Therapeutically active compounds and methods of use thereof

Abstract

Provided are compounds useful for the treatment of cancer and methods of treating cancer comprising administering to a subject in need of cancer treatment a purified compound described herein.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 149,075, filed February 12, 2021, and U.S. Provisional Application No. 63 / 217,843, filed July 2, 2021, the contents of which are incorporated herein by reference in their entireties.

[0002] 2. Background of the Invention Isocitrate dehydrogenases (IDHs) catalyze the oxidative decarboxylation of isocitrates to 2-oxoglutarate (i.e., α-ketoglutarate). These enzymes belong to two distinct subclasses; one of them utilizes NAD(+) as the electron acceptor, and the other utilizes NADP(+). Five isocitrate dehydrogenases have been described; three are NAD(+)-dependent isocitrate dehydrogenases localized in the mitochondrial matrix, and two are NADP(+)-dependent isocitrate dehydrogenases; one is located in the mitochondria and the other is located primarily in the cytosol. Each NADP(+)-dependent isozyme is a homodimer.

[0003] IDH1 (Isocitrate dehydrogenase 1 (NADP+), cytosol) is also known as IDH; IDP; IDCD; IDPC or PICD. The protein encoded by this gene is an NADP(+)-dependent isocitrate dehydrogenase found in the cytoplasm and peroxisomes. This protein contains the PTS-1 peroxisomal targeting signal sequence. The presence of this enzyme in peroxisomes suggests a role in intraperoxisomal reduction, e.g., regeneration of NADPH for the conversion of 2,4-dienoyl-CoA to 3-enoyl-CoA, and in the peroxisomal reaction consuming 2-oxoglutarate, i.e., α-hydroxylation of phytanic acid. The cytoplasmic enzyme plays an important role in NADPH production in the cytoplasm.

[0004] The human IDH1 gene encodes a protein of 414 amino acids. The nucleotide and amino acid sequences for human IDH1 can be found in GenBank entries NM_005896.2 and NP_005887.2, respectively. The nucleotide and amino acid sequences for IDH1 are also described in, for example, Nekrutenko et al., Mol. Biol. Evol. 15:1674-1684(1998); Geisbrecht et al., J. Biol. Chem. 274:30527-30533(1999); Wiemann et al., Genome Res. 11:422-435(2001); The MGC Project Team, Genome Res. 14:2121-2127(2004); Lubec et al., Submitted (DEC-2008) to UniProtKB; Kullmann et al., Submitted (JUN-1996) to the EMBL / GenBank / DDBJ databases, and Sjoeblom et al., Science 314:268-274(2006).

[0005] A non-mutated, e.g., wild-type, IDH1, for example, catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate in the forward reaction, thereby reducing NAD + (NADP + ) to NADH (NADPH). Isocitrate + NAD + (NADP + ) → α-KG + CO2 + NADH (NADPH) + H +

[0006] It has been discovered that mutations in IDH1 present in certain cancer cells result in the enzyme's de novo ability to catalyze the NADPH-dependent reduction of α-ketoglutarate to R(-)-2-hydroxyglutarate (2HG), the production of which is believed to contribute to the formation and progression of cancer (Dang, L et al., Nature 2009, 462:739-44).

[0007] IDH2 (isocitrate dehydrogenase 2 (NADP+), mitochondrial) is also known as IDH; IDP; IDHM; IDPM; ICD-M or mNADP-IDH. The protein encoded by this gene is a NADP(+)-dependent isocitrate dehydrogenase found in mitochondria. This protein plays a role in intermediate metabolism and energy production. This protein may tightly associate or interact with the pyruvate dehydrogenase complex. The human IDH2 gene encodes a protein of 452 amino acids. The nucleotide and amino acid sequences for IDH2 can be found as GenBank entries NM_002168.2 and NP_002159.2, respectively. The nucleotide and amino acid sequences for human IDH2 are also described in, for example, Huh et al., Submitted (NOV-1992) to the EMBL / GenBank / DDBJ databases and The MGC Project Team, Genome Res. 14:2121-2127(2004).

[0008] A non-mutated, e.g., wild-type, IDH2, for example, catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG) in the forward reaction, thereby reducing NAD + (NADP + ) to NADH (NADPH). Isocitrate + NAD + (NADP + ) → α-KG + CO2 + NADH (NADPH) + H +

[0009] It has been discovered that mutations in IDH2 present in certain cancer cells result in the enzyme's new ability to catalyze the NADPH-dependent reduction of α-ketoglutarate to R(-)-2-hydroxyglutarate (2HG), which is not formed by wild-type IDH2. The production of R(-)-2-hydroxyglutarate (2HG) is thought to contribute to the formation and progression of cancer (Dang, L et al., Nature 2009, 462:739-44). Thus, inhibition of mutant IDH1 and / or mutant IDH2 and their new activity may be a therapeutic treatment for cancer.

[0010] Vorasidenib (AG-881) is an orally bioavailable, brain-penetrant, second-generation double mutant isocitrate dehydrogenase 1 and 2 (mIDH1 / 2) inhibitor currently in clinical trials for the treatment of gliomas, including low-grade gliomas. [ka]

[0011] Vorasidenib (AG-881) is described in US Pat. No. 9,579,324, which is incorporated herein by reference in its entirety. To better understand the clinical activity of vorasidenib and provide potential novel inhibitors of mutant IDH1 / IDH2, it is necessary to identify potential biologically active metabolites of vorasidenib that remain in the human body upon administration of vorasidenib.

[0012] Summary of the Invention Described herein are Compounds 1-7 (eg, Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, and Compound 7) and pharma- ceutically acceptable salts thereof. [ka]

[0013] At least one of mutant IDH1 or mutant IDH2 is inhibited by a compound selected from compounds 1-7 or a pharmaceutical salt thereof, or as described in any one of the embodiments herein. Also described herein are pharmaceutical compositions comprising a compound selected from compounds 1-7 or a pharmaceutical salt thereof, and methods of using such compositions to treat cancers characterized by the presence of at least one of mutant IDH1 or mutant IDH2.

[0014] In one embodiment, the compound is in purified form. [ka] or a pharma- ceutically acceptable salt thereof.

[0015] In one embodiment, the compound is in purified form. [ka] or a pharma- ceutically acceptable salt thereof.

[0016] In one embodiment, the compound is in purified form. [ka] or a pharma- ceutically acceptable salt thereof.

[0017] In one embodiment, the compound is in purified form. [ka] or a pharma- ceutically acceptable salt thereof.

[0018] In one embodiment, the compound is in purified form. [ka] or a pharma- ceutically acceptable salt thereof.

[0019] In one embodiment, the compound is in purified form. [ka] or a pharma- ceutically acceptable salt thereof.

[0020] In one embodiment, the compound is in purified form. [ka] or a pharma- ceutically acceptable salt thereof.

[0021] In one aspect, the present invention provides a pharmaceutical composition comprising one or more compounds selected from Compounds 1 to 7 or pharma- ceutically acceptable salts thereof, and one or more pharma- ceutically acceptable excipients.

[0022] In one embodiment, the pharmaceutical composition comprises: [ka] or a pharma- ceutically acceptable salt thereof.

[0023] In one embodiment, the pharmaceutical composition comprises: [ka] or a pharma- ceutically acceptable salt thereof.

[0024] In one embodiment, the pharmaceutical composition comprises: [ka] or a pharma- ceutically acceptable salt thereof.

[0025] In one embodiment, the pharmaceutical composition comprises: [ka] or a pharma- ceutically acceptable salt thereof.

[0026] In one embodiment, the pharmaceutical composition comprises: [ka] or a pharma- ceutically acceptable salt thereof.

[0027] In one embodiment, the pharmaceutical composition comprises: [ka] or a pharma- ceutically acceptable salt thereof.

[0028] In one embodiment, the pharmaceutical composition comprises: [ka] or a pharma- ceutically acceptable salt thereof.

[0029] In one aspect, the present invention provides a method of treating cancer, comprising administering a therapeutically effective amount of one or more purified compounds selected from Compounds 1-7, or a pharma- ceutically acceptable salt thereof, to a patient in need of such treatment, wherein the cancer is characterized by the presence of at least one mutation selected from an IDH1 mutation or an IDH2 mutation.

[0030] In one embodiment, the method comprises administering a therapeutically effective amount of purified [ka] or a pharma- ceutically acceptable salt thereof to said patient.

[0031] In one embodiment, the method comprises administering a therapeutically effective amount of purified [ka] or a pharma- ceutically acceptable salt thereof to said patient.

[0032] In one embodiment, the method comprises administering a therapeutically effective amount of purified [ka] or a pharma- ceutically acceptable salt thereof to said patient.

[0033] In one embodiment, the method comprises administering a therapeutically effective amount of purified [ka] or a pharma- ceutically acceptable salt thereof to said patient.

[0034] In one embodiment, the method comprises administering a therapeutically effective amount of purified [ka] or a pharma- ceutically acceptable salt thereof to said patient.

[0035] In one embodiment, the method comprises administering a therapeutically effective amount of purified [ka] or a pharma- ceutically acceptable salt thereof to said patient.

[0036] In one embodiment, the method comprises administering a therapeutically effective amount of purified [ka] or a pharma- ceutically acceptable salt thereof to said patient.

[0037] In one aspect, the invention provides a method of treating cancer comprising administering to a patient in need of cancer treatment a therapeutically effective amount of a composition comprising one or more purified compounds selected from Compounds 1-7, or pharma- ceutically acceptable salts thereof, wherein the cancer is characterized by the presence of at least one mutation selected from an IDH1 mutation or an IDH2 mutation.

[0038] In one embodiment of the methods described herein, the cancer is selected from glioma (including low grade glioma), glioblastoma (including secondary glioblastoma), grade II or III astrocytoma, grade II or III oligodendroglioma, acute myeloid leukemia (AML), sarcoma, melanoma, non-small cell lung cancer (NSCLC), cholangiocarcinoma, chondrosarcoma, myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), colon cancer, and angioimmunoblastic non-Hodgkin's lymphoma (NHL) in the patient.

[0039] In one embodiment of the methods described herein, the cancer is a glioma. In a further embodiment, the glioma is a low-grade glioma or a high-grade glioma.

[0040] In one embodiment, a method of treating glioma in a subject in need thereof is provided, comprising administering to the subject a therapeutically effective amount of one or more purified compounds selected from Compounds 1-7 or a pharma- ceutically acceptable salt thereof.

[0041] In one embodiment, the method of treating glioma comprises administering a therapeutically effective amount of purified [ka] or a pharma- ceutically acceptable salt thereof to a subject in need of treatment for glioma.

[0042] In one embodiment, the method of treating glioma comprises administering a therapeutically effective amount of purified [ka] or a pharma- ceutically acceptable salt thereof to a subject in need of treatment for glioma.

[0043] In one embodiment, the method of treating glioma comprises administering a therapeutically effective amount of purified [ka] or a pharma- ceutically acceptable salt thereof to a subject in need of treatment for glioma.

[0044] In one embodiment, the method of treating glioma comprises administering a therapeutically effective amount of purified [ka] or a pharma- ceutically acceptable salt thereof to a subject in need of treatment for glioma.

[0045] In one embodiment, the method of treating glioma comprises administering a therapeutically effective amount of purified [ka] or a pharma- ceutically acceptable salt thereof to a subject in need of treatment for glioma.

[0046] In one embodiment, the method of treating glioma comprises administering a therapeutically effective amount of purified [ka] or a pharma- ceutically acceptable salt thereof to a subject in need of treatment for glioma.

[0047] In one embodiment, the method of treating glioma comprises administering a therapeutically effective amount of purified [ka] or a pharma- ceutically acceptable salt thereof to a subject in need of treatment for glioma.

