutant IDH1 and IDH2.
[005] One aspect of the invention is to provide enzyme inhibiting compounds of IDH1 and IDH2 of the Formula:
on what:
R ¹ is -CH ₂ CH (CH ₃ ) 2, -CH2CH3, -CH2CH2OCH3, or -CH2cyclopropyl;
R ² is -CH3 or -CH2CH3;
X is N or CH; or
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3/88 a pharmaceutically acceptable salt thereof.
[006] Another aspect of the invention is to provide enzyme inhibiting compounds of IDH1 and IDH2 of the Formula:
Where
R ¹ is -CH ₂ CH (CH ₃ ) 2, -CH2CH3, -CH2CH2OCH3, or -CH2cyclopropyl;
R ² is -CH3 or -CH2CH3; or a pharmaceutically acceptable salt thereof.
[007] Another aspect of the present invention provides a compound of Formula I or Ia which is:
7 - [[(1 S) -1 - [4 - [(1 R) -2-Cyclopropyl-1 - (4-prop-2-enoylpiperazin-1 -yl) ethyl] phenyl] ethyl] amin no] -1 -ethyl-4H-pyramid [4,5-d] [1,3] oxazin-2-one;
7 - [[(1 S) -1 - [4 - [(1 S) -2-cyclopropyl-1 - (4-prop-2-enoylpiperazin-1-yl) ethyl] phenyl] ethyl] ami no] -1 -ethyl-4H-pyramid [4,5-d] [1,3] oxazin-2-one;
-ethyl-7 - [[(1 S) -1 - [4- [1 - (4-prop-2-enoylpiperazin-1-yl) propyl] phenyl] ethyl] amino] -4H-pyrimido [4, 5-d] [1,3] oxazin-2-one, isomer 1;
-ethyl-7 - [[(1 S) -1 - [4- [1 - (4-prop-2-enoylpiperazin-1-yl) propyl] phenyl] ethyl] amino] -4H-pyrimido [4, 5-d] [1,3] oxazin-2-one, isomer 2;
or a pharmaceutically acceptable salt of any of these.
[008] Another aspect of the present invention is a compound of Formula I or Ia which is 7 - [[(1S) -1- [4 - [(1S) -2-cyclopropyl-1- (4-prop-2enoylpiperazin-1 -yl) ethyl] phenyl] ethyl] amino] -1-ethyl-4H-pyrimido [4,5-d] [1,3] oxazin-2-one or a pharmaceutically acceptable salt thereof.
[009] Another aspect of the present invention provides a pharmaceutical composition comprising a mu HDI inhibitor compound
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4/88 of Formula I or la, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
[0010] Another aspect of the present invention provides a method of treating a cancer expressing mutant IDH1 or mutant IDH2 that is glioma, glioblastoma, glioblastoma multiforme, astrocytomas, oligodendrogliomas, paraganglioma, fibrosarcoma, angioimmunoblastic T cell lymphoma (AITL) (MDS), acute B-cell 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 of the even a therapeutically effective amount of a compound of Formula I or Ia, or a pharmaceutically acceptable salt thereof.
[0011] Another aspect of the present invention provides a method of treating a cancer expressing mutant IDH1 or 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 Ia, or a pharmaceutically acceptable salt thereof.
[0012] Another aspect of the present invention provides a compound of Formula I or Ia, or a pharmaceutically acceptable salt thereof, for use in therapy.
[0013] Another aspect of the present invention provides a compound of Formula I or Ia, 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), acute B-cell lymphoblastic leukemia (B-ALL), cancer of the
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5/88 thyroid, colorectal cancer, acute myeloid leukemia (AML), melanoma, prostate cancer, chondrosarcoma or cholangiocarcinoma.
[0014] Another aspect of the present invention provides a compound of Formula I or Ia, or a pharmaceutically acceptable salt thereof, for use in the treatment of a cancer expressing mutant IDH1 or mutant IDH2 which is fibrosarcoma, acute myeloid leukemia, glioma or glioblastoma .
[0015] Another aspect of the present invention provides use of a compound of Formula I or Ia, 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), acute B-cell lymphoblastic leukemia (B-ALL), thyroid cancer, acute leukemia, colorectal cancer AML), melanoma, prostate cancer, chondrosarcoma or cholangiocarcinoma.
[0016] Another aspect of the present invention provides use of a compound of Formula I or Ia, 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 fibrosarcoma, acute myeloid leukemia, glioma or glioblastoma.
[0017] The term patient means mammal and mammal includes, but is not limited to, a human.
[0018] Therapeutically effective amount means the dosage of the compound of Formula I or Ia, or pharmaceutically acceptable salt thereof, or pharmaceutical composition containing the compound, or pharmaceutically acceptable salt thereof, necessary to inhibit mutant HDI1 or mutant HDI2 in a patient with cancer, leading to the release of the block in differentiation with inhibition resulting from growth
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6/88 tumor cell and eliminates or reduces or stops the progression of cancer in a patient. Advance dosages of a compound of Formula I or Ia, 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. More 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 due to the stage and severity of the disease as well as the specific needs and response of the individual patient. Although expressed as a dosage on a daily basis, dosage administration can be adjusted to provide a more ideal therapeutic benefit to a patient and to administer or ameliorate any drug-related toxicities. In addition to daily dosage, twice daily dosage (B.I.D.); dosage three times a day (T.I.D.); dosage every two days (Q2D); every two days for a period of five days followed by two days without dosing (T.I.W.); or every three days (Q3D) may be appropriate.
[0019] The terms treatment, treat and treating, are intended to include the full spectrum of cancer intervention from which the patient is suffering, such as administration of the active compound to relieve, delay, or reverse one or more of the symptoms and delay the cancer progression even if the cancer is not actually eliminated.
[0020] The term -CH ₂ CH (CH ₃ ) ₂ means 2-methylpropyl, the term -CH ₂ CH ₂ OCH ₃ means 2-methoxyethyl, and the term -CH ₂ -cyclopropyl means cyclopropylmethyl.
[0021] A compound of Formula I or Ia, or a pharmaceutically acceptable salt thereof, is preferably formulated as a pharmaceutical composition using a pharmaceutically acceptable carrier
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7/88 manageable and administered by a variety of routines. Preferably, such compositions are for oral administration. Such pharmaceutical compositions and processes for preparing them are well known in the art. See, eg, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY, LV Allen, Editor, 22 ^â Edition, Pharmaceutical Press, 2012.
[0022] In a particular embodiment, the pharmaceutical composition comprises 7 - {[(1 S) -1 - {4 - [(1 S) -1 - (4-acrylic pipe razi n-1-yl) -2- ci clopropyl leti I] phenyl} ethyl] amino} -1-ethyl-1,4-dihydro-2H-pyrimido [4,5-d] [1,3] oxazin-2-one or a pharmaceutically acceptable salt of even, together with a pharmaceutically acceptable carrier and optionally other therapeutic ingredients particularly for treating cancer generally or a specific type of cancer.
A compound of Formula I or Ia, or a pharmaceutically acceptable salt, can be administered simultaneously with, or before, or after, one or more other therapeutic agents. The compound of Formula I or Ia, or a pharmaceutically acceptable salt, when administered with one or more other therapeutic agents, can be administered separately, by the same or different administration routine, or together in the same pharmaceutical composition as the other ( s) therapeutic agent (s). Where one or more additional therapeutic agents are administered, the administration of each therapeutic agent can be simultaneous, separate or sequential.
[0024] A compound of Formula I or Ia is capable of reaction with various 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, and another, HANDBOOK OF PHARMACEUTICAL SALTS: PROPERTIES, SELECTION AND USE,
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8/88 (VCHA / Wiley-VCH, 2002); S.M. Berge, et al., Pharmaceutical Salts, Journal of Pharmaceutical Sciences, Vol. 66, No. 1, January 1977.
[0025] A compound of Formula I or la, or a pharmaceutically acceptable salt thereof, can be prepared by a variety of procedures known in the art, as well as those described below. The specific synthetic steps can be combined in a different order to prepare a compound of Formula I or Ia, or a pharmaceutically acceptable salt thereof.
[0026] In addition, certain intermediates described in the following preparations may contain one or more nitrogen protecting groups. It is understood that protection groups can be varied when appreciated by someone with experience in the technique depending on the particular reaction conditions and the particular transformations to be carried out. Protection and deprotection conditions are well known to the skilled technician and are described in the literature (See for example Greene's Protective Groups in Organic Synthesis, Fifth Edition, by Peter GM Wuts and Theodora W. Greene, John Wiley and Sons, Inc. 2014) .
[0027] Compounds of Formula I or la are named according to IUPAC, and can likewise be named according to CAS, and other names can be used to unambiguously identify a compound of Formula I or Ia, or a pharmaceutically acceptable salt the same.
[0028] It will be understood that a compound of Formula I or Ia can be described as a single stereoisomer. There are two chiral centers giving rise to four diastereomers. When used herein, references to a single stereoisomer are similarly intended to include stereoisomeric mixtures including the named or described compound of Formula I or Ia. Here, the Cahn-lngoldPrelog designations of (R) - and (S) - can be used to refer to stereoisomers
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Specific 9/88. Specific stereoisomers can be prepared by stereospecific synthesis using enantiomerically pure or enriched starting materials. Specific stereoisomers of 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 Comounds, E., I. Eliel and SH Wilen ( Wiley 1994) and Enantiomers, Racemates, and Resolutions, J., Jacques, A., Collet, and SH Wilen (Wiley 1991), including chiral stationary phase chromatography, 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 the stereocenters shown, substantially enantiomerically pure means the isomeric purity is greater than 90% enantiomeric excess. In another embodiment, a compound of Formula I or isomeric purity is greater than 95% enantiomeric excess. In another modality, a compound of Formula I or isomeric purity is greater than 98% of enantiomeric excess. In yet another embodiment, a compound of Formula I or 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 compounds that elute from chiral chromatography first and second, respectively, and if chiral chromatography is started early in the synthesis, the same designation is applied to subsequent intermediates and examples.
[0029] The compounds used as starting starting materials in the synthesis of the compounds of Formula I or la are well known
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10/88 and, for the non-commercially available extension, are easily synthesized using specific references provided, by standard procedures generally employed by those with ordinary experience in the technique or are found in general reference texts.
[0030] 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, 5 ^th Edition, Michael B. Smith and Jerry March, Wiley Interscience, 2001; Advanced Organic Chemistry, 4 ^th Edition, Part B, Reactions and Synthesis, Francis A. Carey and Richard J. Sundberg, Kluwer Academic / Plenum Publishers, 2000, etc., and references cited here.
[0031] Certain abbreviations are defined as follows: ACN means acetonitrile; aKG means Alpha-ketoglutarate or 2-ketoglutarate; alloc means allyloxycarbonyl; ATCC stands for American Type Culture collection; BCA means bicinconinic acid; BSA stands for Bovine Serum Albumin; CDI means 1,1'-carbonyladiimidazole; 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-isopropylpropan-2-amine; DMAP means dimethylaminopyridine; DMF means dimethylformamide; DMSO means dimethyl sulfoxide; DTT means dithiothreitol; EDC means EDAC, EDCI, or 1- (3dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride; EDTA means ethylenediaminetetraacetic acid; EGTA means ethylene glycol tetraacetic acid; EtOAc means ethyl acetate; EtOH means ethanol or ethyl alcohol; Ex means example; HATU means hexafluorophosphate
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11/88 of (dimethylamino) -N, N-dimethyl (3 / 7- [1,2,3] triazolo [4,5-b] pyridin-3-yloxy) metanimity; HBTU means 2- (1/7-benzotriazol1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate; 2HG means 2-hydroxyglutarate; ds3HG means 3-hydroxy-1,5-pentanedioic acid-2,2,3,4,4-ds; HILIC means hydrophilic interaction liquid chromatography; HOAt means 1-hydroxy-7-azobenzotriazole; HOBt means 1hydroxylbenzotriazole hydrate; HPLC 'means high performance liquid chromatography; IC50 means the concentration of an agent that produces 50% of the maximum possible inhibitory response for that agent; mCPBA means meta-chloroperbenzoic acid; MeOH means methanol or methyl alcohol; NADP ⁺ and NADPH means the oxidized and reduced forms of nicotinamide phosphate adenine dinucleotide respectively; NMP means N-methyl-2-pyrrolidone; PG means protection group; Prep means preparation; PyBOP means benzotriazole-1-yloxytripyrrolidine-phosphonium hexafluorophosphate; PyBrop means bromo-tris-pyrrolidinophosphonium hexafluorophosphate; rpm means revolutions per minute; (F ) - RUCY®-XylBINAP means RuCI [(R) daipena] [(R) -xilbinap; SNAr means nucleophilic aromatic substitution; TEA means triethylamine; TFA means trifluoroacetic acid; THF means tetrahydrofuran; and Tris means tris (hydroxymethyl) aminomethane.
[0032] The compounds of Formula I or Ia, or pharmaceutically acceptable salts thereof, can be prepared by a variety of procedures known in the art, some of which are illustrated in the Schemes, Preparations, and Examples below. Specific synthetic steps for each of the routines described in different ways, or together with steps of different schemes, for preparing compounds of Formula I or Ia, or pharmaceutically acceptable salts thereof. The products of each stage in the diagrams below can be recovered by conventional methods as well as 870190044593, from 05/13/2019, p. 11/195
12/88 known in the art, including extraction, evaporation, precipitation, chromatography, filtration, grinding and crystallization. In the schemes below, all substituents, unless otherwise indicated, are as previously defined. Reagents and starting materials are readily available to someone with ordinary experience in the art.
Alkali n * í ⁵ 'X- YL 0 L ---- ~ x 1, l I _BaBaB -S' Μ- Ύ Í2 '(3} Y
Oxidation | Sapa C
Layout 1
In a series of reactions leads to a substituted 1-7- (methylsulfonyl) -1,4-dihydro-2H-pyrimido [4,5-d] [1,3] oxazin-2one, (4), the product of Step C where R ² 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 carbamoyl conditions to oxazine-2-one using triphosgene and an organic base such as DIPEA or TEA at a temperature of about -30 to -35 ° C to produce Compound (2), the product of Step A. Alternatively a carbonyl dialect or a carbonyl di-pseudoalide such as CDI, phosgene or diphosgene can be used instead of triphosgene to complete carbamoylation. The oxazine amine can be alkylated with the appropriate substituted alkyl halide such as an iodine reagent in a solvent such as NMP and an inorganic base such as K2CO3 at a time
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13/88 ration of about 50-65 ° C to produce Compound (3), the product of Step B. Alternatively, a Mitsunobu reaction can be performed to alkylate the amine from the oxazine using an appropriate alcohol such as MeOH. Mitsunobu reactions are well known in the art and can convert a hydroxyl group to 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 produce Compound (3). The sulfide can be oxidized to the sulfone under conditions well known in the art such as mCPBA or potassium peroxymonosulfate at a temperature of about 10 to 25 ° C in a solvent such as ACN or DCM to produce Compound (4), the product of Step C.
Layout 2
PG = Protection group o-u H
1. Protection
2. Cafhoniiação
EíapaGÍ Grignard reaction
í 1. Protection ·
2. Ownership
3. Desprot8Ç30
naked
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14/88 [0034] In Scheme 2, a (4- (1-aminoethyl) phenyl) methanol (7) can be generated during several steps from an aryl halide such as a bromide (5) using procedures well known to the versed technician. The amine can be protected in sub-step 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 conditions of lithium carbonylation to produce the aryl ketone (6) in sub-step 2, Step D. The ketone can then be reduced using a reducing agent such as as sodium borohydride in a solvent such as MeOH in sub-step 1, Step E. The amine can be deprotected in this step (sub-step 2, Step E) or at a later point in the synthesis to produce 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 at about 60 ° C followed by cooling and the addition of MeOH and a reducing agent such as sodium cyanoborohydride to produce 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 DCM to produce the compound (10), Step I. Compound (10) can be protected if necessary and alkylated in Step J. For example, the amine, (10) can be protected in Sub-step 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 oxidizing the corresponding benzyl alcohol under such conditions as Dess Martin periodinane in a solvent such as DCM to produce an aldehyde (8). A Grignard reaction can be performed in Step G on the aldehyde
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15/88 to produce Compound (7). Compound (7) from Step G can be taken in Step H to produce Compound (10) as discussed above for Step I. The chloride of Compound (10) can be displaced with a monoprotected piperazine in a two-step procedure, one Bowl. It is not always necessary to protect 1-phenylethylamine but if the protection is chosen, it is advantageous to use a different protection group in 1-phenylethylamine than the piperazine amine product (11), Step J, sub-step 1, to selectively unprotect one or the other PG at the desired step. For example, 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 produce the protected sub-step 1 amine product, Step J Someone skilled in the art would understand that other protecting groups could be used in the amine such as CBZ. Chloride displacement can then be carried out under conditions well known to someone 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 using Kl, 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 produce 0 Protected compound (11) from sub-step 2, Step J. The protecting group on 1-phenylethylamine can be removed with a base such as aqueous potassium hydroxide to produce Compound (11) from sub-step 3, Step J.