[0048] In one embodiment, a method of treating glioma is provided, comprising administering to a patient in need of treatment for glioma a therapeutically effective amount of a composition comprising one or more compounds selected from compounds 1-7, or a pharma- ceutically acceptable salt thereof, wherein the glioma is characterized by the presence of at least one mutation selected from an IDH1 mutation or an IDH2 mutation.

[0049] In one embodiment, a method of treating low-grade glioma is provided, comprising administering to a patient in need of treatment for low-grade glioma a composition comprising a therapeutically effective amount of one or more compounds selected from compounds 1-7, or a pharma- ceutically acceptable salt thereof, wherein the glioma is characterized by the presence of at least one mutation selected from an IDH1 mutation or an IDH2 mutation.

[0050] In one embodiment of the methods for treating glioma described herein, the glioma is characterized by the presence of at least one mutation selected from an IDH1 mutation and an IDH2 mutation.

[0051] In certain embodiments, the mutation is an IDH1 mutation. In some embodiments, the IDH1 mutation is an R132X mutation. In further embodiments, the IDH1 mutation is an R132H or R132C mutation.

[0052] In some embodiments, the mutation is an IDH2 mutation. In further embodiments, the mutation is an R140X or R172X mutation. In certain embodiments, the mutation is an R140Q, R140W or R140L mutation. In other embodiments, the mutation is an R172K or R172G mutation.

[0053] In some embodiments of the methods described herein, the amount of the compound or a pharma- ceutically acceptable salt thereof administered to the patient is between 1 and 5000 mg / day. In certain embodiments, the amount of the compound or a pharma- ceutically acceptable salt thereof administered to the patient is between 1 and 2000 mg / day. In certain embodiments, the amount of the compound or a pharma- ceutically acceptable salt thereof administered to the patient is between 1 and 1000 mg / day. In some embodiments, the amount of the compound or a pharma- ceutically acceptable salt thereof administered to the patient is between 1 and 500 mg / day.

[0054] In certain embodiments of the methods described herein, the compound or composition is administered orally.

[0055] In certain embodiments of the methods described herein, the compound or composition is administered in combination with an additional therapeutic modality. In certain embodiments, the additional therapeutic modality is selected from radiation, surgical resection, anti-cancer drugs, anti-epileptic drugs, anti-convulsants, and anti-emetic drugs.

[0056] In some embodiments, the anti-cancer agent is selected from cytotoxic or cytostatic chemotherapy, targeted agents, antibody therapy, immunotherapy, and hormonal therapy. [Brief description of the drawings]

[0057] [Figure 1] FIG. 1 depicts multiple proposed theoretical biotransformation pathways for AG-881 (ivosidenib) in humans.

[0058] Detailed Description The details of construction and arrangement of components set forth in the following description or illustrated in the drawings are not meant to be limiting. Other embodiments and different ways of carrying out the subject matter of this application are expressly included. Also, the language and terminology used herein are for purposes of description and should not be considered as limiting. The use of "including," "comprising," "having," "containing," "involving," and variations thereof herein means the inclusion of the items listed thereafter and equivalents thereof as well as additional items.

[0059] definition The term "body fluid" includes amniotic fluid surrounding a fetus, aqueous humor, blood (e.g., whole blood or plasma), cerebrospinal fluid, earwax, chyme, Cowper's fluid, feces, female ejaculate, interstitial fluid, lymphatic fluid, breast milk, mucus (e.g., nasal secretions or phlegm), pleural fluid, pus, saliva, sebum, semen, serum, sweat, tears, urine, vaginal secretions or vomit.

[0060] As used herein, the terms "inhibit" or "prevent" include both complete and partial inhibition and prevention. An inhibitor can completely or partially inhibit the intended target.

[0061] The term "treat" means to reduce, suppress, attenuate, arrest or stabilize the onset or progression of a disease / disorder (e.g., cancer), reduce the severity of a disease / disorder (e.g., cancer), or ameliorate the symptoms associated with a disease / disorder (e.g., cancer).

[0062] As used herein, the term "daily dose" refers to the total amount of therapeutic agent administered in any 24-hour period, and is used interchangeably with "amount / day." By way of example, a "100 mg daily dose" or a "100 mg / day dose" or an "amount / day of 100 mg" refers to administering a total of 100 mg of therapeutic agent to a patient in any 24-hour period. The daily dose can be administered once a day (i.e., QD or once every 24 hours or every day) or can be divided into multiple doses to be administered at different times during the 24-hour period (e.g., BID or twice a day or every 12 hours; TD or three times a day or every 8 hours; QID or four times a day or every 6 hours, etc.). Each dose (e.g., daily dose or divided dose) can be administered as a single dosage form (e.g., a single tablet or capsule) or as multiple dosage forms (e.g., two or more tablets or capsules). By way of example, a daily dose of 1000 mg (i.e., a dose of 1000 mg / day) administered BID (i.e., twice daily) can be administered as two dosage forms (e.g., capsules or tablets), each containing, for example, 250 mg of therapeutic agent (e.g., a purified compound selected from Compounds 1-7 or a pharma- ceutically acceptable salt thereof) every 12 hours.

[0063] As used herein, an amount of a compound effective to treat a disorder or a "therapeutically effective amount" refers to an amount of a compound that, upon single or multiple administrations to a subject, is effective to treat a cell or cure, palliate, relieve or improve a subject having a disorder beyond that expected in the absence of such treatment.

[0064] As used herein, the term "subject" is intended to include humans and non-human animals. Exemplary human subjects include human patients (referred to as patients) with a disorder, e.g., a disorder described herein, or normal subjects. In some embodiments, a human patient is a child (defined as a person under 18 years of age). In other embodiments, a human patient is an adult (defined as a person 18 years of age or older). The term "non-human animal" in one aspect of the invention includes all vertebrates, e.g., non-mammals (e.g., chickens, amphibians, reptiles) and mammals, e.g., non-human primates, domesticated and / or agriculturally useful animals, e.g., sheep, dogs, cats, cows, pigs, etc.

[0065] As used herein, the terms "purified," "in purified form," or "in isolated and purified form" in reference to a compound refer to the physical state of said compound after it has been physically separated from a synthetic process (e.g., a reaction mixture), a natural source, or a body fluid, or a combination thereof, and / or after it has been subjected to a purification process or step. The above-mentioned "one or more purification processes" are either described herein or known to those of skill in the art (e.g., chromatography, recrystallization, etc.), and the purity of the compound obtained by such one or more purification processes is determined by standard analytical techniques described herein or known to those of skill in the art. In some embodiments, the purified compound does not need to be physically separated from the reaction mixture or body fluid prior to purification, e.g., the reaction mixture or body fluid is subjected to a purification process, and the desired compound is isolated in purified form after completion of the purification process. In further embodiments, the compound can optionally be subjected to multiple purification processes. By way of example, the purification techniques disclosed herein result in isolated and purified forms of the desired compounds 1-7. Such isolation and purification techniques would be expected to result in compound purity of about 90 wt% or greater (e.g., greater than 90 wt%, greater than 95 wt%, greater than 97 wt%, greater than 98 wt%, or greater than 99 wt% purity).

[0066] compound Compounds 1 to 7 or pharma- ceutically acceptable salts or hydrates thereof are provided. [ka]

[0067] Compounds 1-7 are either metabolites observed in one or more body fluids or proposed to be obtained upon oral administration of vorasidenib (AG-881) to humans, or are synthesized de novo. 14 C]AG-881 administered orally once and [ 13 C3 15 A study to profile and identify vorasidenib and its metabolites in selected plasma, urine and fecal samples collected from human subjects following simultaneous intravenous administration of N3]AG-881 is described in Example 8.

[0068] Compounds 1-7 can be synthetically prepared from commercially available materials using methods and combinations of methods known in the art. Exemplary methods for the synthesis of compounds 1-7 are described in Examples 1-7.

[0069] For example, compound 1 and compound 2 can be prepared according to Scheme 1 from 6-(6-chloropyridin-2-yl)-N2,N4-bis((R)-1,1,1-trifluoropropan-2-yl)-1,3,5-triazine-2,4-diamine (vorasidenib, AG-881). [ka]

[0070] Treatment of vorasidenib with thiomethoxide (e.g., sodium thiomethoxide) in a suitable organic solvent (e.g., a polar aprotic organic solvent, such as dimethylsulfoxide) results in the formation of compound 1. The reaction can be carried out at a temperature between 0 and 30° C., and can optionally be carried out under an inert atmosphere (e.g., under a nitrogen atmosphere).

[0071] Additionally, compound 2 can be synthetically prepared from compound 1 by oxidation using an oxidizing agent (e.g., oxone) in a suitable organic solvent (e.g., a polar organic solvent, such as a polar protic organic solvent, e.g., methanol).

[0072] Compounds 3, 4, 5, and 6 can be prepared from vorasidenib (AG-881) by the method generally depicted in Scheme 2. [ka]

[0073] Vorasidenib (AG-881) can be dissolved in a suitable organic solvent (e.g., a polar aprotic organic solvent, e.g., dimethylsulfoxide) and treated with a sulfide reagent (e.g., sodium sulfide) to provide compound 3. The reaction can be carried out at a temperature of 0 to 30° C., and optionally under an inert atmosphere (e.g., under a nitrogen atmosphere). Compound 4 can then be obtained by treating compound 3 with an oxidizing agent (e.g., trichloroisocyanuric acid) in an organic solvent (e.g., a polar aprotic organic solvent, e.g., acetonitrile). The reaction can be carried out at a temperature of −40° C. to 0° C. (e.g., −20° C.), and optionally under an inert atmosphere (e.g., under a nitrogen atmosphere). Compounds 5 and 6 can be obtained by treating compound 3 with an appropriate optically pure 2-amino-3-chloropropanoic acid in an organic solvent (e.g., a polar aprotic organic solvent, e.g., N,N-dimethylformamide) in the presence of a base (e.g., an organic amine base, e.g., N,N-diisopropylethylamine). The reaction can be carried out at a temperature between 20°C and 100°C (e.g., 40°C to 80°C, e.g., about 60°C). In some embodiments, the reaction can be carried out under an inert atmosphere (e.g., under a nitrogen atmosphere).

[0074] Compound 7 can be prepared from vorasidenib (AG-881) by the method generally depicted in Scheme 3. [ka]

[0075] Treatment of vorasidenib with thiomethoxide (e.g., sodium thiomethoxide) in a suitable organic solvent (e.g., a polar aprotic organic solvent, such as dimethylsulfoxide) results in the formation of compound 1. The reaction can be carried out at a temperature between 0 and 30° C., and can optionally be carried out under an inert atmosphere (e.g., under a nitrogen atmosphere).

[0076] Additionally, compound 7 can be synthetically prepared from compound 1 by oxidation using an oxidizing agent (e.g., oxone) in the presence of water in a suitable organic solvent (e.g., a polar organic solvent, such as a polar protic organic solvent, e.g., methanol).

[0077] Compounds 1-7 contain one or more asymmetric centers and thus may occur or be isolated or synthesized as racemates, racemic mixtures, scalenic mixtures, and diastereomeric mixtures, as well as single enantiomers or individual stereoisomers substantially free of other possible enantiomers or stereoisomers. As used herein, the term "substantially free of other stereoisomers" refers to a preparation that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% enriched in a compound having a selected stereochemistry at one or more selected stereocenters. The term "enriched" means that at least a specified percentage of the preparation is a compound having a selected stereochemistry at one or more selected stereocenters. Methods for obtaining or synthesizing individual enantiomers or stereoisomers for a given compound are known in the art and can be practically applied to final compounds or starting materials or intermediates.

[0078] In certain embodiments, compounds 1-7 are enriched in one or more structures having a selected stereochemistry at one or more carbon atoms, e.g., the compounds are at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% enriched in a particular stereoisomer.