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16/88
Layout 3
[0035] In Scheme 3, Compound (11) can be reacted with Compound (4), Scheme 1, in an 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 produce the product from Step L. In Step M, sub-step 1, a tert-butoxy-protected piperazine can be deprotected using an acid such as HCI in dioxane and MeOH or TFA in DCM since an alloc-protected piperazine can be deprotected in the presence of a palladium source such as catalytic tetracis (triphenylphosphine) in a solvent such as THF using a soft nucleophile such as dimedone to produce the unprotected sub-step piperazine 1, Step M. In sub-step 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 a acid salt in a solvent such as DCM to produce the compounds of Formula Ia Alternatively, an amide coupling can be carried out 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. Someone skilled in the art will recognize that there are several methods and reagents for amide formation resulting in the reaction of acids
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17/88 carboxylics 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 Ia. Other coupling reagents include carbodiimides, such as DCC, DIC, or a carbonyladimidazole such as CDI. Other amide coupling additives, such as HOAt, can likewise be used to enhance the reaction. Additionally, uronium or phosphonium salts from non-nucleophilic anions, such as HBTU, HATU, PyBOP, and PyBrOP could be used in place of more traditional coupling reagents. An additive such as DMAP can be used to accelerate the desired amidation reaction. Layout 4
[0036] Alternatively in Scheme 4, the amine of the unprotected product of Compound (7), Scheme 2 can be reacted with Compound (4) as described in Scheme 3, Step L in an SNAr alkylation to produce Compound 13. Hydroxyl can be chlorinated as
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18/88 described in Step I, Scheme 2 to produce 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, sub-step 2 to produce Compound (5). The protected piperazine can be deprotected and the piperazine then amidated as described in Scheme 3, Step M to produce compounds of Formula Ia.
Layout 5 f 1. Protection
H - Carbonization
(5a) G = protection clamp
(6th)
ReSucso rj;
„ _N ,„ | Step 2. De protection;
$;
R ^!
Step i [Ctoraçâo
I
. I 1. ' ^apa ' alteration | (PG-piperazifla) [2.Despratection í
PG
RH-,
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19/88 [0037] In scheme 5, a chiral (4- (1-aminoethyl) phenyl) methanol (7a) can be generated during several stages of a chiral aryl halide such as a bromide (5a) using procedures well known to the versed technician. 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. Aryl bromide (5a) can be converted to a ketone under conditions of lithium carbonylation to produce aryl ketone (6a) in sub-step 2, Step D. The ketone can then be asymmetrically reduced using a chiral reducing agent such as (F ) - RUCY-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 halogenation conditions standards using a halogenating agent such as benzoyl chloride in a solvent such as t-butyl ether to produce the 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 produce 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 for provide compound compound (11a), sub-step 2, Step J.
Layout 6
X · VA ⁰ Ci · -G 1 (15; Anwiaçãc. Rafeçâs Cicíízação ΓΥΊ - * ... ¼ v - * _ç .àA «- BapaA ElagaB Stage C itõ} (W ϊ1Βΐ [0038] No Scheme 6, a series of reactions leads to a 1-
substituted-7-chloro-4H-pyrido [4,3-d] [1,3] oxazin-2-one (18), the product of Step C where R ² is as previously defined. For example, ethyl 4,6dichloropyridine-3-carboxylate (15) can be reacted with a
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20/88 amine under standard conditions to produce 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 provides (4-amino-6-chloro-3-pyridyl) methanol (17). Compound such as 17 can be cyclized under standard carbamoylation conditions to a triphosgene using and an organic base such as DIPEA or TEA at a temperature of about -20 ° C to produce Compound (18), the product of Step C. Alternatively a carbonyl dialect or a carbonyl di-pseudoalide such as CDI, phosgene, or diphosgene can be used instead of triphosgene to complete carbamoylation. Compound 11, prepared as shown above in Schemes 2 or 5, or as described in alternatives to Scheme, can be reacted with Compound 18 under standard alkylation conditions. The resulting intermediate compound is then deprotected and amidated as described above in Schemes 3 or 4 to provide compounds of Formula I where X is CH.
[0039] In an optional step, a pharmaceutically acceptable salt of a compound of Formula I or Ia can be formed by reacting an appropriate free base of Formula I or Ia with a suitable pharmaceutically acceptable acid in a suitable solvent under standard conditions. In addition, the formation of such salts can occur simultaneously under deprotection of a nitrogen protecting group. The formation of such salts is well known and appreciated in the art. For example, see 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., and another, Pharmaceutical Salts, Journal of Pharmaceutical Sciences, 66: 1-19, (1977). Someone from ex
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Ordinary skill in the art will appreciate that a compound of Formula I or Ia is easily converted to and can be isolated as a pharmaceutically acceptable salt.
Preparation 1
7-Methylsulfanyl-1,4-dihydropyrimido [4,5-d] [1,3] oxazin-2-one
H [0040] Triphosgene (859 g, 2.9 mmol) is added to a solution of (4-amino-2-methylsulfanyl-pyrimidin-5-yl) methanol (900 g, 5.26 mmol) in THF (22, 5 L) for 15 minutes at -30 ° C. DIPEA (2.499 g, 18.92 moles) is added over one hour, maintaining the reaction temperature between -35 and -30 ° C. The reaction mixture is then poured into 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 Na2SC> 4 and is concentrated to dryness. The crude product is suspended with petroleum ether / EtOAc (1: 1), filtered, and concentrated to produce a yellow solid that is transported without further purification (890.5 g, 1.62 mol, 83% pure, 86% income). MS (m / z): 198 (M + H).
Preparation 2
-ethyl-7- (methylthio) -1,4-dihydro-2H-pyrido [4,5-d] [1,3] oxazin-2-one [0041] In a solution of 7-methylsulfanyl-1 , 4-dihydropyrimido [4,5d] [1,3] oxazin-2-one (280 g, 1.42 mol) in NMP (2.24 L) is added K2CO3 (294.2 g, 2.13 mols) 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.
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22/88 to produce the crude title compound (286 g, 1.27 mol, 83% purity, 91% yield). MS (m / z): 226 (M + H).
Preparation 3
-methyl-7-methylsulfanyl-4H-pyrimido [4,5-d] [1,3] oxazin-2-one
[0042] In a solution of triphenylphosphine (1.61 g, 6.08 mmol) and 7 methylsulfanyl-1,4-dihydropyrimido [4,5-d] [1,3] oxazin-2-one (1.00 g, 5.07 mmols) in THF (25 ml) is added MeOH (0.248 ml, 6.08 mmols) followed by dropwise addition of DIAD (1.21 ml, 6.08 mmols) at room temperature. After stirring overnight the solvent is removed in vacuo and the resulting yellow oil is purified by chromatography on silica gel (40-50% EtOAc / hexanes) to yield the title compound as a white solid (1.08 g, 5 , 11 mmols, quantitative). MS (m / z): 212 (M + H).
Preparation 4
-ethyl-7- (methylsulfonyl) -1,4-dihydro-2H-pyrimido [4,5-d] [1,3] oxazin-2-one .VVkAo '0 Q [0043] In a stirred solution of 1-ethyl-7- (methylthio) -1,4-dihydro-2Hpirimido [4,5-d] [1,3] oxazin-2-one (286 g, 1.24 mol) in ACN (2 , 8 L) and water (1.4 L) potassium peroxymonosulfate (1526 g, 2.48 moles) is added as a solid for 20 minutes, and the resulting mixture is stirred for 16 hours at 10-20 ° C. The reaction mixture is filtered and the filtrate obtained is washed with DCM. The combined filtrate and DCM are washed with 5% Na ₂ SOs, water and brine. The organic phase
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23/88 single is dried over Na2SC> 4 and concentrated to provide the title compound (133.8 g, 93% pure, 41% yield). MS (m / z): 258 (M + H).
[0044] The following compound is prepared essentially by the method of Preparation 4.
Table 1
Prep. N ^Q. Chemical name Structure ES / MS (m / z) (M + H) 5 1-methyl-7-methylsulfoni I4H-pyrimido [4,5-d] [1,3] oxazin-2-one N ^ h ^^ O JL Jl L _X S ^ N ^ N ^ O OI 244
Preparation 6
N - [(1 S) -1 - (4-Bromophenyl) ethyl] -2,2,2-trifluoro-acetamide
[0045] Trifluoroacetic anhydride (165 ml_, 1.17 mol) is added dropwise to a solution of (1S) -1- (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 moles) over one hour. After 30 minutes, water (3 L) and brine (1 L) are added resulting in the formation of a colorless precipitate. The suspension is stirred for 15 minutes and then the solid is filtered, washed with water and hexane, and air-dried followed by drying at 40 ° C under vacuum to yield the title compound (290 g, 92%). ¹ H 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, 1H, J = 7.6 Hz) .
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Preparation 7
N - [(1S) -1- [4- (2-cyclopropylacetyl) phenyl] ethyl] -2,2,2-trifluoro-acetamide [0046] n-Butyl lithium (2.5 M in hexane, 53 ml_, 130 mmols) is added dropwise to a solution of N - [(1S) -1- (4-bromophenyl) ethyl] -2,2,2trifluoroacetamide (18.00 g, 60.79 mmols) in THF (600 ml_) at -78 ° C 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 mmols) 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 heated to room temperature. The layers are separated and the organic layer is dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. A small amount of DCM is added to the solid and the mixture is briefly heated to dissolve the solids. The mixture is concentrated until just before precipitation and then hexane (150 ml) is added dropwise with vigorous stirring to produce a colorless solid. The solid is collected by filtration, washed with a small amount of hexane, and dried under reduced pressure to produce the title compound (13.82 g, 76%) as a colorless solid. MS (m / z): 298.3 (M-H).
[0047] The following compound is prepared essentially by Preparation method 7.
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Table 2
Prep. N ^Q. Chemical name Structure ES / MS (m / z) (M + H) 8 2,2,2-Trifluoro-N - [(1 S) -1 - [4- (3methoxypropanoyl) phenyl] ethyl] acetamide, ^{H f} 304
Preparation 9
- [4 - [(1 S) -1 -Aminoethyl] phenyl] -2-cyclopropyl-ethanol
[0048] Sodium borohydride (0.1411 g, 2 equiv., 3.729 mmols) is added to a solution of N - [(1S) -1- [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 mmols) in water (3 ml) is added. The mixture is stirred at room temperature for about 20 hours. The mixture is concentrated and divided between DCM and water. The organic layer is washed with saturated aqueous sodium chloride, dried over sodium sulfate, filtered, and concentrated to yield 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).
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Preparation 10
2,2,2-Trifluoro-N - [(1S) -1- (4-formylphenyl) ethyl] acetamide o
H [0049] Dess-Martin periodinane (20.9 g, 49.3 mmols) is added to a 0 ° C solution of 2,2,2-trifluoro-N - [(1S) -1- [4 (hydroxymethyl ) phenyl] ethyl] acetamide (11.1 g, 44.9 mmols) 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 NaHCOs, saturated aqueous Na ₂ S _{2 0} , and brine. The combined organics are dried (Na ₂ SO4), filtered, and concentrated to produce a residue that is purified by silica gel chromatography eluting with a 0-50% EtOAc / hexanes gradient to produce the title compound as a white solid ( 9.5 g, 39 mmols, 86%). ES / MS (m / z): 244 (MH).
Preparation 10a
2,2,2-Trifluoro-N - [(1S) -1- [4- (hydroxymethyl) phenyl] ethyl] acetamide
- O
[0050] Trifluoroacetic anhydride (12 ml_, 85.4 mmols) is added to a 0 ° C solution of [4 - [(1S) -1-aminoethyl] phenyl] methanol (10.8 g, 71.4 mmols) in CH ₂ CI ₂ (150 ml). After 10 minutes, triethylamine (24 ml_, 172 mmoles) in CH ₂ CI ₂ (8 ml_) is added dropwise over 30 minutes, the cooling bath is removed and the reaction is stirred overnight. The reaction mixture is concentrated in vacuo, diluted with additional CH ₂ CI ₂ , and washed with 1 N aqueous HCI and water. The organic phase is dried (Na ₂ SC> 4), filtered and concentrated. The raw material is purified by chromatography on silica gel eluting with a gradient of
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0-50% EtOAc / hexanes to yield 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 - [(1 S) -1 - [4- (1-hydroxy-3-methyl-butyl) phenyl] ethyl] acetamide 'Γ · [0051] In a solution of 2.2 , 2-trifluoro-N - [(1 S) -1- (4-formylphenyl) ethyl] acetamide (1.72 g, 7.01 mmols) in THF (35 ml_) cooled in an ice water bath bromide is added of isobutylmagnesium (2 M in diethyl ether, 7.0 ml, 14.0 mmols) 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 produce 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).
[0052] The following compound is prepared essentially by the method of Preparation 11.
Table 3
Prep. N ^Q. Chemical name Structure ES / MS (m / z) (M + H) 12 2,2,2-trifluoro-N - [(1S) -1- [4- (1 hydroxypropyl) phenyl] ethyl] acetamide 0 Hoi) ^HF T 293 (M + NH ₄ )
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Preparation 13
4- [2-Cyclopropyl-1 - [4 - [(1 S) -1 - [(2,2,2-trifluoroacetyl) amino] ethyl] phenyl] ethyl] piperazine-1-carboxylate tert-butyl
[0053] Titanium (IV) isopropoxide (60 ml_, 200 mmols) is added to a solution of N - [(1S) -1- [4- (2-cyclopropylacetyl) phenyl] ethyl] -2,2,2trifluoro- acetamide (12.0 g, 40.1 mmols) and tert-butyl piperazine-1-carboxylate (17.9 g, 96.1 mmols) in THF (80 ml) and the mixture is stirred at 60 ° C during night. The mixture is cooled to room temperature and MeOH (80 ml) is added followed by the dropwise addition of sodium cyanoborohydride (5.3 g, 80 mmols). The mixture is stirred at room temperature for 8 hours and then water and MeOH are added and the mixture is stirred overnight at room temperature. 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 (2x). The combined organic extracts are dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude material is purified by column chromatography eluting with a gradient of 0% to 30% EtOAc in solvent B where solvent B is 1: 1 hexanes: DCM to produce the title compound (10.5 g, 56%) as a colorless solid. MS (m / z): 470.3 (M + H).
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Preparation 14
4- [1 - [4 - [(1 S) -1 -aminoethyl] phenyl] -2-cyclopropyl-ethyl] piperazine-1-tert-butyl carboxylate o Ο'-Χν-Α.-A k λ .. 00 [0054] Aqueous potassium hydroxide (5 M, 69 ml_, 350 mmols) is added to a solution of 4- [2-cyclopropyl-1- [4 - [(1S) -1 - [(2,2,2trifluoroacetyl ) amino] ethyl] phenyl] ethyl] piperazine-1-tercbutyl-1-carboxylate (32.24 g, 68.67 mmols) in EtOH (350 ml) and the resulting mixture is stirred at room temperature for 4 hours. 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 produce the title compound (24.33 g, 96.5% pure containing 3.5% residual DCM, 92% yield) as a colorless viscous oil. MS (m / z): 374.3 (M + H).
[0055] The following compound is prepared essentially by Preparation method 14.
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Table 4
Prep. N ^s . Chemical name Structure ES / MS (m / z) (M + H) 15 4- [1 - [4 - [(1 S) -1 -am inoethyl] phenyl] -3m ethyl-butyl] piperazine-1 tert-butyl carboxylate k  K 1 376 16 4 - [(1 R) -1 - [4 - [(1 S) -1 -aminoethyl] phenyl] propyl] piperazine-1 tert-butyl carboxylate, isomer 1, x ' ^J z / ___ 7. Λ / / C ò 348 17 4 - [(1R) -1- [4 - [(1S) -1aminoethyl] phenyl] propyl] tert-butyl piperazine1-carboxylate, isomer 2, ΐ 'O _r < ^: ^ X _T - ^x H- 348
Alternate preparation 14 [0056] A solution of potassium hydroxide (1.28 g, 22.9 mmol) in water (4 mL) is added to a solution of 4- [2-cyclopropyl-1- [4 [(1 s ) Tert-Butyl -1 ([2,2,2-trifluoroacetyl) amine] ethyl] phenyl] ethyl] piperazine-1-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 divided 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 produce the title compound as an oil which is used without purification (1.73 g, 4.49 mmols, 98%) . ES / MS (m / z): 374.2 (M + H).
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Preparation 18
4 - [(1 R) -1 - [4 - [(1 S) -1 -ami noethyl] phenyl] -2-cyclopropyl-ethyl] piperazin-1 tert-butyl carboxylate, diastereomer 1,
Preparation 19
4 - [(1 S) -1 - [4 - [(1 S) -1 -ami noethyl] phenyl] -2-cyclopropyl-ethyl] piperazin-1 tert-butyl carboxylate, diastereomer 2,
[0057] A 1: 1 mixture of tert-butyl 4 - [(1 R) -1- [4 - [(1S) -1-aminoethyl] phenyl] 2-cyclopropyl-ethyl] piperazine-1-carboxylate, diastereomer 1 and 4 - [(1 S) -1 - [4 - [(1 S) -1 -aminoethyl] phenyl] -2-cyclopropyl-ethyl] piperazine-1 tert-butyl carboxylate, diastereomer 2 (3.23 g) is dissolved in MeOH (40 ml) and separated into individual diastereomers by chiral preparative HPLC chromatography using the following conditions: Chiralpak DC column, 20 pm, (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, 4 - [(1 R) -1 [4 - [(1 S) -1-aminoethyl] phenyl] -2-cyclopropyl-ethyl] piperazine-1-carboxylate, tert-butyl, diastereomer 1, is obtained at from the first elution peak as a clear viscous oil (1.50 g, 46%,> 99%). MS (m / z): 374.3 (M + H). Preparation 13, 4 - [(1S) -1- [4 - [(1S) -1-aminoethyl] phenyl] -2cyclopropyl-ethyl] piperazine-1-carboxylate, tert-butyl, diastereomer 2,
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32/88 is obtained from the second elution peak as a clear viscous oil (1.46 g, 45%,> 98.2%). MS (m / z): 374.3 (M + H).
Preparation 20
N, 3-Dimethoxy-N-methyl-propanamide [0058] A solution of 3-methoxypropanoic acid (62 g, 577.7 mmols) in DCM (1200 mL) is treated portion by portion slowly with 1,1'-carbonyladiene. imidazole (103 g, 635.2 mmol) and stirred at room temperature for two hours. N hydrochloride, Odimethylhydroxylamine (62 g, 635.6 mmols) is added and the mixture is stirred overnight at room temperature. The mixture is washed with water (2x), 0.1 M aq HCl (2x), and with saturated aqueous sodium bicarbonate (2x), dried over magnesium sulfate, filtered and concentrated to dryness to produce the raw material. The raw material is chromatographed on silica gel eluting with a gradient of 20-40% acetone in hexane. The resulting oil is dried overnight under vacuum to produce the title compound (69.5g, 81.7%). ¹ H NMR (CDCh) δ 2.72 (t, 2H), 3.2 (s, 3H), 3.38 (s, 3H), 3.7 (m, 5H).
Preparation 21
2,2,2-Trifluoro-N - [(1S) -1- [4- (1-hydroxy-3-methoxy-propyl) phenyl] ethyl] acetamide
HO [0059] 2,2,2-Trifluoro-N - [(1S) -1- [4- (3-methoxypropanoyl) phenyl] ethyl] acetamide (23.62 g, 73.98 mmol, 95% by weight) it is dissolved in MeOH (700 ml) and treated with sodium borohydride (5.6 g, 150 mmols). After
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33/88 of stirring at room temperature for two hours the mixture is treated with saturated aqueous ammonium chloride and the MeOH is evaporated. The resulting material is divided between water and EtOAc, separated and the combined organics are dried over Na ₂ SÜ4, filtered and concentrated to dryness. The crude material is chromatographed on silica gel eluting with 40% EtOAc in hexane to produce the title compound as a white solid (17.82g, 79%). MS (m / z): 306 (M + H).
Preparation 22
4- [3-Methoxy-1 - [4 - [(1 S) -1 - [(2,2,2-trifluoroacetyl) amino] ethyl] phenyl] propyl] tert-butyl piperazine-1-carboxylate
O [0060] 2,2,2-trifluoro-N - [(1 S) -1 - [4- (1 - hydroxy-3-methoxy-propyl) phenyl] ethyl] acetamide (21.76g, 71, 27 mmols) is dissolved in DCM (350ml_) and cooled to -10 ° C. Thionyl chloride (26.12 g, 16 ml, 219.6 mmols) is added dropwise and the reaction is stirred for two hours. The mixture is concentrated to dryness, redissolved in DCM, and reconcentrated. The crude material is dissolved in ACN (300 ml) and t-butyl piperazine-1-carboxylate (26.55 g, 142.6 mmols), potassium carbonate (39.5 g, 286 mmols) and potassium iodide ( 12.0 g, 72.3 mmols) 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 hexane to produce the title compound as a white foam (29.63g, 88%). MS (m / z): 474 (M + H).
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Preparation 23
4- [1 - [4- (1 S) -1 -aminoethyl] phenyl] -3-methoxy-propyl] piperazine-1-tert-butylcarboxylate, diastereomer 1,
Preparation 24
4- [1 - [4- (1 S) -1 -aminoethyl] phenyl] -3-methoxy-propyl] tert-butyl piperazine-1-carboxylate, diastereomer 2,
[0061] In a solution of tert-butyl 4- [3-methoxy-1- [4 - [(1S) -1 - [(2,2,2trifluoroacetyl) amino] -ethyl] propyl] piperazine-1-carboxylate (29.60 g, 62.5 mmols) in EtOH (310 ml) aq potassium hydroxide (63 ml, 5 Μ) is added. 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 (3x). The combined organic extracts are dried over sodium sulfate, filtered and concentrated to produce the title compound (23.6 g) 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> 2, detection wavelength 230 nm; flow rate 300 g / min; column temperature: 40 ° C; BPR Fixing point: 100 bar; BPR temperature: 40 ° C. The title compound from Preparation 28 is
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35/88 obtained from the first elution peak as a light viscous yellow oil (10.1 g, 42.8%, 96.6%). MS (m / z): 378 (M + H). The compound from Preparation 29 is isolated as the second elution peak as a light viscous yellow oil (10.3 g, 43.6%, 95.2%). MS (m / z): 378 (M + H).
Preparation 25
7 - [[(1 S) -1 - [4- (1-Chloro-2-cyclopropyl-ethyl) phenyl] ethyl] amino] -1-ethyl-4H-pyrid [4,5-d] [ 1.3] oxazin-2-one
[0062] Thionyl chloride (0.23 ml, 3.219 mmols) is added to a mixture of 7 - [[(1 S) -1- [4- (2-cyclopropyl-1-hydroxyethyl) phenyl] ethyl] amino] -1-ethyl-4H-pyrimido [4,5-d] [1,3] 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 produce 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).
[0063] The following compounds are prepared essentially by Preparation method 25.
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Table 5
Prep. N ^Q. Chemical name Structure ES / MS (m / z) (M + H) 26 N - [(1 S) -1- [4- (1-Chlorine-2-cyclopropyl-ethyl) phenyl] ethyl] -2,2,2-trifluoroacetamide the ^HF f 318 27 N - [(1 S) -1 - [4- (1-Chloro-3-methylbutyl) phenyl] ethyl] -2,2,2-trifluoroacetamide 0 (YÃA < ^f Αχ / F 320 28 N - [(1S) -1- [4- (1-Chloropropyl) phenyl] ethyl] -2,2,2-trifluoroacetamide _z O 311 (M + NH ₄ )
Preparation 29
4 - [(1 R) -2-cyclopropyl-1 - [4 - [(1 S) -1 - [(1-ethyl-2-oxo-4H-pyrimido [4,5-d] [1,3] oxazin-7-yl) amino] ethyl] phenyl] ethyl] tert-butyl piperazine-1-carboxylate
[0064] In a solution of tert-butyl 4 - [(1 R) -1- [4 - [(1 S) -1-aminoethyl] phenyl] -2cyclopropyl-ethyl] piperazine-1-carboxylate (864 mg, 3.35 mmols), and 1-ethyl-7- (methylsulfonyl) -1,4-dihydro-2H-pyrimido [4,5-d] [1,3] oxazin-2-one (1.14 g , 3.05 mmols), in DMSO (15 ml), CsF (1.39 g, 9.15 mmols) and DIPEA (0.80 ml, 4.6 mmols) are added. The mixture is agi
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37/88 at 60 ° C for 1.5 hours. The mixture is cooled to room temperature, diluted with EtOAc, and washed with water (2x). The combined aqueous washes are extracted with EtOAc and the combined organic extracts are dried (Na ₂ SO4), filtered and concentrated to dryness. The resulting crude product is purified by chromatography on silica gel eluting with a gradient of 55% to 95% EtOAc in hexane to yield the title compound as a colorless solid (1.47 g, 88%). MS (m / z): 551.3 (M + H).
[0065] The following compounds are prepared essentially by Preparation method 29.
Table 6
Prep. N ^s . Chemical name Structure ES / MS (m / z) (M + H) 30 4 - [(1 S) -2-cyclopropyl-1 - [4 - [(1 S) -1 [(1-ethyl-2-oxo-4H-pyrimido [4,5-d] [1,3] oxazin -7-yl) amino] ethyl] phenyl] ethyl] tercbutyl piperazine-1-carboxylate, ⁰ : N ^ O ^H k 551 31 4- [1 - [4 - [(1 S) -1 - [(1-ethyl-2-oxo-4Hpyrimido [4,5-d] [1,3] oxazin-7-yl) amino] ethyl] phenyl ] Tert-butyl -3-methoxy-propyl] piperazine-1-carboxylate (diastereomer 1) -o A) = o 0 C0> = ⁷ Q IZ o 555 32 4- [1 - [4 - [(1 S) -1 - [(1-ethyl-2-oxo-4Hpyrimido [4,5-d] [1,3] oxazin-7-yl) amino] ethyl] phenyl ] -3-methoxy-propyl] tert-butyl piperazine-1-carboxylate (diastereomer 2) Ο _Ξ ΝγΌ x kW ^H k 555 33 4- [2-cyclopropyl I-1 - [4 - [(1 S) -1 - [(1 methyl-2-oxo-4H-pyrimido [4,5-d] [1,3] oxazin-7-yl ) amino] ethyl] phenyl] ethyl] tert-butyl piperazine-1-carboxylate (diastereomer 1) Ο _Ξ Νγγο Y kkU Η I 537
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Prep. N ^s . Chemical name Structure ES / MS (m / z) (M + H) 34 4- [2-cyclopropi 1-1 - [4 - [(1 S) -1 - [(1 methyl-2-oxo-4H-pyrimido [4,5-d] [1,3] oxazin-7-yl ) amino] ethyl] phenyl] ethyl] tert-butyl piperazine-1-carboxylate (diastereomer 2) the nZI0 ° A 537 35 4- [1 - [4 - [(1 S) -1 - [(1-ethyl-2-oxo-4Hpyrimido [4,5-d] [1,3] oxazin-7-yl) amino] ethyl] phenyl ] Tert-butyl -3-methyl-butyl] piperazine1-carboxylate 0 - cAn ^> Y ^ f ^ hArTAtION TO YUU ^{H 1} 553 36 4 - ((1 R / S) -1 - (4 - ((1 S) -1 - [(1-ethyl-2-oxo-4H-pyrimido [4,5-d] [1,3] oxazin-7-yl) amino] ethyl] phenyl] propyl] piperazine-1 tert-butyl carboxylate, isomer 1 1 O. N ^ '. Z'O l II XL 1 L ^ ON ^ I N- ^ N ^ O BLUE ^H k 525 37 4 - ((1 R / S) -1 - (4 - ((1 S) -1 - [(1-ethyl-2-oxo4H-pyrimido [4,5-d] [1,3] oxazin-7- il) amino] ethyl] phenyl] propyl] piperazine-1 tert-butyl carboxylate, isomer 2 > ^ cAn ^ Y ^ Y ^ rAhAhAo AAJ ^h < 525 38 7 - ((((1 S) -1 - [4- (2-Cyclopropyl-1 hydroxy-ethyl) phenyl] ethyl] amino] -1-ethyl4H-pyrimido [4,5-d] [1,3] oxazin- 2-one âTX HO LJ ^H Ιχ 383
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Preparation 39
7 - [[(1 S) -1 - [4 - [(1 R) -2-Cyclopropyl-1-piperazin-1-yl-ethyl] phenyl] ethyl] amine] -1 ethyl-4H-pyrimido [ 4,5-d] [1,3] oxazin-2-one
N [0066] Hydrochloric acid (95 ml_, 5.5 M in isopropanol, 520 mmols) is added dropwise to a solution of 4 - [(1 R) -2-cyclopropyl-1- [4 [(1 S) -1 - tert- [(1-ethyl-2-oxo-4H-pyrimido [4,5-d] [1,3] oxazin-7-yl) amino] ethyl] phenyl] ethyl] piperazine-1-carboxylate butyl (29.45 g, 53.48 mmols) in EtOAc (570 ml) at 40 ° C. The mixture is allowed to stir for 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 x 150 ml). The pH of the combined aqueous extracts is adjusted to pH 10 by adding 5 N NaOH resulting in the formation of a colorless solid. The solid is collected by filtration, washed with water, and air-dried to produce the title compound (25.18 g, 99%) as a colorless solid. MS (m / z): 451.2 (M + H).
[0067] The following compound is prepared essentially by Preparation method 39.
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Table 7
Prep. N ^a . Chemical name Structure ES / MS (m / z) (M + H) 40 7 - [[(1S) -1- [4 - [(1S) -2Cyclopropyl-1-piperazin-1 yl-ethyl] phenyl] ethyl] amine] -1 ethyl-4H-pyrimido [4,5d] [1.3] oxazin-2-one OO— / tíZI..... OT 451
Preparation 41
7 - [[(1 S) -1 - [4- (2-Cyclopropyl-1-piperazin-1-yl-ethyl) phenyl] ethyl] amine] -1-methyl4H-pyrimido [4,5-d] [ 1.3] oxazin-2-one, diastereomer 1,
[0068] 4- [2-cyclopropyl-1 - [4 - [(1 S) -1 - [(1-methyl-2-oxo-4H-pyrimido [4,5d] [1,3] oxazin-7- il) amino] ethyl] phenyl] ethyl] piperazine-1-tercbutyl-1-carboxylate, diastereomer 1 (245 mg, 0.46 mmol) is dissolved in DCM (2.5 ml). TFA (0.7 ml, 9 mmols) is added and the reaction is stirred at room temperature for 90 minutes. The mixture is quenched with 20% K2CO3 aq and extracted with DCM (3x). The combined organic extracts are dried over Na2SO4, filtered and concentrated to dryness in a high vacuum overnight to produce the title compound as a white foam, (196 mg, 88.5%). MS (m / z): 437 (M + H).
[0069] The following compounds are prepared essentially by Preparation method 41.