[0079] Compounds 1-7 can also be prepared with one or more isotopic substitutions. For example, H is: 1 H, 2 H (D or deuterium) and 3 H can be in any isotopic form, including T or tritium, and C can be 11 C. 12 C. 13 C and 14 C can be in any isotopic form, including N. 13 N, 14 N and 15 It can be any isotopic form including N, and O is 15 O. 16 O and 18 O can be in any isotopic form, including F. 18 and the like. For example, the compound is enriched at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% in a particular isotopic form of H, C, N, O and / or F. Particular isotopically labeled compounds (e.g., 3 H and 14 C-labeled compounds) are useful in compound and / or substrate tissue distribution assays. 3 H) and carbon-14 isotopes (i.e., 14 C is particularly preferred due to its ease of preparation and detection. Additionally, heavier isotopes such as deuterium (i.e. 2H) may confer certain therapeutic advantages resulting from greater metabolic stability (e.g., increased half-life in vivo or reduced dosage requirements) and therefore may be preferred in some circumstances. Isotopically labeled compounds can generally be prepared by techniques similar to those disclosed in the Examples, by substituting the appropriate isotopically labeled reagent for a non-isotopically labeled reagent.

[0080] It may be convenient or desirable to prepare, purify and / or handle corresponding salts of the active compounds, e.g., pharma- ceutically acceptable salts. Examples of pharma-ceutically acceptable salts are discussed in Berge et al., 1977, "Pharmaceutically Acceptable Salts." J. Pharm. Sci. Vol. 66, pp. 1-19.

[0081] For example, a compound may contain anionic or potentially anionic functional groups (e.g., -COOH, -COO - In the case where the cation has a molecular weight of 1,000 or more, the salt can be formed with a suitable cation. Examples of suitable inorganic cations are alkali metal ions, such as Na + and K + , alkaline earth cations, e.g., Ca 2+ and Mg 2+ and other cations, e.g., Al 3+ Examples of suitable organic cations include, but are not limited to, ammonium ions (i.e., NH + ) and substituted ammonium ions (e.g., NHR + , NH2R2 + , NHR3 + , NR4 +Examples of some suitable substituted ammonium ions include, but are not limited to, ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as those derived from amino acids such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3)4 + It is.

[0082] The compound may have a cationic or potentially cationic functional group (e.g., -NH2, -NH3 + In some cases, the salt may be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfurous acid, nitric acid, nitrous acid, phosphoric acid, and phosphorous acid.

[0083] Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetoxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid, edetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxynaphthalenecarboxylic acid, isethionic acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, methanesulfonic acid, mucoic acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, pantothenic acid, phenylacetic acid, phenylsulfonic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, toluenesulfonic acid, and valeric acid. The mesylates of each compound in Table 1 are expressly included herein. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.

[0084] Thus, the compounds provided herein include the compounds themselves, as well as their salts, hydrates, and prodrugs, if applicable. The compounds provided herein can be modified and converted into prodrugs by adding appropriate functional groups to enhance selected biological properties, such as targeting to specific tissues. Such modifications (i.e., prodrugs) are known in the art and include those that improve biological penetration into a given biological compartment (e.g., blood, lymphatic system, central nervous system), improve oral availability, improve solubility to allow administration by injection, alter metabolism, and alter excretion rate. Examples of prodrugs include esters (e.g., phosphates, amino acid (e.g., valine) esters), carbamates, and other pharma- ceutically acceptable derivatives that can provide active compounds upon administration to a subject. Calcium and sodium phosphates of each of Compounds 1-7, if applicable, are expressly included herein. Amino acid (e.g., valine) esters of each of Compounds 1-7, if applicable, are expressly included herein.

[0085] Compositions and routes of administration The compounds utilized in the methods described herein can be formulated into a pharma- ceutically acceptable composition with a pharma- ceutically acceptable carrier or adjuvant prior to administration to a subject. In one embodiment, such a pharma- ceutically acceptable composition further comprises an additional therapeutic agent in an amount effective to achieve modulation of a disease or disease symptom, including those described herein.

[0086] The term "pharmaceutically acceptable carrier or adjuvant" refers to a carrier or adjuvant that may be administered to a subject, together with a compound of one aspect of the invention, which does not destroy its pharmacological activity and which is non-toxic when administered in a dosage sufficient to deliver a therapeutic amount of the compound.

[0087] Pharmaceutically acceptable carriers, adjuvants and vehicles that can be used in the pharmaceutical composition of one aspect of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS), such as d-α-tocopherol polyethylene glycol 1000 succinate, surfactants used in pharmaceutical dosage forms, such as Tween or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. Cyclodextrins, such as α-, β-, and γ-cyclodextrin or chemically modified derivatives, such as hydroxyalkyl cyclodextrins including 2- and 3-hydroxypropyl-β-cyclodextrin or other solubilizing derivatives, may also be advantageously used to enhance delivery of the compounds of the formulations described herein.

[0088] The pharmaceutical composition of one aspect of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally or by implanted reservoir, preferably orally or by injection.The pharmaceutical composition of one aspect of the present invention can contain any conventional non-toxic pharmaceutically acceptable carrier, adjuvant or vehicle.Optionally, the pH of the formulation can be adjusted with a pharmaceutically acceptable acid, base or buffer to enhance the stability of the formulated compound or its delivery form.As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal and intracranial injection or infusion techniques.

[0089] The pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, a solution in 1,3-butanediol. Among the acceptable vehicles and solvents, mannitol, water, Ringer's solution and isotonic sodium chloride solution may be used. In addition, sterile, fixed oils are conventionally used as a solvent or suspending medium. For this purpose, any sterile, volatile oil may be used, including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives, are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially those polyoxyethylated. These oil solutions or suspensions may also contain a long chain alcohol diluent or dispersant or carboxymethylcellulose or similar dispersing agents commonly used in formulating pharma- ceutically acceptable dosage forms, such as emulsions and / or suspensions. Other commonly used surfactants, such as Tween or Span, and / or other similar emulsifiers or bioavailability enhancers commonly used in producing pharma- ceutically acceptable solid, liquid or other dosage forms, may also be used for formulation purposes.

[0090] In certain embodiments, any one of compounds 1-7 or a pharma- ceutically acceptable salt thereof is made into a composition comprising one of the compounds described herein (e.g., one or more of compounds 1-7) or a pharma- ceutically acceptable salt thereof and one or more polymers as part of a solid dispersion (e.g., an amorphous solid dispersion). In some embodiments, the solid dispersion comprises a compound selected from compounds 1-7 or a pharma- ceutically acceptable salt thereof and one or more polymers. In some embodiments, the solid dispersion comprises one of compounds 1-7 or a pharma- ceutically acceptable salt thereof, one or more polymers, and one or more surfactants. In some embodiments, the solid dispersion comprises any one of compounds 1-7 or a pharma- ceutically acceptable salt thereof and one polymer. In some embodiments, the solid dispersion comprises any one of compounds 1-7 or a pharma- ceutically acceptable salt thereof, one polymer, and a surfactant.

[0091] In some embodiments, at least a portion of the active ingredient (e.g., one of Compounds 1-7 or a pharma- ceutically acceptable salt thereof) in the solid dispersion is in an amorphous state (e.g., at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%). In other embodiments, the solid dispersion is substantially free of crystalline compounds.

[0092] In some embodiments, the composition is an amorphous solid (e.g., spray-dried) dispersion comprising one of Compounds 1-7 or a pharma- ceutically acceptable salt thereof and a polymer. The amorphous solid dispersion may contain, for example, less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% crystalline compound (e.g., substantially free of crystalline compounds selected from Compounds 1-7 or a pharma- ceutically acceptable salt thereof).

[0093] Examples of polymers in solid dispersions are cellulose derivatives (e.g. hydroxypropyl methylcellulose (HPMC), also known as hypromellose, hydroxypropyl methylcellulose phthalate (HPMCP), also known as hypromellose phthalate, hydroxypropyl methylcellulose acetate succinate (HPMCAS), also known as hypromellose acetate succinate, hydroxypropyl cellulose (HPC)), ethyl cellulose or cellulose acetate phthalate); polyvinylpyrrolidone (PVP); polyethylene glycol (PEG); polyvinyl alcohol (PVA); polyvinyl esters, For example, polyvinyl acetate phthalate (PVAP); acrylates, such as polymethacrylates (e.g., Eudragit.RTM.E); cyclodextrins (e.g., beta-cyclodextrin); poly(D,L-lactide) (PLA), poly(D,L-lactide, co-glycolic acid) (PLGA) and copolymers and derivatives thereof, such as polyvinylpyrrolidone-vinyl acetate (PVP-VA), polyvinylcaprolactam-polyvinyl and acetate-polyethylene glycol copolymers, methyl acrylate / methacrylic acid copolymers; Soluplus; copovidone and mixtures thereof.

[0094] In some embodiments, the solid dispersion comprises at least one water-soluble polymer. In some embodiments, the solid dispersion comprises at least one partially water-soluble polymer. In some embodiments, the polymer is a cellulose derivative polymer. In other embodiments, the polymer is copovidone. In yet other embodiments, the polymer is cyclodextrin. In some embodiments, the solid dispersion comprises two or more polymers.

[0095] In some embodiments, the polymer is HPMCAS (e.g., various grades of HPMCAS: HPMCAS-M, HPMCAS-MG, or HPMCAS-HG). In some embodiments, the polymer is PVAP. In some embodiments, the polymer is HPMC (e.g., various grades of HPMC: HMPC60SH50, HPMCE50, or HPMCE15). In some embodiments, the polymer is HPMCP (e.g., various grades of HPMCP: e.g., HMPCP-HP55).

[0096] In some embodiments, the polymer is a pH-dependent enteric polymer. Such pH-dependent enteric polymers include, but are not limited to, cellulose derivatives (e.g., cellulose acetate phthalate (CAP)), HPMCP, HPMCAS, carboxymethylcellulose (CMC) or its salts (e.g., sodium salt, e.g., (CMC-Na)); cellulose acetate trimellitate (CAT), hydroxypropylcellulose acetate phthalate (HPCAP), hydroxypropylmethylcellulose acetate phthalate (HPMCAP) and methylcellulose acetate phthalate (MCAP)), polymethacrylates (e.g., Eudragit S) or mixtures thereof.

[0097] In some embodiments, the polymer is hydroxypropyl methylcellulose acetate succinate (HPMCAS), also known as hypromellose acetate succinate, for example, HMPCAS-HG.

[0098] In another embodiment, the polymer is an insoluble cross-linked polymer, such as polyvinylpyrrolidone (e.g., crospovidone). In another embodiment, the polymer is polyvinylpyrrolidone (PVP).

[0099] In some embodiments, the compound (e.g., a compound selected from Compounds 1-7) or a pharma- ceutically acceptable salt thereof is present in the solid dispersion in an amount of about 10% w / w to about 90% w / w (e.g., about 20% w / w to about 80% w / w; about 30% w / w to about 70% w / w; about 40% w / w to about 60% w / w, or about 15% w / w to about 35% w / w). In some embodiments, the compound (e.g., a compound selected from Compounds 1-7) or a pharma- ceutically acceptable salt thereof is present in the solid dispersion in an amount of about 10% w / w to about 80% w / w, e.g., about 30% w / w to about 75% w / w, or about 40% w / w to about 65% w / w, or about 45% w / w to about 55% w / w, e.g., about 46% w / w, about 47% w / w, about 48% w / w, about 49% w / w, about 50% w / w, about 51% w / w, about 52% w / w, about 53% w / w, or about 54% w / w. In the above embodiments, the remaining weight of the composition is represented by one or more polymers. In some embodiments, the solid dispersion also includes a surfactant or an inert pharma- ceutically acceptable substance. Examples of surfactants in the solid dispersion include sodium lauryl sulfate (SLS), vitamin E or its derivatives (e.g., vitamin E TPGS), docusate sodium, sodium dodecyl sulfate, polysorbates (e.g., Tween 20 and Tween 80), poloxamers (e.g., poloxamer 335 and poloxamer 407), glyceryl monooleate, Span 65, Span 25, Capryol 90, pluronic copolymers (Pluronic F108, Pluronic P-123), and mixtures thereof. In some embodiments, the surfactant is SLS. In some embodiments, the surfactant is vitamin E or its derivatives (e.g., vitamin E TPGS).