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Table 8
Prep. N ^s . Chemical name Structure ES / MS (m / z)(M + H) 42 7 - [[(1 S) -1 - [4- (2-Cyclopropyl1-piperazin-1-yl-ethyl (l) phenyl] ethyl] amine] -1-methyl4H-pyrimido [4,5-d] [1,3] oxazin-2-one, diastereomer 2, ι αΠ ΗΝΛ ΑΥΝ ^Α Ν ^Α Ν ^Λ (I ^ n ^{H 1} 437 43 1-ethyl 1-7 - [[(1 S) -1 - [4- (3-methyl ethyl 1 -piperazin-1-yl butyl) phenyl] ethyl] amino] -4H pyrimido [4,5-d] [1,3 ] oxazin-2one, isomer 1, XBQ. 1 1 ^H | 453 44 1-ethyl 1-7 - [[(1 S) -1 - [4- (3-methyl ethyl 1 -piperazin-1-yl butyl) phenyl] ethyl] amino] -4H pyrimido [4,5-d] [1,3 ] oxazin-2-one, isomer 2, t JOQ I 1 ^H | 453 45 7 - [[(1 S) -1 - [4- (2-Cyclopropyl-1- piperazin-1-yl-eti l) phenyl] ethyl] amine] -1-methyl4H-pyrido [4,3-d] [1,3] oxazin-2- one, isomer 1, ZI 0 X 436 46 7 - [[(1 S) -1 - [4- (2-Cyclopropyl-1- piperazin-1-yl-eti l) phenyl] ethyl] amine] -1-methyl4H-pyrido [4,3-d] [1,3] oxazin-2- one, isomer 2, pA ^z— \ # ^z ZI in ·· / 0 X 436
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Prep. N ^s . Chemical name Structure ES / MS (m / z) (M + H) 47 1-ethyl-7 - [[(1S) -1- [4 - [(1 R / S) 1 -piperazin-1 - ilpropyl] phen i I] ethyl] amino] -4H-pyrimido [4,5d] [ 1.3] oxazin-2-one, isomer 1, N ^x y '' ^x O ... 1 J.>! LX MN ” ¹ SV 'Ν''N * 1 Λ * Η H * 425 48 1-ethyl-7 - [[(1S) -1- [4 - [(1 R / S) 1 -piperazin-1 - ilpropyl] fen ii] ethyl] amino] -4H-pyrimido [4,5d] [1 , 3] oxazin-2-one, isomer 2, MH '' A '' NO in x. y ^H i 425
Preparation 49
7 - [[(1 S) -1 - [4- (2-Cyclopropyl-1-piperazin-1-yl-ethyl) phenyl] ethyl] amine] -1-ethyl-4Hpyrimido [4,5-d] [ 1.3] oxazin-2-one
[0070] A mixture of 7 - [[(1 S) -1- [4- (1-chloro-2-cyclopropylethyl) phenyl] ethyl] amino] -1-ethyl-4H-pyrimido [4,5-d] [1.3] 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 mmols) in ACN (3 mL) is heated in a sealed vial at 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 NH ₃ / MeOH in DCM to yield the title compound (306 mg, 0.66 mmol, 62%) as a white solid. ES / MS (m / z): 451.2 (M + H).
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43/88 [0071] The following compounds are prepared essentially by the method of Preparation 49 using the appropriate protected piperazine.
Table 9
Prep. N ^Q. Chemical nameStructure ES / MS (m / z) (M + H) 50 4- [2-cyclopropyl-1 - [4 - [(1 S) -1 [(2,2,2-trifluoroacetyl) amino] ethyl] phenyl] ethyl] piperazine-1 tert-butyl carboxylateZIzZ— 'o _l_ 470 51 4- [3-methyl-1- [4 - [(1S) -1 - [(2,2,2-trifluoroacetyl) amino] ethyl] phenyl] butyl] tert-butyl piperazine-1-carboxylate/O> °OIZ 472 52 4- [tert-Butyl 4- [1- [4 - [(1S) -1 - [(2,2,2trifluoroacetyl) amino] ethyl] phenyl] propyl] piperazine-1carboxylate0 = ^ 5ZIt — Z Z— 'o__Z— 444
Preparation 53
4- [1 - [4 - [(1 S) -1 - [(2,2,2-trifluoroacetyl) amine] ethyl] phenyl] propyl] piperazine-1 tert-butyl carboxylate, isomer 1, o
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Preparation 54
4- [1 - [4 - [(1 S) -1 - [(2,2,2-trifluoroacetyl) amine] ethyl] phenyl] propyl] piperazine-1 tert-butyl carboxylate, isomer 2,
[0072] tert-butyl 4- [1 - [4 - [(1 S) -1 - [(2,2,2-trifluoroacetyl) amino] ethyl] -phenyl] propyl] piperazine-1-carboxylate ( 29.2 g, 65.8 mmols) is dissolved in MeOH (584 mL) and resolved by chiral SFC chromatography using the following conditions: column: Chiralpak DC-H, 5x25 cm; eluent 85/15 CO2 / MeOH with 0.5% dimethylethylamine; flow rate 300 g / min; detection wavelength 230 nm; column temperature 40 ° C; BPR 100 bar attachment point; 40 ° C solvent temperature. Isomer 1 is isolated as the first elution peak (14.15 g, 31.9 mmols). ES / MS (m / z): 444 (M + H). Isomer 2 is isolated as the second elution peak (13.87 g, 31.3 mmols). ES / MS (m / z): 444 (M + H).
Preparation 55
4- [1 - [4 - [(1 S) -1 - [(1-ethyl-2-oxo-4H-pyrimido [4,5-d] [1,3] oxazin-7-yl) amino] ethyl] phenyl] -3-methyl-butyl] tert-butyl piperazine-1-carboxylate, isomer 1
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Preparation 56
4- [1 - [4 - [(1 S) -1 - [(1-ethyl-2-oxo-4H-pyrimido [4,5-d] [1,3] oxazin-7-yl) amino] ethyl ] tert-butyl phenyl] -3-methyl-butyl] piperazine-1-carboxylate, isomer 2 [0073] 4- [1 - [4 - [(1 S) -1 - [(1-ethyl-2-oxo -4H-tert -butyl [4,5-d] [1,3] oxazin-7yl) amino] ethyl] phenyl] -3-methyl-butyl] piperazin-1-carboxylate (2.6 g, 4.70 mmols) is dissolved in 4: 1 isopropanoklorchloroform (50 ml) and resolved by chiral SFC chromatography using the following conditions: column: Chiralpak DC-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 100 bar attachment point; 40 ° C solvent temperature. Preparation 45 is isolated as the first elution peak (1.02 g, 1.89 mmol). ES / MS (m / z): 553.4 (M + H). Preparation 46 is isolated as the second elution peak (1.05 g, 1.90 mmol). ES / MS (m / z): 553.4 (M + H).
Example 1
7 - [[(1 S) -1 - [4 - [(1 R) -2-Cyclopropyl-1 - (4-prop-2-enoylpiperazin-1 yl) ethyl] phenyl] ethyl] amino] -1-ethyl -4H-pyrimido [4,5-d] [1,3] oxazin-2-one
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46/88 [0074] Acryloyl chloride (305 μΙ_, 3.75 mmols, in 2 ml_ DCM) is added dropwise to a solution of 7 - [[(1S) -1- [4 - [(1 R) -2cyclopropyl-1-piperazin-1-yl-ethyl] phenyl] ethyl] amino] -1-ethyl-4H-pyramid [4,5-d] [1,3] oxazin-2-one (1, 73 g, 3.26 mmols) 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 (Na ₂ SO4), 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 hexane) to produce the title compound as a colorless solid (1 , 16 g, 70%). MS (m / z): 505.3 (M + H).
[0075] The following compounds are prepared essentially by the method of Example 1.
Table 11
Ex. N ^s . Chemical name Structure ES / MS(m / z)(M + H) 2 7 - [[(1S) -1- [4 - [(1S) -2cyclopropyl-1 - (4-prop-2enoylpiperazin-1 - yl) ethyl] phenyl] ethyl] amino] -1-ethyl-4Hpirimido [4, 5-d] [1,3] oxazin2-one O - rí ^ν Ά “CnJU 7 505 3 7 - [[(1 S) -1 - [4- [2-cyclopropyl1 - (4-prop-2-enoylpiperazin1-yl) ethyl] phenyl] ethyl] amino] -1methyl-4H-pyrimido [4,5-d ] [1,3] oxazin-2-one, diastereomer 1, 0 _Ξ An '-' J óyAFiA) II LvU ^{H 1} 491
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4 7 - [[(1 S) -1 - [4- [2-cyclopropyl1 - (4-prop-2-enoylpiperazin1-yl) ethyl] phenyl] ethyl] amino] -1 methyl-4H-pyrimido [4,5- d] [1,3] oxazin-2-one, diastereomer 2, 0 OH ^{H 1} 491 5 7 - [[(1 S) -1 - [4- [2-cyclopropyl-1 - (4-prop-2-enoylpiperazin1 -yl) ethyl] phenyl] ethyl] amine] -1 ethyl-4H-pyrimido [4,5d] [1,3] oxazin-2-one O ₌ NNNO H 505 6 1-ethyl-7 - [[(1S) -1- [4- [3-methyl-1 - (4-prop-2-enoylpiperazin-1 -yl) butyl] phenyl] ethyl] amino] -4Hpyrimido [4,5-d] [1,3] oxazin-2one Or < o O— / H ^z z 507 7 1-ethyl-7 - [[(1S) -1- [4- [3-methyl-1 - (4-prop-2-enoylpiperazin-1 -yl) butyl] phenyl] ethyl] amino] -4Hpyrimido [4,5-d] [1,3] oxazin-2one, isomer 1, Opi o 0—4 /HZlO , 507 8 1-ethyl-7 - [[(1S) -1- [4- [3-methyl-1 - (4-prop-2-enoylpiperazin-1 -yl) butyl] phenyl] ethyl] amino] -4Hpyrimido [4,5-d] [1,3] oxazin-2one, isomer 2, O i QC 1] ^H 507 9 7 - [[(1 S) -1 - [4- [2-cyclopropyl1 - (4-prop-2-enoylpiperazin1 -yl) ethyl] phenyl] ethyl] amine] -1 methyl-4H-pyrido [4 , 3-d] [1,3] oxazin-2-one, isomer 1, O OZIThe rx 490 10 7 - [[(1 S) -1 - [4- [2-C iclopropyl1 - (4-prop-2-enoylpiperazin1 -yl) ethyl] phenyl] ethyl] amine] -1 methyl-4H-pyrido [ 4,3-d] [1,3] oxazin-2-one, isomer 2, 0Y-N X J ^{H 1} 490
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11 1-ethyl-7 - [[(1S) -1- [4 - [(1 R / S) -1 - (4-prop-2-enoylpiperazin1 -yl) propyl] phenyl] ethyl] amino] 4H-pyrimido [4,5-d] [1,3] oxazin-2-one, isomer 1, O JOCXH 479 12 1-ethyl-7 - [[(1S) -1- [4 - [(1 R / S) 1 - (4-prop-2-enoylpiperazin1 -yl) propyl] phenyl] ethyl] amine] 4H-pyrimido [4,5-d] [1,3] oxazin-2-one, isomer 2, O f jOCl ^H J 479
[0076] Determination of the crystalline structure of X-ray IDH1 in complex with 7 - [[(1 S) -1 - [4 - [(1 S) -2-cyclopropyl-1 - (4-prop-2-enoylpiperazin1 -il ) ethyl] phenyl] ethyl] amino] -1-ethyl-4H-pyrimido [4,5-d] [1,3] oxazin-2-one.
[0077] The crystal structure of IDH1 in complex with 7 - [[(1 S) -1 [4 - [(1 S) -2-cyclopropyl-1 - (4-prop-2-enoylpiperazi n-1 -yl) ethyl] phenyl] ethyl] amine] 1-ethyl-4H-pyrimido [4,5-d] [1,3] oxazin-2-one is determined from X-ray diffraction data collected on the beam line of APS 31-ID synchrotron operated at the Advanced Photon Source at Argonne National Laboratory, Argonne, IL 60439. IDH1 protein with an R132H mutation is commercially available from multiple sources. Alternatively, IDH1 R132H protein can be isolated from a commercially available cell line harboring the mutation by well known techniques and can usually be used by those skilled in the art. Crystals are obtained from loose trays balanced at 21 ° C with IDH1 protein with the R132H mutation 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 - [[(1S) -1- [4 - [(1S) -2-cyclopropyl-1- (4prop-2-enoylpiperazin-1-yl) ethyl] phenyl ] ethyl] amino] -1-ethyl-4H-pyrimido [4,5d] [1,3] oxazin-2-one, and mixed with an equal volume of reservoir solution containing 100mM Bis Tris pH 5, 5% DMSO, 22%
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PEG 3350 and 200mM Ammonium Sulfate. Crystals are soaked overnight in a solution containing 3 mM of 7 - [[(1S) -1- [4 - [(1S) 2-cyclopropyl-1 - (4-prop-2-enoylpiperazin-1-yl) ethyl ] phenyl] ethyl] amine] -1-ethyl4H-pyrimido [4,5-d] [1,3] oxazin-2-one, before being transferred into a solution supplemented with 22% Ethylene Glycol and frozen for collection of data. Diffraction data for 2.8 Á resolution is collected with X-ray radiation of 0.9793 Á wavelength. The crystals belong to Group Spacer P4 ₃ 2i2 with cell parameters a = 82.74 Á, b = 82.74 Á, c = 299.4À, α = β = γ = 90 °. The structure is determined by Molecular Replacement and contained an molecule of IDH1 dimer. The electron difference density maps calculated after modeling the IDH1 protein have clear density per two 7 - [[(1S) -1- [4 - [(1S) -2-cyclopropyl-1- ( 4prop-2-enoylpiperazin-1-yl) ethyl] phenyl] ethyl] amino] -1-ethyl-4H-pyrimido [4,5d] [1,3] oxazin-2-one.
[0078] The stereochemistry of 7 - [[(1S) -1- [4 - [(1S) -2-cyclopropyl-1- (4prop-2-enoylpiperazin-1 -yl) ethyl] phenyl] ethyl] amino] - 1-ethyl-4H-pyrimido [4,5-d] [1,3] oxazin-2-one is determined from the electron density, and both molecules of 7 - [[(1S) -1- [4 - [(1S) -2-cyclopropyl-1- (4-prop-2enoylpiperazin-1-yl) ethyl] phenyl] ethyl] amino] -1-ethyl-4H-pyrimido [4,5-d] [1,3 ] oxazin-2-one are modeled and the co-complex structure refined to R factors of Rwork = 0.192 and Rfree = 0.228.
Õ24 ^ θ252θ27 ^C 26
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Table 12 [0079] Coordinates of 7 - [[(1 S) -1 - [4 - [(1 S) -2-cyclopropyl-1 - (4-prop2-enoylpiperazin-1 -yl) ethyl] phenyl] ethyl] amino] -1-ethyl-4H-pyrimido [4,5d] [1,3] 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
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ATOM X Y Z C11 55,701 0.819 -3.988 C14 54,812 3,365 -3,432 C15 55,295 3,057 -4,691 C10 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 C1 58,372 0.310 -5.968 N 59,104 -0.809 -6.086 N1 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 Ç 60,369 -0.717 -5.655
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Example 13
-ethyl-7 - [[(1 S) -1 - [4- [3-methoxy-1 - (4-prop-2-enoylpiperazin-1 yl) propyl] phenyl] ethyl] amino] -4H-pyrimido [4 , 5-d] [1,3] oxazin-2-one, diastereomer 1,
Chiral
[0080] 4 - [(1 S) -1 - [4 - [(1 S) -1 - [(1-ethyl-2-oxo-4H-pyrimido [4,5-d] [1,3] oxazin -7-yl) amino] ethyl] phenyl] -3-methoxy-propyl] tert-butyl piperazine-1-carboxylate (1.372 g, 2.473 mmol) is dissolved in DCM (15 ml_) and TFA (10 ml_, 15, 08 g, 132.3 mmols) is added. The reaction is stirred for 1 hour and then concentrated to dryness. The raw material is dissolved in DCM (12 ml_) and DIPEA (1.25 ml_, 7.17 mmols) 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 extinguish the remaining acryloyl chloride and the reaction is concentrated to dryness (cold). The crude material is chromatographed on silica gel eluting with a gradient of 50-70% acetone in hexane to produce the title compound as a white foam (867mg, 73%). MS (m / z): 509 (M + H) [0081] The following compound is prepared essentially by the method of Example 13.