[0100] In some embodiments, the surfactant is present in the solid dispersion in an amount of about 0.1% w / w to about 10% w / w, e.g., about 0.5% w / w to about 2% w / w, or about 1% w / w to about 3% w / w, about 1% w / w to about 4% w / w, or about 1% w / w to about 5% w / w, such that the sum of the weights of the active ingredient (e.g., a compound selected from Compounds 1-7, or a pharma- ceutically acceptable salt thereof), polymer, and surfactant equals 100%.

[0101] In some embodiments, solid dispersions can be prepared according to the processes described herein. Generally, methods that can be used include rapidly removing the solvent or solvent mixture from the mixture or cooling the molten sample. Such methods include, but are not limited to, rotary evaporation, lyophilization (i.e., freeze drying), vacuum drying, melt congealing, and melt extrusion. One embodiment of the present disclosure includes solid dispersions obtained by spray drying. In one embodiment, the product obtained by spray drying is dried to remove the solvent or solvent mixture.

[0102] The preparations disclosed herein, e.g., pharmaceutical compositions, can be obtained by spray drying a mixture comprising a compound selected from compounds 1-7 or a pharma- ceutically acceptable salt thereof, one or more polymers and a suitable solvent or solvent mixture. Spray drying includes, for example, atomization of a liquid mixture comprising a solid and a solvent or solvent mixture, and removal of the solvent or solvent mixture. The solvent or solvent mixture can also include a non-volatile solvent, e.g., glacial acetic acid. Atomization can be carried out, for example, through a two-fluid nozzle or a pressure nozzle or an electrosonic nozzle or on a rotating disk.

[0103] Techniques and methods for spray drying can be found in Perry's Chemical Engineering Handbook, 6th Ed., RH Perry, DW Green & JO Maloney, eds., McGraw-Hill Book Co. (1984) and Marshall "Atomization and Spray-Drying" 50, Chem. Eng. Prog. Monogr. Series 2 (1954). In one embodiment, the spray drying is fluidized spray drying (FSD).

[0104] In certain embodiments, a method for preparing a solid dispersion of a compound selected from Compounds 1-7, or a pharma- ceutically acceptable salt thereof, comprises: a) forming a mixture of a compound (e.g., a compound selected from Compounds 1-7) or a pharma- ceutically acceptable salt thereof, a polymer, and a solvent; b) spray drying the mixture to form a solid dispersion comprising the compound and a polymer.

[0105] Post-drying and / or grinding of the wet spray dried dispersion can optionally be performed to bring it below ICH or a given specification for residual solvent.

[0106] These methods can be used to prepare the pharmaceutical compositions disclosed herein. The amounts and characteristics of the ingredients used in the methods can be as disclosed herein or as determined by one of skill in the art.

[0107] In certain embodiments, pharmaceutical compositions comprising a solid dispersion can be prepared by the methods described herein. For example, the pharmaceutical composition can comprise a solid dispersion comprising (a) a compound selected from Compounds 1-7 or a pharma- ceutically acceptable salt thereof, and (b) one or more polymers and, optionally, one or more surfactants and, optionally, one or more additional excipients.

[0108] The pharmaceutical compositions disclosed herein can be orally administered in any orally acceptable dosage form, including (but not limited to) capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. For oral tablets, commonly used carriers include lactose and corn starch. Lubricants, such as magnesium stearate, are also typically added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. For oral administration of aqueous suspensions and / or emulsions, the active ingredient can be suspended or dissolved in an oily phase and combined with emulsifying and / or suspending agents. If necessary, certain sweeteners and / or flavors and / or colorants can be added.

[0109] In some embodiments, a compound selected from compounds 1-7 or a pharma- ceutically acceptable salt thereof is orally administered in an amount of 1-5000 mg / day (e.g., 1-1000 mg / day, 1000-2000 mg / day, 2000-3000 mg / day, 3000-4000 mg / day, or 4000-5000 mg / day). In one embodiment, the compound is administered in an amount of 1-1000 mg / day (e.g., 1-500 mg / day, 500-1000 mg / day). In one embodiment, the compound is administered in an amount of 1-500 mg / day (e.g., 1-100 mg / day, 100-200 mg / day, 200-300 mg / day, 300-400 mg / day, 400-500 mg / day). In one embodiment, the compound is administered in an amount of 500-1000 mg / day (e.g., 500-750 mg / day, 750-1000 mg / day). In one embodiment, the compound is administered in an amount of 1000-2000 mg / day (e.g., 1000-1500 mg / day, 1500-2000 mg / day). In one embodiment, the compound is administered in an amount of 2000-3000 mg / day (e.g., 2000-2500 mg / day, 2500-3000 mg / day). In one embodiment, the compound is administered in an amount of 3000-4000 mg / day (e.g., 3000-3500 mg / day, 3500-4000 mg / day). In one embodiment, the compound is administered in an amount of 4000-5000 mg / day (e.g., 4000-4500 mg / day, 4500-5000 mg / day). In some embodiments, a compound selected from compounds 1-7 or a pharma- ceutically acceptable salt thereof is orally administered in an amount of 1-500 mg / day, 1-250 mg / day, 5-100 mg / day, 8-75 mg / day, 10-50 mg / day, 15-40 mg / day, 20-30 mg / day, or about 25 mg / day. In some embodiments, a compound selected from compounds 1-7 or a pharma- ceutically acceptable salt thereof is orally administered in an amount of 1-500 mg / day, 10-250 mg / day, 20-100 mg / day, 30-80 mg / day, 40-60 mg / day, 45-55 mg / day, or about 50 mg / day.In some embodiments, the compound selected from compounds 1-7 or a pharma- ceutically acceptable salt thereof is orally administered in an amount of 1-500 mg / day, 20-400 mg / day, 40-200 mg / day, 50-150 mg / day, 75-125 mg / day, 85-115 mg / day, 90-110 mg / day, or about 100 mg / day. In some embodiments, the compound selected from compounds 1-7 or a pharma-ceutically acceptable salt thereof is orally administered in an amount of 1-500 mg / day, 50-400 mg / day, 100-300 mg / day, 150-250 mg / day, 175-225 mg / day, 185-215 mg / day, 190-210 mg / day, or about 200 mg / day. In some embodiments, the compound selected from compounds 1-7 or a pharma- ceutically acceptable salt thereof is orally administered in an amount of 1-500 mg / day, 100-500 mg / day, 200-400 mg / day, 250-350 mg / day, 275-375 mg / day, 285-315 mg / day, 290-310 mg / day, or about 300 mg / day. In some embodiments, the daily amount of the compound selected from compounds 1-7 or a pharma- ceutically acceptable salt thereof is administered once (i.e., administered in a single dosage form) or in one or more divided doses over a 24-hour period (i.e., administered in two or more dosage forms). In some embodiments, each of the daily doses or divided doses can be administered as a single dosage form or as multiple dosage forms (e.g., administering two or more dosage forms at each administration) for ease of administration and patient compliance. In some embodiments, the dosage form is a tablet. In other embodiments, the dosage form is a capsule.

[0110] In other embodiments, a compound disclosed herein (e.g., a compound selected from Compounds 1-7) or a pharma- ceutically acceptable salt thereof is administered once daily in an amount of about 1 mg, about 5 mg, about 10 mg, about 25 mg, about 50 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 750 mg, about 1000 mg, about 1250 mg, about 1500 mg, about 2000 mg, about 2500 mg, about 3000 mg, about 3500 mg, about 4000 mg, or about 5000 mg per administration.

[0111] The pharmaceutical composition of one aspect of the present invention can also be administered in the form of a suppository for rectal administration. These compositions can be prepared by mixing the compound of one aspect of the present invention with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and will therefore melt in the rectum to release the active ingredient. Such materials include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.

[0112] Topical administration of the pharmaceutical composition of one aspect of the present invention is useful when the desired treatment involves areas or organs readily accessible by topical application. For topical application to the skin, the pharmaceutical composition should be formulated in a suitable ointment containing the active ingredient suspended or dissolved in a carrier. Carriers for topical administration of the compounds of one aspect of the present invention include, but are not limited to, mineral oil, liquid petroleum oil, white petroleum oil, propylene glycol, polyoxyethylene polyoxypropylene compounds, emulsifying wax and water. Alternatively, the pharmaceutical composition should be formulated in a suitable lotion or cream containing the active compound suspended or dissolved in a carrier with suitable emulsifying agents. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The pharmaceutical composition of one aspect of the present invention may also be topically applied to the lower intestinal tract by rectal suppository formulation or suitable enema formulation. Topically transdermal patches are also included in one aspect of the present invention.

[0113] The pharmaceutical compositions described herein may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as solutions in saline utilizing benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons and / or other solubilizing or dispersing agents known in the art.

[0114] Alternatively, the compounds described herein can be administered, for example, by injection, intravenously, intraarterially, subcutaneously, intraperitoneally, intramuscularly or subcutaneously, or orally, bucally, intranasally, transmucosally, topically, in ophthalmic preparations, or by inhalation, with dosages ranging from about 0.5 to about 100 mg / kg body weight, alternatively, dosages ranging from 1 mg to 1000 mg / administration every 4 to 120 hours or according to the requirements of the particular agent. The methods herein contemplate administration of an effective amount of the compound or composition of the compound to achieve the desired or described effect. Typically, the pharmaceutical composition of one aspect of the invention will be administered about 1 to about 6 times per day, or alternatively as a continuous infusion. Such administration can be used as a chronic or acute therapy. The amount of active ingredient which can be combined with the carrier materials to produce a single dosage form will vary depending on the host treated and the particular mode of administration. A typical preparation will contain from about 5% to about 95% active compound (w / w). Alternatively, such preparations contain from about 20% to about 80% active compound.

[0115] Lower or higher doses than those mentioned above may be necessary. The specific dosage and treatment regimen for any particular subject will depend on a variety of factors, including the activity of the specific compound employed, age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combinations, the severity and course of the disease, condition or symptom, the predisposition of the subject to the disease, condition or symptom, and the judgment of the treating physician.

[0116] Once the subject's condition has improved, a maintenance dose of a compound, composition or combination of an aspect of the invention can be administered, if necessary. Thereafter, the dose or frequency of administration, or both, can be reduced as a function of symptoms, to a level at which the improved condition is maintained when the symptoms have been alleviated to a desired level. However, subjects may require intermittent treatment on a long-term basis in the event of a recurrence of disease symptoms.

[0117] The pharmaceutical compositions described above, comprising a compound selected from compounds 1-7 or a pharma- ceutically acceptable salt thereof, or a compound described in any one of the embodiments herein, may further comprise another therapeutic agent useful in the treatment of cancer.

[0118] When the compositions of the present disclosure include a combination of a compound of the formulations described herein with one or more additional therapeutic or prophylactic agents, both the compound and the additional agent should be present at dosage levels of about 1-100%, more preferably about 5-95%, of the dose normally administered in a monotherapy regimen. The additional agent can be administered separately from the compound of one aspect of the present invention as part of a multiple dose regimen. Alternatively, these agents can be part of a single dosage form and mixed together with the compound of one aspect of the present invention in a single composition.

[0119] How to use Provided is a method for inhibiting the activity of mutant IDH1 and / or mutant IDH2, comprising contacting a subject in need of inhibition of said activity with a compound selected from compounds 1-7 or a pharma- ceutically acceptable salt thereof.

[0120] Also provided is a method of treating a cancer characterized by the presence of a mutant allele of IDH1, comprising administering to a subject in need of treatment for the cancer (a) a compound of the disclosure (e.g., a compound selected from compounds 1-7) or a pharma- ceutically acceptable salt thereof, or (b) a pharmaceutical composition comprising (a) and a pharma- ceutically acceptable carrier.

[0121] In one embodiment, the cancer to be treated is characterized by a mutant allele of IDH1, where the IDH1 mutation results in a de novo ability of the enzyme to catalyze the NADPH-dependent reduction of α-ketoglutarate to R(-)-2-(2HG) in the patient. In one aspect of this embodiment, the IDH1 mutation is an R132X mutation. In another aspect of this embodiment, the R132X mutation is selected from R132H, R132C, R132L, R132V, R132S and R132G. In another aspect, the R132X mutation is R132H or R132C. The cancer can be analyzed by sequencing cell samples to determine the presence and specific nature of the mutation at amino acid 132 of IDH1 (e.g., the altered amino acid present therein).

[0122] Without wishing to be bound by theory, Applicants believe that mutant alleles of IDH1, in which IDH1 mutations confer a new ability of the enzyme to catalyze the NADPH-dependent reduction of α-ketoglutarate to R(-)-2-hydroxyglutarate (2HG), characterize a subset of all types of cancer, regardless of their cellular nature or location in the body. Thus, the compounds and methods of the present disclosure are useful for treating any type of cancer characterized by the presence of mutant alleles of IDH1 that confer such activity, in particular the IDH1 R132H or R132C mutations.

[0123] In one embodiment, the cancer is a tumor in which at least 30, 40, 50, 60, 70, 80 or 90% of the tumor cells have an IDH1 mutation, in particular an IDH1 R132H or R132C mutation, at the time of diagnosis or treatment.

[0124] IDH1R132X mutations are known to occur in certain types of cancer, as shown in Table 1 below.

[0125] [Table 1]

[0126] IDH1R132H mutations have been identified in gliomas (including low-grade gliomas), glioblastomas (including secondary glioblastomas), acute myeloid leukemia, sarcomas, melanomas, non-small cell lung cancer, cholangiocarcinoma, chondrosarcomas, myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), colon cancer, and angioimmunoblastic non-Hodgkin lymphoma (NHL). Thus, in one embodiment, the methods described herein are used to treat glioma (including low grade glioma), glioblastoma (including secondary glioblastoma), grade II and III astrocytoma, grade II and III oligodendroglioma, acute myeloid leukemia, sarcoma, melanoma, non-small cell lung cancer (NSCLC), cholangiocarcinoma, chondrosarcoma, myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), colon cancer or angioimmunoblastic non-Hodgkin's lymphoma (NHL) in a patient.

[0127] In another embodiment, the methods described herein are used to treat glioma (including low-grade glioma), glioblastoma (including secondary glioblastoma), grade II and III astrocytoma, grade II and III oligodendroglioma, acute myeloid leukemia, sarcoma, melanoma, non-small cell lung cancer (NSCLC), cholangiocarcinoma (e.g., intrahepatic cholangiocarcinoma (IHCC)), chondrosarcoma, myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPN), prostate cancer, chronic myelomonocytic leukemia (CMML), B-acute lymphoblastic leukemia (B-ALL), myeloid sarcoma, multiple myeloma, lymphoma colon cancer, or angioimmunoblastic non-Hodgkin's lymphoma (NHL) in a patient.

[0128] In another embodiment, the methods described herein are used to treat advanced hematological malignancies. In one embodiment, the advanced hematological malignancies treated are lymphomas (e.g., non-Hodgkin's lymphoma (NHL), such as B-cell lymphomas (e.g., Burkitt's lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, and mantle cell lymphoma) and T-cell lymphomas (e.g., mycosis fungoides, anaplastic large cell lymphoma, and precursor T-lymphoblastic lymphoma).

[0129] Thus, in one embodiment, the cancer is a cancer selected from any one of the cancer types listed in Table 1 or further described herein, and the IDHR132X mutation is one or more of the IDH1R132X mutations listed in Table 1 for that particular cancer type.

[0130] Also provided is a method for inhibiting mutant IDH2 activity, comprising contacting a subject in need of such inhibition with a compound of the present disclosure (e.g., a compound selected from compounds 1-7) or a pharma- ceutically acceptable salt thereof.

[0131] Also provided is a method of treating a cancer characterized by the presence of a mutant allele of IDH2, comprising administering to a subject in need of such treatment (a) a compound of the disclosure (e.g., a compound selected from compounds 1-7) or a pharma- ceutically acceptable salt thereof, or (b) a pharmaceutical composition comprising (a) and a pharma- ceutically acceptable carrier.

[0132] In one embodiment, the cancer to be treated is characterized by a mutant allele of IDH2, where the IDH2 mutation results in a de novo ability of the enzyme to catalyze the NADPH-dependent reduction of α-ketoglutarate to R(-)-2-hydroxyglutarate (2HG) in the patient. In one aspect of this embodiment, the mutant IDH2 has a R140X mutation. In another aspect of this embodiment, the R140X mutation is a R140Q mutation. In another aspect of this embodiment, the R140X mutation is a R140W mutation. In another aspect of this embodiment, the R140X mutation is a R140L mutation. In another aspect of this embodiment, the mutant IDH2 has a R172X mutation. In another aspect of this embodiment, the R172X mutation is a R172K mutation. In another aspect of this embodiment, the R172X mutation is a R172G mutation. Cancers can be analyzed by sequencing cell samples to determine the presence and specific nature of mutations at amino acids 140 and / or 172 of IDH2 (eg, the altered amino acids present).

[0133] Without wishing to be bound by theory, Applicants believe that mutant alleles of IDH2, in which IDH2 mutations confer a new enzymatic ability to catalyze the NADPH-dependent reduction of α-ketoglutarate to R(-)-2-hydroxyglutarate (2HG), characterize a subset of all types of cancer, regardless of their cellular nature or location in the body. Thus, the compounds and methods of one aspect of the invention are useful for treating any type of cancer characterized by the presence of mutant alleles of IDH2 that confer such activity, in particular the IDH2 R140Q and / or R172K mutations.

[0134] In one embodiment, the cancer is a tumor in which at least 30, 40, 50, 60, 70, 80 or 90% of the tumor cells have an IDH2 mutation, in particular an IDH2 R140Q, R140W or R140L and / or an R172K or R172G mutation, at the time of diagnosis or treatment.

[0135] In another embodiment, an aspect of the present invention provides a method of treating a cancer selected from glioma (including low grade glioma), glioblastoma (including secondary glioblastoma), grade II and III astrocytoma, grade II and III oligodendroglioma, myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), acute myeloid leukemia (AML), sarcoma, melanoma, non-small cell lung cancer, chondrosarcoma, cholangiocarcinoma, or angioimmunoblastic lymphoma in a patient by administering to the patient a compound of the present disclosure (e.g., a compound selected from Compounds 1-7) or a pharmaceutically acceptable salt thereof in an amount effective to treat the cancer. In more specific embodiments, the cancer to be treated is glioma (including low grade glioma), glioblastoma (including secondary glioblastoma), grade II and III astrocytoma, grade II and III oligodendroglioma, myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), acute myeloid leukemia (AML), melanoma, chondrosarcoma, or angioimmunoblastic non-Hodgkin's lymphoma (NHL).

[0136] The treatment methods described herein can further include various evaluation steps before and / or after treatment with a compound of the present disclosure (e.g., a compound selected from Compounds 1-7) or a pharma- ceutically acceptable salt thereof.

[0137] In one embodiment, before and / or after treatment with a compound of the disclosure (e.g., a compound selected from Compounds 1-7) or a pharma- ceutically acceptable salt thereof, the method further comprises assessing the growth, size, weight, invasiveness, stage, and / or other phenotype of the cancer using one or more techniques known and used by those of skill in the art.

[0138] In one embodiment, the method further comprises assessing the IDH1 or IDH2 genotype of the cancer before and / or after treatment with a compound of the disclosure (e.g., a compound selected from Compounds 1-7) or a pharma- ceutically acceptable salt thereof. This can be accomplished by methods routine in the art, such as DNA sequencing, immunoassay, and / or assessing the presence, distribution, or levels of R(-)-2-hydroxyglutarate (2HG).

[0139] In one embodiment, the method further comprises determining R(-)-2-hydroxyglutarate (2HG) levels in the subject before and / or after treatment with a compound of the disclosure (e.g., a compound selected from Compounds 1-7) or a pharma- ceutically acceptable salt thereof. This can be accomplished by spectroscopic analysis, e.g., magnetic resonance-based analysis, e.g., MRI and / or MRS measurements, analysis of a sample of a bodily fluid, e.g., serum or spinal fluid analysis, or by analysis of surgical material, e.g., by mass spectroscopy.

[0140] In one aspect of this embodiment, the efficacy of cancer treatment is monitored by measuring the level of R(-)-2-hydroxyglutarate (2HG) in the subject. Typically, the level of R(-)-2-hydroxyglutarate (2HG) is measured before treatment, where an elevated level of R(-)-2-hydroxyglutarate (2HG) (along with a confirmed IDH mutation status) is used to confirm the eligibility of using a compound of the present disclosure (e.g., a compound selected from compounds 1-7) or a pharma- ceutically acceptable salt thereof to treat cancer. Once an elevated level is established, the level of R(-)-2-hydroxyglutarate (2HG) is determined during and / or after the course of treatment to establish target engagement (i.e., inhibition of mutant IDH by administration of a compound of the present disclosure or a pharma- ceutically acceptable salt thereof). In certain embodiments, the level of R(-)-2-hydroxyglutarate (2HG) is determined only during and / or after the course of treatment. A decrease in R(-)-2-hydroxyglutarate (2HG) levels during and after the course of treatment indicates target engagement. Typically, measurement of R(-)-2-hydroxyglutarate (2HG) will be utilized in conjunction with other known determinations of the effectiveness of cancer treatment, such as a reduction in the number and size of tumors and / or other cancer-related lesions, an improvement in the general health of the subject, and changes in other biomarkers associated with the effectiveness of cancer treatment.

[0141] R(-)-2-hydroxyglutarate (2HG) can be detected in the samples by LC / MS. Samples are mixed 80:20 with methanol and centrifuged at 3,000 rpm at 4°C for 20 min. The resulting supernatant can be collected and stored at -80°C prior to LC-MS / MS to assess 2-hydroxyglutarate (2HG) levels. Various liquid chromatography (LC) separation methods can be used. Each method can be coupled with a triple quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) mode by negative electrospray ionization (ESI, -3.0 kV), with MS parameters optimized with injected metabolite standard solutions. Metabolites can be separated by reversed-phase chromatography using 10 mM tributyl-amine as an ion-pairing agent in the aqueous mobile phase according to a modification of a previously reported method (Luo et al. J Chromatogr A 1147, 153-64, 2007). One method allows the separation of TCA metabolites: t=0, 50% B; t=5, 95% B; t=7, 95% B; t=8, 0% B, where B refers to an organic mobile phase of 100% methanol. Another method is specific for 2-hydroxyglutarate (2HG) and uses a fast linear gradient from 50% to 5% B (buffer defined above) over 5 min. The column can be a Synergi Hydro-RP, 100 mm x 2 mm, 2.1 μm particle size (Phenomonex), as described above. Metabolites can be quantified by comparing peak areas with pure metabolite standards of known concentration. 13 Metabolic flux studies from C-glutamine can be performed, for example, as described in Munger et al. Nat Biotechnol 26, 1179-86, 2008.