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Table 13
Ex. N ^Q. Chemical name Structure ES / MS (m / z) (M + H) 14 1-ethyl-7 - [[(1S) -1- [4- [3methoxy-1- (4-prop-2enoylpiperazin-1yl) propyl] phenyl] ethyl] amino] 4H-pyrimido [4,5d] [1, 3] oxazin-2-one, diastereomer 2, co OA ZI O ' ^X A ° 0 ° Á_ 509
Example 15
7 - [[(1 S) -1 - [4 - [(1 S) -2-cyclopropyl-1 - (4-prop-2-enoylpiperazin-1 yl) ethyl] phenyl] ethyl] amino] -1 -ethyl-4H-pyrimido [4,5-d] [1,3] oxazin-2-one
H ₂ SO ₄ [0082] 7 - [[(1 S) -1 - [4 - [(1 S) -2-Cyclopropyl-1 - (4-prop-2-enoylpiperazin1 -yl) ethyl] phenyl] ethyl] amino] -1-ethyl-4H-pyrido [4,5-d] [1,3] 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 in 2 ml_ of acetone). A thick white suspension results after a few drops. After adding half of the sulfuric acid, the consistency of the suspension is changed. Addition of the second half of the sulfuric acid is done slowly, drop by drop. The suspension breaks slightly before converting to a shiny, white free-flowing suspension of the solid. Heat is turned off
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54/88 on the plate after 30 minutes, and the sample cooled to room temperature, producing a thick suspension of white solid. The white solid is isolated by vacuum filtration. The resulting mass is bright white solid. The sample is dried in place of the filter under a draft for 20 minutes, then in the vacuum oven at 70 ° C overnight. 266 mg recovered (96.9% yield).
Example 16 7 - [[(1 S) -1 - [4- [2-cyclopropyl-1 - (4-prop-2-enoylpiperazin-1-yl) ethyl] phenyl] ethyl] amino] -1-ethyl- 4H-pyrimido [4,5-d] [1,3] oxazin-2-one-4-hydroxybenzoic o
the ^N Ί
L [0083] 4-Hydroxybenzoic acid (0.023 g, 0.165 mmol) is added to a solution of 7 - [[(1S) -1- [4- [2-cyclopropyl-1- (4-prop-2-enoylpiperazin1 - il) ethyl] phenyl] ethyl] amino] -1-ethyl-4H-pyrimido [4,5-d] [1,3] oxazin-2-one (84 mg, 0.165 mmol) in dichloromethane (5 ml). After stirring 5 minutes, the solvent is evaporated slowly under a stream of nitrogen. The resulting solid is also dried under vacuum to produce 4 - [[(1S) -1- [4- [2-cyclopropyl-1- (4-prop-2enoylpiperazin-1-yl) ethyl] phenyl] ethyl] amino 4-hydroxybenzoate ] -1-ethyl-4H-pyrimido [4,5d] [1,3] oxazin-2-one (105 mg, 0.1617 mmol) as a white solid. MS (m / z): 423.2 (M + H).
Preparation 57
2-Cyclopropyl-N-methoxy-N-methylacetamide
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55/88 [0084] Dichloromethane (160 ml, 8 volumes) and 1,1'-carbonyladimidazole (35.63g, 1.1 equiv.) Are loaded into a stirred 500 ml round-bottom flask. The heterogeneous mixture is cooled to 15 ° C and the mixture is charged with a solution of 2-cyclopropylacetic acid (20, Og, 1.0 equiv.) In dichloromethane (40 ml_, 2 volumes) at a rate controlling the internal temperature below 20 ° C. The resulting solution is heated to 25 ° C and stirred for two hours. The solution is then cooled to 15 ° C and to this is added Ν, Ο-dimethyl hydroxylamine hydrochloride (21.43g, 1.1 equiv.) In portions, keeping the internal temperature below 20 ° C. The resulting heterogeneous mixture is heated to 25 ° C and stirred for 15 hours. The reaction mixture is then diluted with water (160 ml, 8 volumes) and stirred for 15 minutes. Stirring is stopped and the lower aqueous layer is separated. The resulting aqueous layer is extracted with dichloromethane two more times (100 ml, 5 volumes x 2) and the organic layers are combined. The combined organic layer is washed twice with 1.5 N HCI (100 ml, 5 volumes x 2). The organic layer is then washed twice with 10% aqueous sodium bicarbonate (100 ml, 5 volumes x 2). The organic layer is then washed with water (100 ml, 5 volumes) followed by saturated aqueous sodium chloride (100 ml, 5 volumes). The organic layer is dried over anhydrous sodium sulfate (1.0% w / w). The mass is filtered and washed with dichloromethane (20 ml, 1 volume) and then concentrated in vacuo. The resulting solid is dried under high vacuum for 5 hours to obtain the title compound (25.9g, 90.5% yield). ES / MS m / z 144.1 (M + H).
Alternative Synthesis of Preparation 6 (S) -N- (1- (4-bromophenyl) ethyl) -2,2,2-trifluoroacetamide
Έ The
II J H
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56/88 [0085] Dichloromethane (100 mL, 10 volumes) is loaded into a 250 ml round-bottomed flask followed by (S) - (-) - 1- (4bromophenyl) ethylamine (10, Og, 1.0 equiv.). The solution is cooled to 0 ° C and the cooled solution is loaded with trifluoroacetic anhydride slowly (13.12g, 1.25 equiv.), Keeping the internal temperature below 5 ° C. The resulting heterogeneous mixture is stirred at 0 ° C for two hours during which time trimethylamine (12.64g, 2.5 equiv.) Is slowly charged, maintaining the internal temperature below 5 ° C. The mixture is stirred for an additional hour and then quenched by the addition of water (30 ml, 3 volumes). The biphasic mixture is heated to 25 ° C and stirred for 30 minutes. The layers are then separated and the aqueous layer is also extracted twice with dichloromethane (50 ml, 5 volumes x 2). The combined organic layers are washed with saturated aqueous sodium chloride (50 ml, 5 volumes) and dried over anhydrous sodium sulfate (1% by weight). The dried solution is filtered and washed with dichloromethane (10 ml, 1 volume) and the solution is concentrated in vacuo to produce a raw white solid. The crude solid is suspended in petroleum ether (100 mL, 10 volumes for two hours at 25 ° C and the solid is collected by filtration. The wet solid is dried under vacuum at 40 ° C for 8 hours to obtain the title compound ( 13.3 g, 90% yield) ES / MS m / z 295.3 (M + H).
Preparation 58 (S) -N- (1- (4- (2-cyclopropylacetyl) phenyl) ethyl) -2,2,2-trifluoroacetamide
[0086] A tert-butyl methyl ether (1500 mL, 15 volumes) is loaded into a 10 L reaction vessel with superior stirring. The stirred solution is charged (S) -N- (1- (4-bromophenyl) ethyl) -2,2,2
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57/88 trifluoroacetamide (100g, 1.0 equiv.). The heterogeneous mixture is stirred at 25 ° C for 30 minutes and then tetrahydrofuran (500 ml, 5 volumes) is loaded. The resulting homogeneous solution is cooled to 83 ° C. n-Butyl lithium (297 ml, 2.2 equiv.) is added slowly to the solution keeping the temperature below -78 ° C and the resulting solution is stirred at -83 ° C for 1.5 hours. To the cooled solution is added a solution of 2-cyclopropyl-N-methoxy-N-methylacetamide (53.19g, 1.1 equiv.) In tert-butyl methyl ether (200 ml_, 2 volumes), keeping the internal temperature below -78 ° C. The resulting solution is stirred at -83 ° C for 1.5 hours during which time the solution is heated to -30 ° C and quenched by the addition of saturated aqueous ammonium chloride (5 L, 5 volumes). The quenched reaction mixture is heated to 25 ° C and the layers are separated. The aqueous layer is extracted with methyl tert-butyl ether (500 ml, 5 volumes). The combined organic layers are washed with water (500 ml_, 5 volumes), followed by saturated aqueous sodium chloride (500 ml_, 5 volumes) and then dried over anhydrous sodium sulfate (50g, 0.5% w / w ). The mass is filtered and washed with tert-butyl methyl ether (50 ml, 0.5 volumes). The resulting solution is concentrated in vacuo until approximately 1 volume of solution remains. Petroleum ether (1500 ml, 15 volumes) is charged to the concentrated mixture and the resulting suspension is stirred below 30 ° C for two hours. The solid is collected by filtration and dried under vacuum at 40 ° C for 8 hours to obtain the title compound (65.9 g, 67% yield). ES / MS m / z 298.0 (M-H).
Preparation 59
N - (((S) -1 - (4 - ((S) -2-cyclopropyl-1-hydroxyethyl) phenyl) ethyl) -2,2,2-trifluoroacetamide 3 o
N CF H
N CF ₃
H ³
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58/88 [0087] In a hydrogenator reactor absolute ethanol (7.89 kg, 10 volumes) is loaded. To the stirred solution is charged (S) -N- (1- (4- (2-cyclopropylacetyl) phenyl) ethyl) -2,2,2-trifluoroacetamide (1.0 kg, 1.0 equiv.). The solution is charged with a solution of potassium tert-butoxide (1.0 M in tBuOH, 0.41 kg, 0.5 volumes) keeping the internal temperature below 30 ° C. The solution is then charged (R) -RUCY®XylBINAP (0.0675 kg, 0.017 equiv.). The hydrogenator is purged twice with hydrogen gas, stirring at 25 ° C. After purging, the solution is stirred under 4.5 kg of hydrogen pressure at 25 ° C for 5 hours. After 5 hours, the solution is vented and then concentrated in an oil with a vacuum below 42 ° C. The oil is dissolved in methyl tert-butyl ether (11,115 kg, 15 volumes) and then concentrated in an oil with a vacuum below 45 ° C. The oil is dissolved in methyl tert-butyl ether (11,115 kg, 15 volumes) and then concentrated in an oil with a vacuum below 45 ° C. The oil is charged with methyl tert-butyl ether (22.23 kg, 30 volumes) at 25 ° C. The resulting solution is washed with water (15.0 kg, 15 volumes) followed by a solution of NaCI in water (5.4 kg NaCI in 15.0 L of water). The organic layer is dried over anhydrous sodium sulfate (1.5 kg, 1.5 w / w) and then filtered and washed with tert-butyl methyl ether (1.112 kg, 1.5 volumes). The filtered solution is charged with activated carbon (0.3 kg, 0.3 w / w) and the mixture is stirred and heated at 40 ° C for two hours. The mixture is then filtered and washed with tert-butyl methyl ether (1.112 kg,
1.5 volumes). The filtered solution is concentrated in an oil with a vacuum below 45 ° C. The oil is dissolved in petroleum ether (9.84 kg, 15 volumes) and concentrated in an oil with a vacuum below 45 ° C. The oil is dissolved in petroleum ether (9.84 kg, 15 volumes) and concentrated in an oil with a vacuum below 45 ° C. Petroleum ether (19.68 kg, 30 volumes) is charged to the oil and the mixture is stirred at 30 ° C for 3 hours and the resulting solid is collected by filtration and washed with
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59/88 petroleum ether (9.84 kg, 15 volumes). The isolated solid is dried under vacuum at 40 ° C for 5 hours to obtain the title compound (0.79 kg, 79%). ES / MS m / z 300.0 (M-H).
Preparation 60
N - ((S) -1 - (4 - ((R) -1-chloro-2-cyclopropylethyl) phenyl) ethyl) -2,2,2trifluoroacetamide
[0088] In a reactor, tert-butyl methyl ether (4.45 kg, 6 volumes) and N - ((S) -1 - (4 - ((S) -2-cyclopropyl-1-hydroxyethyl) phenyl) is loaded into a reactor ethyl) -2,2,2trifluoroacetamide (1.0 kg, 1.0 equiv.). The solution is cooled to 10 ° C and 1-formylpyrrolidine is slowly charged (0.066 kg, 0.2 equiv.) Maintaining the internal temperature below 13 ° C. The solution is then slowly charged with benzoyl chloride (0.56 kg, 1.2 equiv.) Keeping the internal temperature below 13 ° C. The resulting solution is heated to 25 ° C and stirred for up to 36 hours (reaction can be monitored during the course of the reaction and if stopped, additional charges of 1-formylpyrrolidine and benzoyl chloride can be made). The completed reaction is then concentrated to an oil with a vacuum below 40 ° C. The oil is cooled to 15 ° C and then quenched with a 10% aqueous sodium bicarbonate solution (22.0 kg, 20 volumes) and stirred for 3 hours at 25 ° C. The biphasic mixture is charged with petroleum ether (6.56 kg, 10 volumes). The organic layer is separated. The aqueous layer is extracted with light petroleum (6.56 kg, 10 volumes). The combined organic layers are washed with 10% aqueous sodium bicarbonate (5.0 L, 5 volumes) three times. The organic layer is then washed with water (5.0 L, 5 volumes) followed by brine (5.0 L, 5 volumes). The organic layer is dried over sodium sulfate
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60/88 anhydrous (0.5 kg, 0.5 w / w). The mixture is then filtered and washed with petroleum ether (0.33 kg, 0.5 volumes). The filtrate is concentrated in an oil with a vacuum below 40 ° C. The oil is dissolved in acetonitrile (3.93 kg, 5 volumes) and concentrated in an oil with a vacuum below 40 ° C. The oil is dissolved in acetonitrile (3.93 kg, 5 volumes) and concentrated in an oil with a vacuum below 40 ° C. The oil is dissolved in acetonitrile (7.86 kg, 10 volumes) and the solution obtained from the title compound is used raw in the next step. ES / MS m / z 318.0 (M-H).
Preparation 61
4 - ((S) -2-cyclopropyl-1 - (4 - ((S) -1 - (2,2,2-trifluoroacetamido) ethyl) phenyl) ethylpiperazine-1-carboxylate o
[0089] To the solution previously obtained from N - ((S) -1- (4 - ((R) -1chloro-2-cyclopropylethyl) phenyl) ethyl) -2,2,2-trifluoroacetamide is loaded NBoc-piperazine (0.924 kg, 1.5 equiv.) Followed by sodium bicarbonate (1.1 kg, 4.0 equiv.). The resulting mixture is heated to 85 ° C for 60 hours. Note: the reaction is tested every 24 hours and after each sample is used, the reaction is charged with an additional amount of N-Boc-piperazine (0.31 kg, 0.5 equiv.). Once complete, the reaction is concentrated in vacuo below 45 ° C to provide an oil. The oil is diluted with water (10.0 kg, 10 volumes) and methyl tert-butyl ether (7.41 kg, 10 volumes). The resulting biphasic mixture is separated and the aqueous layer is extracted with tert-butyl methyl ether (7.41 kg, 10 volumes). The combined organic layers are extracted with 30% citric acid (5.0 L, 5 volumes) five times. The combined aqueous layers are washed twice with light petroleum
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61/88 (6.56 kg, 10 volumes). The aqueous layer is brought to a pH of 9 with the addition of sodium carbonate (approximately 25.0 kg, 25 w / w) at 15 ° C. The basified aqueous layer is extracted with methyl tert-butyl ether (7.41 kg, 10 volumes). The combined organic layers are washed with water (5.0 L, 5 volumes) and brine (5.0 L, 5 volumes). The organic layer is dried over anhydrous sodium sulfate (0.5 kg, 50% w / w). The mixture is filtered and washed with tert-butyl methyl ether (0.37 kg, 0.5 volumes). The filtrate is concentrated in an oil under vacuum below 40 ° C. The resulting oil is dissolved in absolute ethanol (3.95 kg, 5 volumes) and the crude solution of the title compound is used directly in the next step. ES / MS m / z 374.3 (M + H-CF ₃ CO).
Preparation 62
4 - ((S) -1 - (4 - ((S) -1 -aminoethyl) phenyl) -2-cyclopropylethyl) piperazine-1 tert-butyl carboxylate [0090] To the solution previously obtained from 4 - (( Tert-Butyl S) -2cyclopropyl-1- (4 - ((S) -1- (2,2,2-trifluoroacetamido) ethyl) phenyl) ethylpiperazine1-carboxylate is loaded absolute ethanol (3.7 kg, 3.7 w / w) The solution is cooled to 20 ° C and 1 M aqueous potassium hydroxide (0.168 kg KOH in 3.0 kg water) is charged in. The resulting mixture is stirred at 25 ° C for 10 hours and then quenched by the slow addition of 30% aqueous citric acid (5.0 kg, 5 w / w) keeping the internal temperature below 30 ° C. The extinct solution is charged with methyl tert-butyl ether (7.41 kg , 7.41 w / w) .The layers are separated and the organic layer is extracted with 30% aqueous citric acid (5.0 kg, 5 w / w). The aqueous layer is brought to a pH of 8 with the addition of sodium carbonate (approximately 25.0 kg, 25 w / w) to
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15 ° C. The basified aqueous layer is extracted twice with ethyl acetate (7.41 kg, 7.41 w / w). The combined organic layers are washed with water (5.0 kg, 5 w / w) and then brine (5.0 kg, 5 w / w). The organic layer is dried over anhydrous sodium sulfate (0.5 kg, 50% by weight), then filtered and washed with ethyl acetate (0.37 kg, 0.37 w / w). The solution is concentrated in a vacuum oil below 45 ° C. The resulting oil is dissolved in isopropyl alcohol (2 L, 2 volumes), and the resulting solution is concentrated to an oil under vacuum below 45 ° C. The resulting oil is dissolved in isopropyl alcohol (1.2 L, 1.2 volumes) and the crude solution of the title compound is used directly in the next purification step. ES / MS m / z 374.2 (M + H).