[0142] In one embodiment, the concentration of R(-)-2-hydroxyglutarate (2HG) is assessed prior to treatment with a compound of the present disclosure (e.g., a compound selected from compounds 1-7) or a pharma- ceutically acceptable salt thereof. In another embodiment, the concentration of R(-)-2-hydroxyglutarate (2HG) is assessed after treatment with a compound of the present disclosure or a pharma- ceutically acceptable salt thereof. In some embodiments, the assessment of R(-)-2-hydroxyglutarate (2HG) is performed using a biological fluid from a human patient. In other embodiments, the assessment of R(-)-2-hydroxyglutarate (2HG) is performed using a biological sample from a biopsy or a tissue sample from a human patient. In some aspects, the biopsy or tissue sample is from a brain tumor of a human patient. In some embodiments, the biopsy or tissue sample is taken from a human patient before treatment with a compound of the present disclosure or after treatment with a compound of the present disclosure or both before and after treatment with a compound of the present disclosure.

[0143] In another embodiment, the derivatives of R(-)-2-hydroxyglutarate (2HG) formed during the course of the analytical method are evaluated. For example, such derivatives may be derivatives formed during MS analysis. Derivatives may include salt adducts, e.g., Na adducts, hydrated variants, and hydrogenated variants. These variants may also be salt adducts, e.g., Na adducts, as formed during MS analysis.

[0144] In another embodiment, metabolic derivatives of R(-)-2-hydroxyglutarate (2HG) are evaluated. Examples include species that accumulate or are elevated or decreased as a result of the presence of R(-)-2-hydroxyglutarate (2HG), such as glutaric acid or glutarate or glutamate, which may be related to R(-)-2-hydroxyglutarate (2HG), e.g., R-2HG.

[0145] Exemplary R(-)-2-hydroxyglutarate (2HG) derivatives include dehydrated derivatives, such as the compounds provided below or salt adducts thereof: [ka] Includes.

[0146] Also provided is a method of treating a disease selected from Maffucci syndrome and Ollier disease, characterized by the presence of a mutant allele of IDH1, comprising administering to a subject in need of such treatment (a) a compound of the present disclosure (e.g., a compound selected from compounds 1-7) or a pharma- ceutically acceptable salt thereof, or (b) a pharmaceutical composition comprising (a) and a pharma- ceutically acceptable carrier.

[0147] Brain Tumors Treated by the Methods of the Invention In one embodiment, the method of the present invention is useful for treating brain tumors. This includes all tumors within the human skull (cranium) or central spinal canal. Tumors may originate from the brain itself, but may also originate from lymphatic tissue, blood vessels, cranial nerves, brain envelope (meninges), skull, pituitary gland, or pineal gland. In the brain itself, the cells involved may be neurons or glial cells (including astrocytes, oligodendrocytes, and ependymal cells). Brain tumors may also metastasize from cancers that are primarily present in other organs (metastatic tumors).

[0148] In some embodiments, the brain tumor is a glioma, such as an ependymoma, astrocytoma, oligoastrocytoma, oligodendroglioma, ganglioglioma, glioblastoma (also known as glioblastoma multiforme), or mixed glioma. Gliomas are primary brain tumors and are classified into four grades (I, II, III, and IV) based on their appearance under a microscope, particularly the presence of atypical cells, mitoses, endothelial proliferation, and necrosis. Grade I and II tumors are called "low-grade gliomas" and have none or one of these characteristics, including diffuse astrocytoma, pilocytic astrocytoma, low-grade astrocytoma, low-grade oligoastrocytoma, low-grade oligodendroglioma, ganglioglioma, dysplastic neuroepithelial tumor, polymorphous xanthoastrocytoma, and mixed glioma. Grade III and IV tumors, referred to as "high-grade gliomas," have two or more of these characteristics and include anaplastic astrocytoma, anaplastic oligodendroglioma, anaplastic oligoastrocytoma, anaplastic ependymoma, and glioblastoma (including giant cell glioblastoma and gliosarcoma). In one aspect of these embodiments, the glioma is a low-grade glioma. In another aspect of these embodiments, the glioma is a high-grade glioma. In another aspect of these embodiments, the glioma is a glioblastoma (including secondary glioblastoma). In some embodiments, the brain tumor is a grade II or III astrocytoma. In some embodiments, the brain tumor is a grade II or III oligodendroglioma.

[0149] In further embodiments, the brain tumor (e.g., glioma (including low grade glioma), glioblastoma (including secondary glioblastoma), grade II or III astrocytoma, grade II or III oligodendroglioma) is newly diagnosed. In further embodiments, the brain tumor (e.g., glioma (including low grade glioma), glioblastoma (including secondary glioblastoma), grade II or III astrocytoma, grade II or III oligodendroglioma) has been previously treated with one or more therapeutic modalities including surgery, radiation therapy, or one or more additional therapeutic agents. In other embodiments, the brain tumor (e.g., glioma (including low grade glioma), glioblastoma (including secondary glioblastoma), grade II or III astrocytoma, grade II or III oligodendroglioma) has not been treated with radiation therapy.

[0150] In some embodiments, the brain tumor to be treated (e.g., glioma (including low grade glioma), glioblastoma (including secondary glioblastoma), grade II or III astrocytoma, grade II or III oligodendroglioma) is characterized by the presence of an IDH1 mutation, where the IDH1 mutation results in the accumulation of R(-)-2-hydroxyglutarate (2HG) in the patient. In one aspect of these embodiments, the IDH1 mutation results in the accumulation of R(-)-2-hydroxyglutarate (2HG) in the patient by providing a new ability for the enzyme to catalyze the NADPH-dependent reduction of α-ketoglutarate to R(-)-2-hydroxyglutarate (2HG) in the patient. In another aspect of these embodiments, the IDH1 mutation is an R132X mutation. In another aspect of these embodiments, the R132X mutation is selected from R132H, R132C, R132L, R132V, R132S, and R132G. In another aspect of these embodiments, the R132X mutation is R132H or R132C. In yet another aspect of these embodiments, the R132X mutation is R132H. In yet another aspect of these embodiments, at least 30, 40, 50, 60, 70, 80, or 90% of the cells of a brain tumor (e.g., glioma (including low grade glioma), glioblastoma (including secondary glioblastoma), grade II or III astrocytoma, grade II or III oligodendroglioma) have an IDH1 R132X mutation, e.g., R132H, R132C, R132L, R132V, R132S, or R132G mutation, at the time of diagnosis or treatment. Brain tumors (e.g., glioma (including low grade glioma), glioblastoma (including secondary glioblastoma), grade II or III astrocytoma, grade II or III oligodendroglioma) can be analyzed by sequencing cell samples to determine the presence and specific nature of mutations at amino acid 132 of IDH1 (e.g., the altered amino acid present).

[0151] In other embodiments, the treated brain tumor (e.g., glioma (including low grade glioma), glioblastoma (including secondary glioblastoma), grade II or III astrocytoma, grade II or III oligodendroglioma) is characterized by the presence of an IDH2 mutation, where the IDH2 mutation results in the accumulation of R(-)-2-hydroxyglutarate (2HG) in the patient. In one aspect of these embodiments, the IDH2 mutation results in the accumulation of R(-)-2-hydroxyglutarate (2HG) in the patient by providing a new ability for the enzyme to catalyze the NADPH-dependent reduction of α-ketoglutarate to R(-)-2-hydroxyglutarate (2HG) in the patient. In another aspect of these embodiments, the mutant IDH2 has a R140X mutation. In another aspect of these embodiments, the R140X mutation is a R140Q mutation. In another aspect of these embodiments, the R140X mutation is a R140W mutation. In another aspect of these embodiments, the R140X mutation is a R140L mutation. In another aspect of these embodiments, the mutant IDH2 has a R172X mutation. In another aspect of these embodiments, the R172X mutation is a R172K mutation. In another aspect of these embodiments, the R172X mutation is a R172G mutation. In yet another aspect of these embodiments, at least 30, 40, 50, 60, 70, 80, or 90% of cells of the brain tumor (e.g., glioma (including low grade glioma), glioblastoma (including secondary glioblastoma), grade II or III astrocytoma, grade II or III oligodendroglioma) have an IDH2 R140X and / or R172X mutation, e.g., an R140Q, R140W, or R140L, and / or an R172K or R172G mutation, at the time of diagnosis or treatment. Brain tumors (e.g., gliomas (including low grade gliomas), glioblastomas (including secondary glioblastomas), grade II or III astrocytomas, grade II or III oligodendrogliomas) can be analyzed by sequencing cell samples to determine the presence and specific nature (e.g., the altered amino acids present) of mutations at amino acids 140 and / or 172 of IDH2.

[0152] In yet other embodiments, the brain tumors to be treated (e.g., gliomas (including low-grade gliomas), glioblastomas (including secondary glioblastomas), grade II or III astrocytomas, grade II or III oligodendrogliomas) are characterized by the presence of an IDH1 mutation and an IDH2 mutation, where the IDH1 mutation and the IDH2 mutation collectively result in the accumulation of R(-)-2-hydroxyglutarate (2HG) in the patient. In one aspect of these embodiments, the IDH1 and IDH2 mutations result in the accumulation of R(-)-2-hydroxyglutarate (2HG) in the patient by providing a de novo ability for the enzyme to catalyze the NADPH-dependent reduction of α-ketoglutarate to R(-)-2-hydroxyglutarate (2HG) in the patient. In various aspects of these embodiments, the IDH1 mutation is an R132X mutation selected from R132H, R132C, R132L, R132V, R132S, and R132G. In various aspects of these embodiments, the IDH2 mutation is an R140Q, R140W, R140L, R172K, or R172G mutation. In various other aspects of these embodiments, the treated brain tumor (e.g., glioma (including low grade glioma), glioblastoma (including secondary glioblastoma), grade II or III astrocytoma, grade II or III oligodendroglioma) is characterized by any combination of the above IDH1 and IDH2 mutations. In still other aspects of these embodiments, at least 30, 40, 50, 60, 70, 80, or 90% of the brain tumor (e.g., glioma (including low grade glioma), glioblastoma (including secondary glioblastoma), grade II or III astrocytoma, grade II or III oligodendroglioma) cells have an IDH1 R132X mutation, e.g., an R132H, R132C, R132L, R132V, R132S, or R132G mutation, and an IDH2 R140X and / or R172X mutation, e.g., an R140Q, R140W, or R140L, and / or an R172K or R172G mutation, at the time of diagnosis or treatment.Brain tumors (e.g., gliomas (including low grade gliomas), glioblastomas (including secondary glioblastomas), grade II or III astrocytomas, grade II or III oligodendrogliomas) can be analyzed by sequencing cell samples to determine the presence and specific nature (e.g., the altered amino acids present) of mutations at amino acid 132 of IDH1 and amino acids 140 and / or 172 of IDH2.

[0153] In yet other embodiments, the brain tumors (e.g., gliomas (including low grade gliomas), glioblastomas (including secondary glioblastomas), grade II or III astrocytomas, grade II or III oligodendrogliomas) to be treated are characterized by the presence of an IDH1 allele that does not contain a R132X mutation and an IDH2 allele that does not contain a R140X or R172X mutation. In one aspect of these embodiments, at least 90% of the cells of the brain tumor (e.g., gliomas (including low grade gliomas), glioblastomas (including secondary glioblastomas), grade II or III astrocytomas, grade II or III oligodendrogliomas) do not contain a mutation at amino acid 132 of IDH1 or amino acids 140 or 172 of IDH2 at the time of diagnosis or treatment. Brain tumors (e.g., gliomas (including low grade gliomas), glioblastomas (including secondary glioblastomas), grade II or III astrocytomas, grade II or III oligodendrogliomas) can be analyzed by sequencing cell samples to determine the presence or absence of mutations at amino acid 132 of IDH1 and amino acids 140 and / or 172 of IDH2.

[0154] Combination therapy In some embodiments, the methods described herein include the additional step of co-administering to a subject in need thereof an additional therapeutic procedure (e.g., an additional cancer therapeutic agent, an additional therapeutic agent or an additional cancer treatment to minimize the symptoms of cancer or the side effects of the cancer treatment). Exemplary additional cancer therapeutic agents (anti-cancer agents) include, for example, chemotherapy with cytotoxic or cytostatic agents, targeted therapy (targeted agents), antibody therapy, immunotherapy, and hormonal therapy. Exemplary additional therapeutic agents (pharmacotherapy) to minimize symptoms and side effects include, for example, anti-epileptic agents, anti-convulsants, and anti-emetic agents. Additional cancer treatments include, for example, surgery and radiation therapy. Examples of each of these treatments are provided below.