[0091] In a 1000 ml round base flask, a solution of 4 - ((S) -1- (4 - ((S) -1-aminoethyl) phenyl) -2cyclopropylethyl) piperazine-1-carboxylate DE terc is loaded -butyl (30.0 g, 1.0 equiv.) in isopropyl alcohol (270 ml, 9 volumes). The solution stirred at 20-30 ° C is charged with L-dibenzoyl tartaric acid (L-DBTA, 34.5 g, 1.2 equiv.). The solution is stirred at 20-30 ° C for 2-4 hours. The resulting suspension is allowed to stir for 8-12 hours at 20-30 ° C. MTBE (300 mL, 10 volumes) is loaded into the clear suspension. The resulting suspension is allowed to stir and thicken at 20-30 ° C for 12-16 hours. The solid is then collected by filtration and washed with MTBE (150 ml, 5 volumes). The solid is dried under reduced pressure at 45-55 ° C for 12 hours to provide 4 - ((S) -1- (4 - ((S) -1-aminoethyl) phenyl) - L-dibenzoyl tartaric salt 2-cyclopropylethyl) piperazine-1-carboxylate tert-butyl as a white solid (48.8 g, 83% yield,> 98: 2 dr).
[0092] 4 - ((S) -1 - (4 - ((S) -1 -aminoethyl) phenyl) -2-cyclopropylethyl) piperazine-1 tert- butyl salt of L-dibenzoyl tartaric acid (40.0 g, 1.0
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63/88 equiv.) Followed by dichloromethane (400 ml, 10 volumes). To the solution stirred at 15-25 ° C is charged an aqueous solution of 10% Na2CC> 3 (enough to bring pH to 8-10, approximately 6-8 volumes). The biphasic mixture is stirred for one hour at 15-25 ° C and then the layers are separated in a separatory funnel. DMSO (240 ml, 6 volumes) is added to the organic layer. The solution is concentrated under reduced pressure below 40 ° C to remove the dichloromethane. The free base tert-butyl solution of 4 - ((S) -1- (4 - ((S) -1-aminoethyl) phenyl) -2cyclopropylethyl) piperazine-1-carboxylate in DMSO is then directly used in the next stage.
Preparation 63
Ethyl 6-chloro-4- (methylamino) pyridine-3-carboxylate
II ^ NH [0093] Methyl amine (16 ml_, 11.6 mol / L in water, 186 mmols) is added dropwise to a solution of ethyl 4,6-dichloropyridine-3-carboxylate (20 g, 92 mmols ) in acetonitrile (300 ml) at 0 ° C. The reaction mixture is allowed to warm to room temperature for two hours. To the reaction mixture, water and ethyl acetate are added and the aqueous layer is extracted with ethyl acetate. The combined organic extracts are dried over anhydrous Na2SO4, filtered and concentrated to dryness. The resulting raw material is purified by chromatography on silica gel (0-100% EtOAc / hexanes) to produce, after concentration of the appropriate fractions, ethyl 6-chloro-4 (methylamino) pyridine-3-carboxylate as a white solid (10.46 g, 52.14 mmols, 57% yield). MS (m / z): 201 (M + H).
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Preparation 64 [6-Chloro-4- (methylamino) -3-pyridyl] methanol [0094] A solution of ethyl 6-chloro-4- (methylamino) pyridine-3-carboxylate (10.46 g, 52.14 mmols ) in THF (100 ml) is added dropwise to a mixture of lithium aluminum hydride (78.2 ml, 1.0 mol / L in THF, 78.2 mmols) at 0 ° C. The reaction mixture is allowed to warm to room temperature for 1.5 hr. To the reaction mixture, water (3 ml), 15% aqueous NaOH (3 ml), and then water (9 ml) are added consecutively. After stirring, the reaction mixture is filtered through a pad of Celite. The filtrate is diluted with water and extracted with dichloromethane. The combined organic extracts are dried over anhydrous Na2SO4, filtered and concentrated to dryness to provide [6-chloro-4- (methylamino) -3-pyridyl] methanol as a pale yellow solid (2.78 g, 27% yield) . The filtered mass of celite is also washed with methanol and the filtrate is concentrated to dryness. The solids from the filtered mass are collected and stirred with dichloromethane and filtered through Celite. The filtrate is combined with the residue from the methanol washes and the material is concentrated to dryness and the material is reserved. To the solids collected from the filtration, 4: 1 chloroform: isopropyl alcohol is added and the mixture is stirred overnight. The mixture is filtered on a Celite pad, the filtrate is combined with the reserved residue and concentrated to dryness to provide an additional amount of [6-chloro-4 (methylamino) -3-pyridyl] methanol as a pale yellow solid ( 5.07 g, 56% yield). The total recovery of [6-chloro-4- (methylamino) -3pyridyl] methanol is 7.85 g, 83% yield). MS (m / z): 173 (M + H).
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Preparation 65
7-Chloro-1-methyl-4H-pyrido [4,3-d] [1,3] oxazin-2-one [0095] Triphosgene (6.04 g, 20.4 ml) is added to a solution of [ 6-chloro-4- (methylamino) -3-pyridyl] methanol (5.07 g, 29.1 ml) and DIPEA (51.2 ml, 291 mmols) in THF (100 ml) at -20 ° C. The cold bath is removed and the mixture is allowed to warm to room temperature. After 30 minutes, water is added and the mixture is extracted with dichloromethane. The combined organic extracts are dried over anhydrous Na2SO4, filtered and concentrated to dryness. The resulting raw material is purified by chromatography on silica gel (0-100% EtOAc / hexanes) to produce, after the concentration of the appropriate fractions, 7-chloro-1-methyl-4H-pyrido [4,3-d] [ 1.3] oxazin-2-one as an orange solid (5.13 g, 24.5 mmols, 84% yield). MS (m / z): 199 (M + H).
Preparation 66
4- [2-cyclopropyl-1 - [4 - [(1 S) -1 - [(1-methyl-2-oxo-4H-pyrido [4,3-d] [1,3] oxazin7-yl) amino ] ethyl] phenyl] ethyl] tert-butyl piperazine-1-carboxylate, diastereomer 1
NH [0096] In a solution of tert-butyl 4- [1- [4 - [(1S) -1-aminoethyl] phenyl] -2cyclopropyl-ethyl] piperazine-1-carboxylate (1.30 g, 3.48 mmols), 7-chloro-1-methyl-4H-pyrido [4,3-d] [1,3] oxazin-2-one (864 mg,
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4.35 mmols), and cesium carbonate (2.27 g, 6.96 mmols) in toluene (17.4 ml) under nitrogen is added dichloro [1,3-bis (2,6-di-3pentylphenyl) imidazole -2-ylidene] (3-chloropyridyl) palladium (ll) (291 mg, 0.348 mmol) and the mixture is heated to 75 ° C overnight. After cooling to room temperature, the mixture is filtered through a plug of silica gel and eluted with ethyl acetate. The filtrate is concentrated under reduced pressure and the acquired residue is purified by chromatography on silica gel (20-100% EtOAc / hexanes). Mixed fractions are repurified by silica gel chromatography (50-100% methyl tert-butyl ether / hexanes) and the combined pure fractions from both columns are concentrated to produce 4- [2-cyclopropyl-1- [4 - [(1S ) 1 - [(1-methyl-2-oxo-4H-pyrido [4,3-d] [1,3] oxazin-7-yl) amino] ethyl] phenyl] ethyl] piperazine-1-carboxylate tert- butyl, diastereomer 1 as an almost white foam (1.312 g, 2,400 mmols, 69% yield). MS (m / z): 536 (M +).
[0097] Cancer is increasingly recognized as a heterogeneous collection of diseases whose initiation and progression are induced by the aberrant function of one or more genes that regulate DNA repair, genome stability, cell proliferation, cell death, adhesion, angiogenesis, invasion, and cell metastasis and tissue microenvironments. Aberrant variant or function of cancer genes can result in naturally occurring DNA polymorphism, changes in genome copy number (through amplification, deletion, chromosome loss, or duplication), changes in gene and chromosome structure (through chromosomal translocation, inversion, or other arrangement leading to unregulated gene expression), and point mutations. Cancerous neoplasms can be induced by an aberrant gene function, and maintained by the same aberrant gene function, or exacerbated maintenance and progression by additional aberrant gene functions.
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67/88 [0098] In addition to the genetic chromosomal aberrations mentioned above, each of the cancers may likewise include epigenetic modifications of the genome including DNA methylation, genomic printing, and histone modification by acetylation, methylation or phosphorylation. An epigenetic modification can play a role in inducing and / or maintaining malignancy.
[0099] Extensive catalogs of cytogenetic aberrations in human cancer have been fulfilled and are maintained and regularly updated online The Mitelman Database of Chromosome Aberrations in Cancer at the US National Cancer Institute (NCI) Cancer Genome Anatomy Project (CGAP) Web site). The Wellcome Trust Sanger Institute Cancer Genome Project maintains a detailed Online Cancer Gene Census of all human genes that have been causally linked to tumorigenesis as well as the COSMIC (Catalog of Somatic Mutations in Cancer) database of somatic mutations in human cancer. Another source containing abundant information on cytogenetic changes casually linked to various cancers is the Atlas of Genetics and Cytogenetics in Oncology and Haematology.
[00100] Diagnosis of cancerous malignancies by biopsy, immunophenotyping and other tests are known and commonly used. In addition to high resolution chromosomal bands and advanced chromosomal imaging technologies, chromosomal aberrations in suspected cases of cancer can be determined by cytogenetic analysis such as fluorescence in situ hybridization (FISH), karyotyping, spectral karyotyping (SKY), multiplex FISH (M- FISH), comparative genomic hybridization (CGH), simple nucleotide polymorphism provisions (SNP Chips) and other diagnostic tests and analysis known and used by those skilled in the art.
[00101] Mutations in HDI1 and HDI2 have been identified in multiple cancer tumor types including, but not limited to, glioma,
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68/88 glioblastoma multiforme, astrocytomas, oligodendrogliomas, paraganglioma, myelodysplastic syndrome (MDS), acute B-cell lymphoblastic leukemia (B-ALL), acute myeloid, colorectal, acute myeloid leukemia (AML), Dang and others, Trends Mol. Med. ., 2010, 16: 387-397; Ward et al., Oncogene, 2012, 31 (19): 2491-2498; melanoma, Shibata et al., Am. J. Pathol., 2010, 178 (3): 1395-1402; prostate, Flaherty and others, J. Clin. Oncol., 2014, 32 (suppl. 4; Abstract 213); Cairns et al., Cancer Discovery, 2013, 3: 730-741; chondrossarcoma and cholangiocarcinoma, Balss et al., Acta Neuropathol., 2012, 124: 883-891; Cairns et al., Cancer Discovery, 2013, 3: 730-741; angioimmunoblastic T-cell lymphoma (AITL), Cairns and another Blood, 2012. 119 (8): 1901-1903. Mutations were found in or near particular residues at the active site: G97D, R100, R132H, R132C, R132S, R132V, R132G, V71I, R132L, and G123R for IDH1, Dang et al, Dang et al, Trends Mol. Med., 2010, 16: 387-397; Ward et al., 2012 and Supplementary Table 2.
[00102] Mutant forms of IDH1 and IDH2 have been shown to have neomorphic activity (gain in function) reducing a-ketoglutarate to 2-hydroxyglutarate. Endogenous production of 2-hydroxyglutarate is enantiospecific resulting in the generation of D-enantiomer (similarly called (R) enantiomer. Normally, cells have low levels of 2-hydroxyglutarate while cells containing IDH1 or IDH2 mutations show significantly high levels of 2hydroxyglutarate. Significantly elevated levels of 2hydroxyglutarate are detected in tumors containing the mutations and in plasma from patients with mutant HDI1 or HDI.2 High levels of 2-hydroxyglutarate are associated with a hypermethylation phenotype resulting in a differentiating block that leads to increased tumorigenesis.
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69/88 [00103] The activity of a specific irreversible covalent inhibitor is defined by its binding to the target (IDH1 or IDH2), defined by Ki, and the maximum potential rate of covalent bond formation, defined by kinact. These two factors are not separate entities, but work closely together to produce the desired covalent bonding effect. This is illustrated by the following 3 points.
[00104] First, the fact that an electrophile, for example, acrylamide, must be positioned correctly relative to a nucleophile, for example, cysteine, is a fundamental component of covalent bond formation in organic chemistry. There is a precise angle and distance at which the nucleophile must approach the electrophile to form the covalent bond. The simple placement of an electrophile close to a nucleophile is not sufficient to form a covalent bond.
[00105] Second, when incorporating a reactive group in a nucleus that contains hydrogen bonding portions to stabilize the inhibitor's binding to the enzyme for example, a guiding nucleus, a skilled technician should consider how the guiding nucleus binds to the target and electrophilic positions relative to the nucleophile taking into account the ideal angle and distance mentioned above. Again, the simple placement of an electrophile close to a nucleophile is not sufficient for covalent bond formation. Changes in the orientation core can impact the ability of an inhibitory compound to form a covalent bond.
[00106] Third, when the above two points are considered together, the mere presence of an electrophilic portion in an orientation nucleus is not sufficient to suggest a covalent bond will be formed.
[00107] The following in vitro and in vivo studies demonstrate that inhibitory activity of mutant IDH1 and IDH2 protein and effectiveness of the tested compounds of Formula I or la against various cell lines
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70/88 specific cancer. These trials are generally recognized by those skilled in the art as indicative of human clinical therapeutic activity. Inhibition of mutant neomorphic IDH1 or IDH2 mutant proteins in the described studies is believed to be effective against other mutant IDH1 and IDH2 neomorphic proteins. Assays showing inhibitory activity of IDH1 or mutant IDH2 and efficacy can be performed substantially as follows or by similar assays providing similar data.
[00108] The results of the following assays demonstrate that the exemplified and tested compounds are useful as inhibitors of mutant IDH1 and IDH2 and may be useful in the treatment of cancers expressing mutant IDH1 or IDH2.
Biochemical Assays for Mutant IDH1 and IDH2 Enzymes [00109] IDH1-R132H, IDH1-R132C, IDH2-R172K and IDH2R140Q mutants catalyze the conversion of aKG to 2HG. 2HG is analyzed using in-line solid phase extraction and mass spectrometry. This analysis is performed on a RapidFire® instrument coupled to a 6460 triple quadrupole mass spectrometer (Agilent G6460A).
[00110] Mutant IDH1 (R132H and R ¹ 32C) and mutant IDH2 (R140Q and R172K) proteins are expressed containing N-terminal His-tag in E.coli and purified using nickel affinity chromatography. Enzyme assays are performed on 96-well base V polypropylene plates containing 100 mM Tris-HCI buffer, 1 mM DTT, 0.005% TRITONTM X-100, 120 mM NaCI. For IDH1 R132H, α-ketoglutarate, NADPH and MnCE are included in final concentrations of 300 μΜ, 2.5 μΜ and 300 μΜ respectively. For IDH1 R132C, α-ketoglutarate, NADPH and MnCE are included in final concentrations of 100 μΜ, 10 μΜ and 100 μΜ respectively. For IDH2 R172K, α-ketoglutarate, NADPH and MnCE are included in concentrations
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71/88 final sections of 150 μΜ, 10 μΜ and 150 μΜ respectively. For IDH2 R140Q, α-ketoglutarate, NADPH and MnCk are included in final concentrations of 3000 μΜ, 10 μΜ and 100 μΜ respectively. Final PH = 7.0. Test compound dissolved in DMSO raw material is diluted in the reaction mixture to a final DMSO concentration of 4%. Compounds are tested in a dose response format. The assay is started by adding an enzyme. Enzymes are used in the following final concentrations: IDH1 R132H, 2 NM; IDH1 R132C, 0.5 nM; IDH2 R172K, 1.2 nM; IDH2 R140Q, 1.2 nM. After 90 minutes the reaction is quenched by adding ACN (50:50) containing 3-hydroxy-1,5-pentanedioic acid-2,2,3,4,4-d5 (5d5-3HG) as an internal standard for analysis of mass spectrometry and quantification of the reaction product. 2-Hydroxyglutarate (2HG) in quenched samples is separated using strong anion exchange column chromatography (Phenomenex Strata-X-A SecurityGuard) and analyzed by mass spectrometry on a 6460 quadrupole triple mass spectrometer (G6460A Agilent). The detected 2HG signal is transformed into a analyzed concentration using a calibration curve generated using known 2HG concentrations. For each compound tested, the% inhibition is calculated using a DMSO control sample as 0% inhibition and no enzyme control as 100% inhibition. IC50 values are obtained from the individual% inhibition values at different compound concentrations using a parameter 4 equation. These calculations are performed using Activity Base (IDBS) or Evaluator (Genedata) data analysis programs.
[00111] The results of this assay demonstrate that the exemplified and tested compounds inhibit mutant IDH1 activity against IDH1 / R132H and IDH1 / R132C and inhibit mutant IDH2 activity against IDH2 / R140Q and IDH2 / R172K.
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72/88 [00112] The following Examples are tested essentially as described above and display activity against mutant IDH1 and mutant IDH2 as shown in Table 14 below.
Table 14
Example # IDH1 / R132H IC50 (μΜ) IDH1 / R132C IC50 (μΜ) IDH2 / R140QIC50 (μΜ) IDH2 / R172KIC50 (μΜ) 1 0.00569 + 0.000766, n = 3, 0.00431 + 0.000895, n = 3, 0.113 0.0328 2 0.00627 + 0.00127, n = 3, 0.00371 + 0.00126, n = 3, 0.0369 0.0115 3 0.0111 +0.0035, n = 2, 0.0156 + 0.0075, n = 2, 0.0648 0.0156 4 0.0137 +0.0030, n = 2, 0.00869 + 0.00280, n = 2, 0.0811 0.028 5 0.00276 0.00249 6 0.00491 0.00505 7 0.00638 <0.00508 0.0743 0.0224 8 <0.00508 <0.00508 0.046 0.017 9 0.00743 +0.00188, n = 2, 0.0151 + 0.0024, n = 2, 10 0.0139 +0.0017, n = 2, 0.0165 + 0.0019, n = 2, 11 0.00978 + 0.00038, n = 2, 0.0156 + 0.0034, n = 2, 0.0543 0.02 12 0.0294 +0.0331, n = 2, 0.0.0390 + 0.0629, n = 2, 0.134 0.0263 13 0.0124 +0.0105, n = 2, 0.0166 + 0.0055, n = 2, 0.239 0.039 14 0.0190 +0.0224, n = 2, 0.0186 + 0.0176, n = 2, 0.746 0.162
Standard deviation Mean ± from the mean.
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Biochemical Assays for IDH1 and IDH2 wild type enzymes [00113] IDH1 and IDH2 enzymes catalyze the conversion of isocitrate to aKG.
[00114] IDH1 wild-type proteins (National Center for Biotechnology Information, Accession: NP_001269316,1) and IDH2 (National Center for Biotechnology Information, Accession: EAX02082.1) containing His terminal N-tag in E. coli and purified using chromatography nickel affinity. Enzyme assays are performed on 96-well base V polypropylene plates containing 100 mM Tris-HCI buffer in pH 7.5, 1 mM DTT, 0.005% TRITONTM X-100, 120 mM NaCI. For the wild type test isocitrate IDH1, NADP ⁺ and MnCE are included in the concentrations of 85 μΜ, 50 μυΜ and 20 μΜ respectively. For the wild type test isocitrate IDH2, NADP ⁺ and MnCI ² are included in the concentrations of 30 μΜ, 50 μΜ and 10 μΜ respectively. Inhibitors dissolved in a DMSO raw material solution are diluted in the reaction mixture to a final 4% DMSO concentration. The enzyme assay is terminated (quenched) by adding ACN (50:50) containing d6-2-ketopentanedioic acid (d6-aKG) as an internal standard for mass spectrometry analysis. Ten microliters of reaction mixture are combined with 100 μΙ_ of water, 50 μΙ_ of 1 M Obenzylhydroxylamine in pyridine buffer (8.6% pyridine, pH 5), and 50 μΙ_ of 1 M N- hydrochloride (3- dimethylaminopropyl) -N-ethylcarbodiimide (EDC) in pyridine buffer. Following derivatization at room temperature for one hour, samples are extracted with 600 μΙ_ of EtOAc. Four hundred μΙ_ of the top layer is removed, dried under heated nitrogen and reconstituted with 100 μΙ_ of MeOH / water (1: 1). Ten μΙ_ of derivatized sample are injected into an LCMS system consisting of a Shimadzu Proeminence 20A HPLC system and a The Thermo Quantum triple quadrupole mass spectrometer
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Ultra ™. Analyzes are separated on a Waters XBridge ™ C18 column (2.1 x 50 mm, 3.5 pm) with a flow rate of 0.6 mL / minute. Mobile phase A is 0.1% formic acid in water and mobile phase B is MeOH. The detected aKG signal is transformed into the analyzed concentration using a calibration curve generated using known aKG concentrations. For each compound tested, the% inhibition is calculated using a DMSO control sample as 0% inhibition is calculated and without enzyme control as 100% inhibition. IC50 values are obtained from the individual% inhibition values at different compound concentrations using a parameter 4 equation. These calculations are performed using Activity Base (IDBS) or Screener (Genedata) data analysis programs.
[00115] The results of this assay demonstrate that the exemplified and tested compounds are less active in inhibiting the wild type enzyme IDH1 compared to the enzymes of IDH1 R132H or mutant R132C and less active in inhibiting the wild type enzyme IDH2 compared to the enzymes of HDI R140Q or R172K mutant.
[00116] The following Examples in Table 15 are tested essentially as described above and are less active in inhibiting wild type enzymes compared to mutant enzymes.
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Table 15
Example # IDH1 wild type IC50 (μΜ) IDH2 wild type IC50 (pM) 1 0.0854 ± 0.107, n = 2, 0.801 ± 0.745, n = 2, 2 0.105 ± 0.113, n = 2, 0.884 ± 0.748, n = 2, 3 0.425 3.37 4 0.302 3.7 6 0.0549 0.493 7 0.233 1.53 8 0.237 1.62 11 0.345 2.08 12 0.277 3.22 13 0.456 7.03 14 0.392 7.4
Biochemical Jump Dilution Assay IDH1 (R132H) [00117] Lyophilized Example Compounds are reconstituted in 10 mM or 100 mM with 100% DMSO and kept at room temperature until testing. IDH1 (R132H) -His protein is expressed and purified by well-known methods and generally used by those skilled in the art. The assay reagents included the following: α-ketoglutaric acid (Sigma Cat # K1875), MnCE - Fisher Scientific Cat # M87-100, NADPH - Sigma-Aldrich Cat # N7505, Tris-HCI (Invitrogen, Cat # 15567-027) , NaCI (Sigma, S3014), dithiothreitol (Sigma, D5545), and TRITON ™ X100 (Peirce, 28314). The H-Glo ™ NAD (P) kit from Promega (G9061).
[00118] The assay buffer used throughout contains 100 mM Tris-HCI pH 7.0, 120 mM NaCI, 1 mM DTT, 0.005%
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TRITON ™ X-100, and 2% DMSO (from the addition of test compound). The IC50 of each compound is determined by incubating a dose response of the compound, prepared in an Echo555, with 1.5 nM IDH1 (R132H), 1 mM a-ketoglutarate, 1 mM MnCE, and 15 μΜ NADPH in assay buffer. The reaction is incubated for 2 hours at room temperature, then stopped using 6-cyclopropyl-5 (isoquinolin-5-yl) -2 - [(3R) -4- (3-methoxypropanoyl) -3-methylpiperazin-1yl] pyridine -3-carbonitrile (10 μΜ). NADPH concentrations are measured using the H-Glo ™ NAD (P) Kit, as specified by the vendor. The luminescent signal is read on the Envision (Perkin Elmer; 0.1 sec / Luminescense Mirror / Lum700 WL400-700 filter). In the subsequent hop dilution experiment, a compound concentration equivalent to 10x 0 IC50 is pre-incubated with 100 nM IDH1 (R132H). The concentration of the compound is always greater than or equal to the concentration of the enzyme. After two hours at room temperature, this mixture is diluted 1: 100 in a solution containing α-ketoglutarate (10 mM), MnCE (10 mM), and NADPH (15 μΜ). This final enzyme reaction contains 1 nM of IDH1 (R132H) and 0.1 χ [ICso]. After a two hour incubation at room temperature, the concentration of NADPH was measured as specified above using 6-cyclopropyl-5 (isoquinolin-5-yl) -2 - [(3R) -4- (3-methoxypropanoyl) -3- methylpiperazin-1yl] pyridine-3-carbonitrile and the NAD (P) H-Glo ™ kit. Three controls are included: 1) ΊΟχ Control containing WxICso compound in the preincubation and enzyme assay except 1 mM a-ketoglutarate, 1 mM MnCE, and 15 μΜ NADPH is used in the final assay measuring enzyme activity, 2) Control of Maximum activity containing DMSO in place of the compound for preincubation and enzyme assay, and 3) 0.1 χ Control containing DMSO in place of compound in the preincubation and 0.1xLC50 compound in the enzyme assay. A Minimal Activity Control missing enzyme, but otherwise equated
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77/88 valiant to Maximum Activity Control is included. A second group of Maximum and Minimum Activity Control is performed using 1 mM of o-ketoglutarate, 1 mM of MnCE, and 15 μΜ of NADPH. Each test condition is tested in triplicate and 32 replicates are performed for Maximum Activity Control (10 mM) and Minimum Activity Control (10 mM) while 16 replicates are performed for Maximum Activity Control (1 mM) and Control of Minimum Activity (1 mM).
[00119] The concentration of NADP (product) produced in each experiment / control is determined using the percentage decrease in the observed signal relative to the Minimum Activity Control, containing 15 μΜ NADPH. The Minimum Activity Control (1 mM and 10 mM) and the Maximum Activity Control (1 mM and 10 mM) are dimensioned and the standard deviation calculated for each. The signal for each jump dilution and for the 0.1 χ Controls are multiplied by 15 then divided by the average accounts for the Minimum Activity Control wells (10 mM). This number is subtracted from 15 to calculate NADP (μΜ Product). The same calculations are used for the 10x Controls but the minimum activity controls (1 mM) are used. The pmols of the product for the maximum Activity controls (1 mM and 10 mM) are calculated by multiplying the average accounts by 15 then dividing by the respective Minimum Activity Control (1 mM and 10 mM). Ο μΜ NADP for each well is divided by the Average Maximum Activity Control (1 mM or 10 mM) then multiplied by 100 to determine% HDI Activity for the compound jump dilution, 10x Control, and 0.1 χ Control. A pass-through compound should show <30% activity for the 10x control-showing that the pre-incubation concentration is sufficient to saturate the enzyme with the compound. In addition, the compound should show> 70-80%
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78/88 activity for the 0.1 χ control confirming that there is no inhibition in the concentration of compound 0.1 x / diluted.
[00120] Compounds of the examples are tested essentially as described above and exhibits% recovery data for IDH1 / R132H in this assay. Exemplified and tested compounds of the present invention inhibit the enzyme two hours after dilution as opposed to compound (s) of the technique that did not inhibit the enzyme two hours after dilution with% recovery. Data from this assay demonstrate that the tested compounds of the present invention act in a manner consistent with covalent inhibition of mutant HDI1 since dilution of the inhibitor does not result in recovery of enzyme activity.
Cell-based assays for Mutant IDH1 Inhibitors [00121] To test cell inhibition of mutant IDH1 R132C, the fibrosarcoma cell line HT1080 (purchased from ATCC) is used. To test cell-based inhibition of the R132H mutation, the U87MG glioma cell line (ATCC) was stably transfected with a DNA construct expressing the mutant R132H enzyme by methods well known and commonly used by those skilled in the art.
HT1080 Cell Assay:
[00122] Fifteen thousand cells are seeded in 96-well plates coated with poly-D-lys (15,000 cells / well) 18-24 hours before treatment with compounds. Four hours prior to compounding, cells are deprived of glutamine by removing normal media and replacing with glutamine-free media. Following deprivation, cells are then treated with different concentrations of test compounds (20 μΜ to 1 nM; or 0.2 μΜ to 0.01 nM) dissolved in glutamine-free media containing DMSO in a final concentration of 0.2% . The initial compound incubation is for one hour at
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37 ° C / 5% CO2. After one hour, glutamine is added in a final concentration of 2 mM and the treated cells are then incubated for another 18 hours at 37 ° C / 5% CO2. Following the 18-hour incubation, 2HG and aKG intracellular are analyzed in cell lysates. Lysates are prepared by removing media and adding buffer containing 25 mM Tris-HCI pH 7.5, 150 mM NaCI, 1 mM EDTA, 1 mM EGTA / 1% TRITON ^TM- X 100 to the cells. An aliquot of lysate is added to a mixture of deaKG and dõ-3HG as internal standards and the mixture is treated with Obenzylhydroxylamine in the presence of N- (3dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (EDC) and pyridine. Analyzed derivatives are then extracted with EtOAc, dried, and then reconstituted with 50% MeOH in H ₂ O. Samples prepared as described are injected on the HPLC to separate 2HG and aKG derivatives (and corresponding internal standards) using chromatography reverse phase in a C18 column. Sample analysis is performed using a 6460 quadrupole triple mass spectrometer (Agilent G6460A). Detected 2HG and aKG signals are transformed into analyzed concentration using the aKG / de-aKG ratio and the 2HG / d5-3HG ratio that is extrapolated within a calibration curve. Percent inhibition for each individual sample is obtained after normalizing the concentration of 2HG or aKG calculated on maximum and minimum references obtained in the presence and absence of glutamine during cell treatment with compounds. IC50 values are obtained from% individual inhibition using a sigmoidal dose response parameter 4 equation. These calculations are performed automatically using Activity Base (IDBS) or Screener (Genedata) data analysis programs.
[00123] The results of this test demonstrate that the Examples tested in Table 15 inhibit 2-hydroxyglutarate production, indican
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80/88 inhibited R132C mutant IDH1 in cells in this assay. oKG, a metabolite generated by wild-type IDH1 is unaffected by the inhibitors, indicating that the compounds are selective for mutant IDH1 in wild-type IDH1 in cells in this assay. The resulting IC50 values for the following Examples are shown in Table 16. For those assays where the inhibition curve did not reach 50%, the highest tested concentration is shown (for example IC50> 20 μΜ or> 0.2 μΜ) .
Table 16
Example # HT1080 (R132C, 2hydroxyglutarate) IC50 (μΜ) HT1080 (R132C, aKG) ICso (μΜ) 1 0.000698 1 0.000352, n = 7, > 20.0 2 0.0012810.00100, n = 8, > 20.0 3 0.00127 ± 0.00044, n = 2, > 0.200 4 0.00334 ± 0.00140, n = 2, > 0.200 5 0.000623 1 0.000745, n = 2, > 20.0 6 0.0011210,00052, n = 4, 19.2 7 0.00062510,000182, n = 2, > 0.200 8 0.00077510,0000981, n = 2, > 0.200 9 0.00275 1 0.00046, n = 3, > 20.0 10 0.00391 1 0.00259, n = 3, > 20.0 11 0.001041 0.00050, n = 4, > 20.0 12 0.00272 1 0.00330, n = 4, > 20.0 13 0.00098710.000009, n = 2, > 20.0 14 0.0013810,00015 n = 3 > 20.0
[00124] Mean ± standard deviation from the mean.