[0155] As used herein with respect to an additional cancer therapeutic agent, the term "co-administration" means that the additional cancer therapeutic agent can be administered together with the compound of an embodiment of the present invention, either as part of a single dosage form (e.g., a composition of an embodiment of the present invention comprising a compound of an embodiment of the present invention and a second therapeutic agent as described above) or as separate multiple dosage forms. Alternatively, the additional cancer therapeutic agent can be administered prior to, sequentially with, or after the administration of the compound of an embodiment of the present invention. In such combination therapy treatments, both the compound of an embodiment of the present invention and the second therapeutic agent are administered by conventional methods. The administration of a composition of an embodiment of the present invention comprising both a compound of an embodiment of the present invention and a second therapeutic agent to a subject does not preclude the separate administration of that same therapeutic agent, any other second therapeutic agent, or any compound of an embodiment of the present invention to the subject at another time during the course of treatment. As used herein with respect to an additional cancer treatment, the term "co-administration" means that the additional cancer treatment can be administered prior to, sequentially with, simultaneously with, or after the administration of the compound of an embodiment of the present invention.

[0156] In some embodiments, the additional cancer therapeutic agent is a chemotherapeutic agent. Examples of chemotherapeutic agents used in cancer treatment include, for example, antimetabolites (e.g., folic acid, purine and pyrimidine derivatives), alkylating agents (e.g., nitrogen mustards, nitrosoureas, platinum, alkylsulfonates, hydrazines, triazenes, aziridines, spindle poisons, cytotoxic agents, topoisomerase inhibitors, etc.), and hypomethylating agents (e.g., decitabine (5-aza-deoxycytidine), zebularine, isothiocyanates, azacitidine (5-azacytidine), 5-fluoro-2'-deoxycytidine, 5,6-dihydro-5-azacytidine, etc.). Exemplary agents include aclarubicin, actinomycin, alitretinoin, altretamine, aminopterin, aminolevulinic acid, amrubicin, amsacrine, anagrelide, arsenic trioxide, asparaginase, atrasentan, belotecan, bexarotene, bendamustine, bleomycin, bortezomib, busulfan, camptothecin, capecitabine, carboplatin, carboquone, carmofur, carmustine, celecoxib, chlorambucil, chlormethine, cisplatin, cladribine, clofarabine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, decitabine, demecolcine, docetaxel, doxorubicin, efaproxiral, elesclomol, elsamitrucin, enocitabine, epirubicin, estramustine, , etoglucide, etoposide, floxuridine, fludarabine, fluorouracil (5FU), fotemustine, gemcitabine, gliadel implant, hydroxycarbamide, hydroxyurea, idarubicin, ifosfamide, irinotecan, irofulven, ixabepilone, larotaxel, leucovorin, liposomal doxorubicin, liposomal daunorubicin, lonidamine, lomustine, leucovorin Cantone, mannosulfan, masoprocol, melphalan, mercaptopurine, mesna, methotrexate, methyl aminolevulinate, mitobronitol, mitoguazone, mitotane, mitomycin, mitoxantrone, nedaplatin, nimustine, oblimersen, omacetaxine, ortataxel, oxaliplatin, paclitaxel, pegaspargase, pemetrexed, pentostatin,Pirarubicin, pixantrone, plicamycin, porfimer sodium, prednimustine, procarbazine, raltitrexed, ranimustine, rubitecan, sapacitabine, semustine, sitimagenseradenovec, strataplatin, streptozocin, talaporfin, tegafur-uracil, temoporfin, temozolomide, teniposide, tesetaxel, testoralactone, tetranitrate, thiotepa, These include tiazofurin, thioguanine, tipifarnib, topotecan, trabectedin, triazicon, triethylenemelamine, triplatin, tretinoin, treosulfan, trofosfamide, uramustine, valrubicin, verteporfin, vinblastine, vincristine, vindesine, vinflunine, vinorelbine, vorinostat, zorubicin and other cytostatic or cytotoxic agents described herein.

[0157] Some drugs work better when used together than alone, so two or more drugs are often given at the same time. Often, two or more chemotherapy drugs are used in combination chemotherapy.

[0158] In some embodiments, the additional cancer therapeutic agent is a differentiation agent. Such differentiation agents include retinoids (e.g., all-trans retinoic acid (ATRA), 9-cis-retinoic acid, 13-cis-retinoic acid (13-cRA) and 4-hydroxy-fenretinamide (4-HPR); arsenic trioxide; histone deacetylase inhibitors; HDACs (e.g., azacytidine (Vidaza) and butyrates (e.g., sodium phenylbutyrate); hybrid polar compounds (e.g., hexamethylene bisacetamide (HMBA)); vitamin D and cytokines (e.g., colony stimulating factors, including G-CSF, GM-CSF, and interferons).

[0159] In some embodiments, the additional cancer therapeutic agent is a targeted therapy. Targeted therapy constitutes the use of drugs specific to deregulated proteins in cancer cells. Small molecule targeted therapy agents are generally inhibitors of enzyme domains on mutated, overexpressed, or other important proteins in cancer cells. Representative examples are tyrosine kinase inhibitors, such as axitinib, bosutinib, cediranib, dasatinib, erlotinib, imatinib, gefitinib, lapatinib, lestaurtinib, nilotinib, semaxanib, sorafenib, sunitinib, and vandetanib, and cyclin-dependent kinase inhibitors, such as alvocidib and seliciclib. Monoclonal antibody therapy is another strategy in which the therapeutic agent is an antibody that specifically binds to a protein on the surface of cancer cells. Examples are trastuzumab (Herceptin®), an anti-HER2 / neu antibody typically used in breast cancer, and rituximab and tositumomab, anti-CD20 antibodies typically used in various B-cell malignancies. Other exemplary antibodies include cetuximab, panitumumab, trastuzumab, alemtuzumab, bevacizumab, edrecolomab, and gemtuzumab. Exemplary fusion proteins include aflibercept and denileukin diftitox. In some embodiments, targeted therapy can be used in combination with a compound described herein, such as a biguanide, such as metformin or phenformin, preferably phenformin.

[0160] Targeted therapies can also include small peptides as "homing devices" that can bind to cell surface receptors or the affected extracellular matrix surrounding the tumor. Radionuclides attached to these peptides (e.g., RGD) ultimately result in cancer cell death as the nuclide decays in the vicinity of the cell. Examples of such therapies include BEXXAR®.

[0161] In some embodiments, the additional cancer therapeutic agent is an immunotherapy agent. Cancer immunotherapy refers to a diverse set of therapeutic strategies designed to induce the subject's own immune system to fight the tumor. Modern methods for generating an immune response against tumors include intrafollicular BCG immunotherapy for superficial bladder cancer and the use of interferon and other cytokines to induce an immune response in subjects with renal cell carcinoma and melanoma.

[0162] Allogeneic hematopoietic stem cell transplantation can be considered a form of immunotherapy, since in many cases the donor's immune cells will attack the tumor in a graft-versus-tumor effect. In some embodiments, immunotherapeutic agents can be used in combination with the compounds or compositions described herein.

[0163] In some embodiments, the additional cancer therapeutic agent is a hormonal therapy agent. The growth of some cancers can be inhibited by providing or blocking certain hormones. Common examples of hormone-sensitive tumors include certain types of breast and prostate cancer. Removal or blocking of estrogen or testosterone is often an important additional treatment. In certain cancers, administration of hormone agonists, such as progestogens, may be therapeutically beneficial. In some embodiments, hormonal therapy agents can be used in combination with the compounds or compositions described herein.

[0164] Other potential additional therapeutic approaches include imatinib, gene therapy, peptide and dendritic cell vaccines, synthetic chlorotoxin and radiolabeled drugs and antibodies.

[0165] Additional Treatments for Brain Cancer Additional therapeutic procedures used in combination with the methods for treating brain cancer described herein include those therapeutic procedures (e.g., surgery, radiation, therapeutic agents / drugs) that are known to be useful in treating brain tumors, i.e., to have a therapeutic effect on brain tumors, to alleviate one or more symptoms of brain tumors, to alter the progression of brain tumors, to eradicate brain tumors, to reduce the size of brain tumors, to slow or inhibit the growth of brain tumors, to delay or minimize one or more symptoms associated with brain tumors, to reduce the aggressiveness of brain tumors or to induce stasis of brain tumors, or to alleviate or minimize one or more side effects associated with another therapy applied or administered to treat brain tumors.

[0166] In some embodiments, the additional therapeutic procedure is surgery.

[0167] In some embodiments, the additional therapeutic modality is radiation therapy. In some embodiments, radiation therapy is administered in a manner consistent with the National Comprehensive Cancer Network Clinical Practice Guidelines in OncoloGy (e.g., Dosage and Administration Schedule), version 1.2016, available at nccn.org. In some embodiments, radiation therapy is administered in fractions of 1.0-5.0 Gy, with a cumulative dose of 20-100 Gy. In some embodiments, radiation therapy is administered in 1.0-5.0 Gy fractions or 1.5-3.0 Gy fractions or 1.0-1.5 Gy fractions or 1.5-2.0 Gy fractions or 2.0-2.5 Gy fractions or 2.5-3.0 Gy fractions or 1.8-2.0 Gy fractions or 1.8 Gy fractions or 2.0 Gy fractions, with a cumulative dose of 20-100 Gy or 30-80 Gy or 30-60 Gy or 40-70 Gy or 40-60 Gy or 30-40 Gy or 40-50 Gy or 50-60 Gy or 45-55 Gy. In some embodiments, radiation therapy is administered in 1.5-2.5 Gy fractions with a cumulative dose of 50-70 Gy or in 2.0 Gy fractions with a cumulative dose of 60 Gy. The cumulative dose refers to the sum of all fraction doses given during the course of treatment.

[0168] The dose of radiation therapy can be selected based on the nature of the brain tumor. In some embodiments where the brain tumor is a low-grade glioma, radiation therapy is administered at a cumulative dose of 40-50 Gy in 1.5-2.5 Gy fractions, or at a cumulative dose of 45-54 Gy in 1.8-2.0 Gy fractions, or at a cumulative dose of 45.5 Gy in 1.8-2.0 Gy fractions. In some embodiments where the brain tumor is a high grade glioma, radiation therapy is administered at a cumulative dose of 50-70 Gy in fractions of 1.5-2.5 Gy, or at a cumulative dose of 59.4 Gy in fractions of 1.8 Gy, or at a cumulative dose of 55.8-59.4 Gy in fractions of 1.8 Gy, or at a cumulative dose of 57 Gy in fractions of 1.9 Gy, or at a cumulative dose of 60 Gy in fractions of 1.8-2.0 Gy, or at a cumulative dose of 25 Gy in fractions of 5.0 Gy. In some embodiments where the brain tumor is a glioblastoma, radiation therapy is administered at a cumulative dose of 30-60 Gy in fractions of 2.0-4.0 Gy, or at a cumulative dose of 34 Gy in fractions of 3.4 Gy, or at a cumulative dose of 35-45 Gy in fractions of 2.5-3.0 Gy, or at a cumulative dose of 50 Gy in fractions of 2.5 Gy.

[0169] In some embodiments, the additional therapeutic procedure is one or more additional therapeutic agents.

[0170] In some embodiments, the one or more additional therapeutic agents include one or more of an additional cancer therapeutic agent (i.e., an anti-cancer agent) (e.g., a DNA-reactive agent, a PARP inhibitor, an immunotherapy (e.g., a checkpoint inhibitor), PVC chemotherapy, an antibody therapy (e.g., bevacizumab), gemcitabine), an antiemetic agent, an anticonvulsant agent, or an antiepileptic agent.