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U87MG / IDH1R132H Cell Assay [00125] Cells are seeded in 96-well plates coated with poly-D-lys (12,000 cells / well) 18-24 hours prior to compound treatment. Four hours before the treatment compound, cells are deprived of glutamine by removing normal media and replacing it with free glutamine media. Following deprivation, cells are then treated with different concentrations of test compounds (20 μΜ at 1 nM) dissolved in glutamine-free media containing DMSO in a final concentration of 0.2%. The incubation of the starting compound is for one hour at 37 ° C / 5% CO2. After one hour, glutamine is added in a final concentration of 2 mM and the treated cells are then incubated for an additional 18 hours at 37 ° C / 5% CO2. Intracellular 2HG is analyzed in Cell Uses obtained after media removal and Use buffer treatment (25 mM Tris-HCI pH 7.5, 150 mM NaCI, 1 mM EDTA, 1 mM EGTA / 1% TRITON ^TM -X 100). Cell lysates are stored at -80 ^Q C until processing. For sample extraction, an aliquot of thawed lysate is transferred to a 96-well plate and treated with cold MeOH containing dõ-3HG as an internal standard followed by chloroform and H ₂ O (1: 4: 3: 2). The upper phase is collected after separation and injected on HPLC to separate 2HG (and internal standard) using hydrophilic interaction chromatography (HILIC) coupled to detection of MS / MS on a 6460 quadrupole triple mass spectrometer. Percent inhibition for each individual sample it is obtained after normalizing 2HG concentration calculated on maximum and minimum references obtained in the presence and absence of glutamine during cell treatment with compounds. IC50 values obtained from% of individual inhibition using a sigmoidal dose response parameter 4 equation. These calcu
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82/88 they are performed automatically using Activity Base (IDBS) or Screener (Genedata) data analysis programs.
[00126] The following Examples are tested essentially as described above and display inhibiting activity against mutant IDH1 / R132H in U87MG cells in this assay as shown in Table 17 below.
Table 17
Example # U87MG (IDH1 / R132H 2hydroxyglutarate IC50 (μΜ) 1 0.000209 ± 0.000100, n = 6, 2 0.000406 ± 0.000375, n = 8, 3 0.000426 ± 0.000188, n = 2, 4 0.000805 ± 0.000187, n = 2, 5 0.000295 ± 0.00200, n = 2, 6 0.000379 ± 0.000202, n = 2, 7 0.000356 ± 0.000178, n = 2, 8 0.000432 ± 0.000016, n = 2, 9 0.000388 ± 0.000703, n = 2, 10 0.000529 ± 0.000356, n = 2, 11 0.000258 ± 0.000209, n = 3, 12 0.00135 ± 0.00151, n = 4, 13 0.000267 ± 0.000227, n = 2, 14 0.000353 ± 0.000483, n = 2,
Mean + standard deviation from the mean.
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2-Hydroxyglutarate In Vivo Assay [00127] For in vivo testing of IDH1 inhibitors, subcutaneous xenograft tumors are cultured in athymic nude mice (20-22g, Harlan Laboratories) following implantation of HT1080 cells (fibrosarcoma carrying R132C IDH1 mutant) or TB08 cells (secondary glioblastoma carrying mutant R132H IDH1). Mice are fed and watered ad libitum and are acclimatized for a week before cell implantation. Tumor cells (HT1080) or tumor fragments (TB08) are implanted in the right rear flank. For HT1080, 5.0 x 10 ⁶ cells are implanted in a 1: 1 mixture with Matrigel in a final volume of 0.2 ml. For TB08, tumor fragments generated from ex-planted tumor samples are implanted directly in the rear flank. Tumor volumes are measured by calibrator twice a week and tumor volume is calculated using 0.536 χ L x W ² where L = length and W = width. When tumor volumes reach 150-400 mm ³ , animals are randomized, placed in groups (n = 3-6 per group) and dosed with IDH1 inhibitors or vehicle control. For IDH1 inhibitors, compounds are formulated in a vehicle containing either 1% hydroxyethylcellulose / 0.25% Tween ™ 80 / 0.05% antifoam or 10% Acacia with 1.1 mol HCI equivalent. Compounds are sonicated in a bath to obtain suspension. Compounds are dosed on a milligram per kilogram (mpk) basis by oral gavage in a final volume of 0.2 ml. To determine inhibition of 2HG, compounds are dosed twice a day (BID) for 3 days (total number of doses = 6). Following compound treatment, mice are euthanized with isofluorane anesthesia and cervical dislocation. Tumors are cut, placed in labeled tubes, and immediately frozen in liquid nitrogen. Tumors are stored at -80 ° C for processing.
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Preparation of tumor lysates [00128] XY Lite buffer is prepared in molecular grade water and contains the following components: 25 mM Tris, pH 7.5, 150 mM NaCI, 1% TRITON ™ X-100, 1 mM EDTA, 1 mM EGTA. XY Lite (40 ml), 800 pl of Stop Protease and Phosphatase Inhibitor cocktail (Halt ™ Protease and Phosphatase Inhibitor Cocktail, Thermo EDTA-Free Scientific, Cat # 78441) is added. Samples are vortexed and then cooled on ice. Orange cap lysing A tubes are labeled and placed on an ice shelf. A mortar and pestle is placed on dry ice to cool. A 2 χ 2 square inch aluminum foil is placed at the base of the mortar. A tumor sample is transferred to the pre-cooled mortar on the plate. Liquid nitrogen (about 5 ml) is added and allowed to evaporate, freezing the tumor. Another piece of sheet metal is placed over the tumor and the tumor is crushed into small pieces with the ceramic pestle. The crushed tumor is quickly transferred to the lysing tube. XY Lite ice-cold (500 pL) is added to each tube and buffered. Tumors are then processed in the FastPrep-24 MP Biomedicals by rotating twice for 35 seconds at each fixing speed 5. Samples are then centrifuged in Beckman Microfuge R at 4 ° C at 14,000 rpm for 30 minutes. Supernatant is transferred to a 96-well deep pre-cooled plate. The pellet is discarded.
Protein assay [00129] A protein assay dilution plate is generated first by generating XY buffer (145 μΙ) in a 96-well round base granular non-sterile plate. To this, tumor lysate (5 µl) is added and gently mixed. The plate is kept on ice. Serial dilutions of standard BSA (Thermo Scientific cat. 23209 2 mg / mL) are fixed as follows: Five 0.5 mL tubes are placed
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85/88 on a shelf and XY buffer (60 μΙ_) is added to each. Raw material BSA (60 μΙ) is added to the first tube and vortexed. 60 μΙ of the first tube is transferred to the next tube, vortexed, and so on, until the dilution series are completed as follows: Tube 1 = raw material BSA, Tubes 2-5 are 1: 2 serial dilutions, Tube 6 = XY buffer only. Thermo BCA Protein Assay reagents are mixed according to the manufacturer's instructions. Mixed BCA reagent (200 μΙ) is added to each sample and incubated for 15 minutes. The results of the protein assay are read in SOFTmax Pro Plate Reader. Based on protein assay results, the appropriate amount of XY buffer is added to each tumor lysate to generate a final protein concentration of 5 mg / ml. All samples are labeled and stored at -80 ° C.
Metabolite Analysis in Tumor Lysates [00130] The in vivo effects of IDH1 inhibition at 2HG and total aKG concentrations are determined by liquid chromatography-mass spectrometry (LC-MS) analysis of tumor xenografts. The method uses derivatization with O-benzylhydroxylamine before analysis by LC-MS. Ten microliters of each tumor lysate is placed in a 96-well deep plate and combined with 100 pL of internal standard solution containing 10 μΜ ds-SHG and 10 μΜ de-aKG. 50 pL of 1 M O-benzylhydroxylamine in pyridine buffer (8.6% pyridine, pH 5) and 50 pL of 1 M N- (3-dimethylaminopropyl) -Netylcarbodiimide hydrochloride (EDC) in pyridine is added to each sample. The derivatization reaction proceeds at room temperature for one hour. Using a Beckman Biomek FX 600 pL liquid EtOAc handler is added to each sample. Plates are sealed and vortexed for 5 minutes, then they are centrifuged for 5 minutes at 4000 rpm in an Eppendorf 5810R centrifuge. 400
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86/88 μΙ_ of the top layer is transferred to a new 96-well plate. Samples are dried under nitrogen heated to 50 ° C and reconstituted with 100 μΙ_ MeOH / water (1: 1). A microliter of the derivatized sample is injected into an LC-MS system consisting of a Shimadzu Prominence 20A HPLC system and a Thermo Quantum Ultra ™ triple quadrupole mass spectrometer. Analyzes are separated on a Water XBridge ™ C18 column (2.1 x 50 mm, 3.5 pm) with a flow rate of 0.6 mL / minute. Mobile phase A is 0.1% formic acid in water and mobile phase B is MeOH. The gradient profile is: 0 minutes, 5% B; 3 minutes, 100% B; 4.00 minutes, 100% B; 4.1 minutes, 5% B; 5.50 minutes, stop. The mass spectrometer uses a HESI-II probe operated in a reaction monitoring mode selected by positive ion. Calibration curves are constructed by plotting concentrations of analyzed vs. peak area relationships of the analyzed / internal standard and performing a quadratic adjustment of the data using a 1 / concentration weighing with Xcalibur ™ software. Analyte concentrations for the unknowns are recalculated from the calibration curves. Metabolite data from the LC-MS assay is expressed in nmol / mg protein. The average 2HG level in the vehicle treated group is used to determine the 0% inhibition control. The% inhibition in each animal treated by inhibitor is then determined relative to vehicle control. Data are analyzed in JMP software to determine the mean% inhibition in each dose group, the standard deviation, and the standard error.
[00131] Data demonstrating in vivo inhibition of 2-hydroxyglutarate in mutant IDH1 xenograft mice by exemplified compounds and tested compounds is shown in Table 18 below.
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Table 18
Xenograft model Treatment or Ex N ^Q. Dose Mice(n) 2HG,% Average Inhibition DetourPattern Mean standard error TB08 (R132H) Vehicle 0 mpk 5 0 30.3 13.55 TB08 (R132H) 1 1 mpk 5 21.4 21 9.38 TB08 (R132H) 1 2 mpk 5 16.1 30.3 13.6 TB08 (R132H) 1 4 mpk 5 41.5 26 11.6 TB08 (R132H) 1 8 mpk 5 78.8 1.8 0.8 TB08 (R132H) 1 16 mpk 5 92.1 1.8 0.8 TB08 (R132H) 1 32 mpk 5 95.8 0.7 0.3 Xenograft model Treatment or Ex N ^Q. Dose Number of Mice 2HG, Average Inhibition% DetourPattern Standard error Medium TB08 (R132H) Vehicle 0 mpk 5 0 39.2 17.5 TB08 (R132H) 2 1 mpk 5 37.4 13.3 5.9 TB08 (R132H) 2 2 mpk 5 23.9 16.2 7.2 TB08 (R132H) 2 4 mpk 5 63.7 12.1 5.4 TB08 (R132H) 2 8 mpk 5 80.95 6.27 2.8 TB08 (R132H) 2 16 mpk 5 92.97 2.63 1.1 TB08 (R132H) 2 32 mpk 5 96.88 0.68 0.3 Xenograft model Treatment or Ex N ^Q. Dose Number of Mice 2HG, Average Inhibition% DetourPattern Mean standard error TB08 (R132H) Vehicle 0.00 mpk 5 0 24.4 10.9 TB08 (R132H) 8 10.0 mpk 5 60.36 10.06 4.5 TB08 (R132H) 7 10.0 mpk 5 69.56 9.15 4.1 TB08 (R132H) 4 10.0 mpk 5 61.82 14.4 6.4 TB08 (R132H) 3 10.0 mpk 5 87.26 3.95 1.77 TB08 (R132H) 14 10.0 mpk 5 86.71 5.27 2.36
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Xenograft model Treatment or Ex N ^Q. Dose Number of Mice 2HG, Average Inhibition% DetourPattern Mean standard error TB08 (R132H) Vehicle 0.00 mpk 5 0 26.87 12.02 TB08 (R132H) 11 10.0 mpk 5 90.63 4.5 2.01 Xenograft model Treatment or Ex N ^Q. Dose Number of Mice 2HG, Average Inhibition% DetourPattern Mean standard error TB08 (R132H) Vehicle 0.00 mpk 5 0 39.6 17.7 TB08 (R132H) 13 10.0 mpk 5 86.3 3.7 1.67 TB08 (R132H) 12 10.0 mpk 5 94.16 0.66 0.3
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权利要求:
Claims (11)
[1]
1. Compound, characterized by the fact that it is of the Formula:

on what:
R ¹ is -CH ₂ CH (CH ₃ ) 2, -CH ₂ CH ₃ , -CH ₂ CH ₂ OCH ₃ , or -CH ₂ cyclopropyl;
R ² is -CH ₃ or -CH ₂ CH ₃ ; or a pharmaceutically acceptable salt thereof.
[2]
2. Compound, according to claim 1, characterized by the fact that it is:
7 - [[(1 S) -1 - [4 - [(1 R) -2-Cyclopropyl-1 - (4-prop-2-enoylpiperazin-1 -yl) ethyl] phenyl] ethyl] amino] -1 - ethi-4H-pyramid [4,5-d] [1,3] oxazin-2-one;
7 - [[(1 S) -1 - [4 - [(1 S) -2-cyclopropyl-1 - (4-prop-2-enoylpiperazin-1 -yl) ethyl] phenyl] ethyl] amino] -1 - ethi-4H-pyramid [4,5-d] [1,3] 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] [1,3] 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] [1,3] oxazin-2-one, isomer 2;
or a pharmaceutically acceptable salt of either.
[3]
3. Compound according to claim 2, characterized by the fact that it is 7 - [[(1S) -1- [4 - [(1S) -2-cyclopropyl-1- (4-prop-2enoylpiperazin-1 - il) ethyl] phenyl] ethyl] amino] -1-ethyl-4H-pyrimido [4,5-
d] [1,3] oxazin-2-one or a pharmaceutically acceptable salt thereof.
[4]
4. Pharmaceutical composition characterized by the fact that it comprises a compound, as defined in claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
Petition 870190044593, of 05/13/2019, p. 97/115
2/3
[5]
5. Method of treating a cancer expressing mutant IDH1 or mutant IDH2 in which it is glioma, glioblastoma, glioblastoma multiforme, astrocytomas, oligodendrogliomas, paraganglioma, fibrosarcoma, angioimmunoblastic T cell lymphoma (AITL), myelodysplastic leukemia syndrome (MDL) B cell (B-ALL), thyroid cancer, colorectal cancer, acute myeloid leukemia (AML), melanoma, prostate cancer, chondrosarcoma or cholangiocarcinoma in a patient, characterized by the fact that it comprises administering to a patient in need of it a therapeutically effective amount of a compound, as defined in any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof.
[6]
6. Method according to claim 5, characterized by the fact that the cancer expressing mutant IDH1 or mutant IDH2 is fibrosarcoma, acute myeloid leukemia, glioma or glioblastoma.
[7]
A compound according to claim 1, or a pharmaceutically acceptable salt thereof, characterized by the fact that it is for use in therapy.
[8]
A compound according to 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 characterized by the fact that it is glioma, glioblastoma, glioblastoma multiforme, astrocytomas , oligodendrogliomas, paraganglioma, fibrosarcoma, angioimmunoblastic T-cell lymphoma (AITL), myelodysplastic syndrome (MDS), acute B-cell lymphoblastic leukemia (B-ALL), thyroid cancer, colorectal cancer, acute myeloid leukemia, AML, melanoma (AML) prostate cancer, chondrosarcoma or cholangiocarcinoma.
Petition 870190044593, of 05/13/2019, p. 98/115
3/3
[9]
9. Use according to claim 8, characterized by the fact that the cancer expressing mutant IDH1 or mutant IDH2 is fibrosarcoma, acute myeloid leukemia, glioma, or glioblastoma.
[10]
10. Use of a compound, as defined in any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, characterized by the fact that it is for the manufacture of a medicament for the treatment of a cancer expressing mutant IDH1 or mutant IDH2 in which it is glioma, glioblastoma, glioblastoma multiforme, astrocytomas, oligodendrogliomas, paraganglioma, fibrosarcoma, angioimmunoblastic T cell lymphoma (AITL), myelodysplastic syndrome (MDS), B-cell cancer, B-cell acute lymphoblastic leukemia, B-cell cancer colorectal cancer, acute myeloid leukemia (AML), melanoma, prostate cancer, chondrosarcoma or cholangiocarcinoma.
[11]
11. Use according to claim 10, characterized by the fact that the cancer expressing mutant IDH1 or mutant IDH2 is fibrosarcoma, acute myeloid leukemia, glioma, or glioblastoma.

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法律状态:
2021-10-05| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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2022-02-08| B06W| Patent application suspended after preliminary examination (for patents with searches from other patent authorities) chapter 6.23 patent gazette]|

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