[0171] In some embodiments, the one or more additional therapeutic agents is an additional cancer therapeutic agent (eg, an anti-cancer agent).

[0172] In some embodiments, the additional cancer therapeutic agent is a DNA-reactive agent. As used herein, a "DNA-reactive agent" is an agent such as alkylating agents, cross-linking agents, and DNA intercalating agents, which interact with cellular DNA either covalently or non-covalently. For example, DNA-reactive drugs include adozelesin, altretamine, bizeresin, busulfan, carboplatin, carboquone, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, estramustine, fotemustine, hepsulfam, ifosfamide, improsulfan, irofulven, lomustine, mechlorethamine, melphalan, mitozolomide, nedaplatin, oxaliplatin, piposulfan, procarbazine, semustine, streptozocin, temozolomide, thiotepa, treosulfan, diethylnitrosamine, benzo(a)pyrene, doxorubicin, mitomycin-C, etc. Many of these DNA-reactive drugs are useful in the treatment of cancer as DNA-reactive chemotherapeutic agents.

[0173] In some embodiments, the DNA-reactive agent is temozolomide (TMZ). In one aspect of these embodiments, TMZ is administered in a manner consistent with the National Comprehensive Cancer Network Clinical Practice Guidelines in OncoloGy (e.g., Dosage and Administration Schedule), version 1.2016, available at nccn.org. In one aspect of these embodiments, TMZ is administered in a manner consistent with the prescribing information for TEMODAR® (temozolomide) Capsules and TEMODAR® (temozolomide) for Injection. In some aspects of these embodiments, TMZ is administered in a daily amount of 100-250 mg / m2, or 100-150 mg / m2, or 150-200 mg / m2, or 200-250 mg / m2, based on the patient's body surface area. In some aspects of these embodiments, TMZ is administered in a daily dose of 50-100 mg / m2, or 50-75 mg / m2, or 75-100 mg / m2, or 60-90 mg / m2, or 65-85 mg / m2, or 70-80 mg / m2 based on the patient's body surface area. In some aspects of these embodiments, TMZ is administered in a daily dose of 125-175 mg / m2 based on the patient's body surface area for 5 consecutive days of a 28-day treatment cycle. In some aspects of these embodiments, TMZ is administered in combination with radiation therapy in a daily dose of 50-100 mg / m2, or 50-75 mg / m2 based on the patient's body surface area. In some aspects of these embodiments, TMZ is administered in combination with radiation therapy at a daily dose of 50-100 mg / m2, 50-75 mg / m2, 75-100 mg / m2, 60-90 mg / m2, 65-85 mg / m2, or 70-80 mg / m2 based on the patient's body surface area. In some aspects of these embodiments, TMZ is administered in combination with radiation therapy at a daily dose of 70-80 mg / m2 based on the patient's body surface area for 42 days. In some aspects of these embodiments where the brain tumor is a high-grade glioma or glioblastoma, TMZ is administered in combination with radiation therapy at a daily dose of 70-80 mg / m2 based on the patient's body surface area for 42 days.In some of these embodiments where the brain tumor is an anaplastic astrocytoma, TMZ is administered in an amount of 125-175 mg / m2 per day based on the patient's body surface area for 5 consecutive days of a 28 day treatment cycle. In some of these embodiments where the brain tumor is an anaplastic astrocytoma, TMZ is administered in an amount of 175-225 mg / m2 per day based on the patient's body surface area for 5 consecutive days of a 28 day treatment cycle.

[0174] In some embodiments, one or more additional cancer therapeutic agents (anti-cancer agents) are PARP inhibitors. As used herein, "PARP inhibitor" refers to an inhibitor of the enzyme poly ADP-ribose polymerase (PARP). Examples of PARP inhibitors include pamiparib, olaparib, rucaparib, veraparib, iniparib, talazoparib, niraparib, etc.

[0175] In some embodiments, the one or more additional cancer therapeutic agents (anti-cancer agents) are immunotherapies, such as checkpoint inhibitors. As used herein, "checkpoint inhibitors" refers to therapeutic agents that inhibit immune checkpoints (e.g., CTLA-4, PD-1 / PD-L1, etc.) that would in some way impede the immune system's attack on cancer cells, thereby allowing the immune system to attack cancer cells. Examples of checkpoint inhibitors include ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, BGB-A317, spartalizumab, etc.

[0176] In some embodiments, the one or more additional cancer therapeutic agents (anti-cancer agents) is PVC chemotherapy. As used herein, "PVC chemotherapy" refers to a chemotherapy regimen that includes the combined administration of procarbazine, lomustine (sold under the trade name CCNU®) and vincristine (sold under the trade name Onocovin®). Typically, vincristine is administered intravenously, while procarbazine and lomustine are administered orally. PCV chemotherapy is often administered cyclically, where each cycle includes a single administration of vincristine and lomustine, and 10 days of treatment with procarbazine.

[0177] In some embodiments, the one or more additional cancer therapeutic agents (anti-cancer agents) is an antibody, such as bevacizumab. Bevacizumab, sold under the trade name Avastin®, is a recombinant humanized monoclonal antibody.

[0178] In some embodiments, the one or more additional cancer therapeutic agents (anti-cancer agents) is gemcitabine. Gemcitabine, sold under the trade name Gemzar®, is a pyrimidine nucleoside analog.

[0179] In some embodiments, the one or more additional therapeutic agents is an antiemetic agent. As used herein, "antiemetic agent" refers to an agent that is effective in relieving the symptoms of vomiting and nausea. Examples of antiemetic agents include 5-HT3 receptor antagonists (e.g., dolasetron, granisetron, ondansetron, tropisetron, palonosetron, mirtazapine, etc.), dopamine agonists (e.g., domperidone, olanzapine, droperidol, haloperidol, chlorpromazine, prochlorperazine, alizapride, prochlorperazine, metoclopramide, etc.), NK1 receptor antagonists (e.g., aprepitant, casopitant, rolapitant, etc.), antihistamines (e.g., cinnarizine, cyclizine, diphenhydramine, benzodiazepine, benzodiazepine, benzoic acid ... These include, for example, benzodiazepines (e.g., midazolam, lorazepam, etc.), anticholinergics (e.g., scopolamine, etc.), steroids (e.g., dexamethasone, etc.), trimethobenzamide, ginger, propofol, glucose / fructose / phosphate (sold under the trade name Emetrol®), peppermint, muscimol, ajwain, etc.

[0180] In some embodiments, the one or more additional therapeutic agents are anticonvulsants or antiepileptic agents. As used herein, "anticonvulsants or antiepileptic agents" refers to agents effective in treating or preventing seizures, including epileptic seizures. Examples of anticonvulsants include paraldehyde, stiripentol, phenobarbital, methylphenobarbital, barbexaclone, clobazam, clonazepam, clorazepate, diazepam, midazolam, lorazepam, nitrazepam, temazepam, nimetazepam, potassium bromide, felbamate, carbamazepine, oxcarbazepine, eslicarbazepine acetate, valproic acid, sodium valproate, divalproex sodium, vigabatrin, progabide, tiagabine, topiramate, gabapentin, valproate ... These include pentin, pregabalin, ethotoin, phenytoin, mephenytoin, fosphenytoin, paramethadione, trimethadione, ethadione, beclamide, primidone, brivaracetam, etiracetam, levetiracetam, seletracetam, ethosuximide, pheneximide, mesuximide, acetazolamide, sulthiame, methazolamide, zonisamide, lamotrigine, pheneturide, phenacemide, valpromide, valoctamide, perampanel, stiripentol, pyridoxine, and the like.

[0181] Working Example General experimental considerations In the following examples, reagents (chemicals) were purchased from commercial sources (e.g., Alfa, Acros, Sigma Aldrich, TCI, and Shanghai Chemical Reagent Company) and used without further purification. Nuclear magnetic resonance (NMR) spectra were obtained on a Brucker AMX-400NMR (Brucker, Switzerland). Chemical shifts are reported in parts per million (ppm, δ) downfield from tetramethylsilane. Mass spectra were obtained by electrospray ionization (ESI) from a Waters LCT TOF mass spectrometer (Waters, USA) or a Shimadzu LCMS-2020 mass spectrometer (Shimadzu, Japan). Microwave reactions were performed on an Initiator 2.5 Microwave Synthesizer (Biotage, Sweden).

[0182] For the exemplary compounds disclosed in this section, the designation of a stereoisomer (e.g., (R) or (S) stereoisomer) indicates preparations of that compound such that the compound is at least about 90%, 95%, 96%, 97%, 98% or 99% enriched at the designated stereocenter. The chemical names of each of the exemplary compounds described below are generated by ChemDraw software.

[0183] List of Abbreviations general anhy. aq. Water-based min hrs time mL Milliliters nmol millimolar mol MS mass spectroscopy NMR nuclear magnetic resonance TLC Thin Layer Chromatography HPLC High Performance Liquid Chromatography satd. saturation

[0184] Spectrum Hz Hertz δ Chemical shift J coupling constant s singlet d doublet t triplet q Quartet m multiplet br Broad qd Quartet of Doublets dquin quintet doublet dd Doublet of Doublets dt Triplet doublet

[0185] Solvents and Reagents DAST Diethylaminosulfur trifluoride CHCl3Chloroform DCM Dichloromethane DMF Dimethylformamide Et2O Diethyl ether EtOH Ethyl alcohol EtOAc Ethyl acetate MeOH Methyl alcohol MeCN Acetonitrile PE Petroleum Ether THF Tetrahydrofuran DMSO Dimethyl sulfoxide AcOH Acetic acid HCl Hydrochloric acid H2SO4 Sulfuric acid NH4Cl Ammonium chloride KOH Potassium hydroxide NaOH Sodium hydroxide K2CO3 Potassium Carbonate Na2CO3 Sodium Carbonate TFA Trifluoroacetic acid Na2SO4 Sodium Sulfate NaBH4 Sodium borohydride NaHCO3 Sodium Bicarbonate NaHMDS Sodium hexamethyldisilylamide LiHMDS Lithium hexamethyldisilylamide LAH Lithium aluminum hydride NaBH4 Sodium borohydride LDA Lithium diisopropylamide Et3N Triethylamine Py Pyridine DMAP 4-(Dimethylamino)pyridine DIPEA N,N-Diisopropylethylamine Xphos 2-Dicyclohexylphosphine-2,4,6-triisopropylbiphenyl BINAP 2,2'-Bis(diphenylphosphanyl)-1,1'-binaphthyl dppf 1,1'-bis(diphenylphosphino)ferrocene TBTU 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate DPPA Phenylphosphoryl Azide NH4OH Sodium hydroxide EDCI 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide HOBt 1-Hydroxybenzotriazole Py Pyridine Dppf 1,1'-Bis(diphenylphosphino)ferrocene HATU O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetra-methyluronium BINAP 2,2'-Bis(diphenylphosphanyl)-1,1'-binaphthyl MTBE Methyl tert-butyl ether NaSMe Sodium thiomethoxide

[0186] Example 1 Preparation of 6-(6-(methylthio)pyridin-2-yl)-N2,N4-bis((R)-1,1,1-trifluoropropan-2-yl)-1,3,5-triazine-2,4-diamine (Compound 1) [ka] DMSO (500 mL) and 6-(6-chloropyridin-2-yl)-N2,N4-bis((R)-1,1,1-trifluoropropan-2-yl)-1,3,5-triazine-2,4-diamine (free base of AG-881, 100 g, 0.241 mol) were charged to a 1 L flask under N2 at 15-20 °C. The resulting solution was stirred at 15-20 °C for 10 min to form a clear brown solution. The reaction solution was cooled to 5 °C and sodium thiomethoxide (NaSMe, 35.6 g, 0.508 mol) was added portionwise over 20 min at 5-10 °C. The reaction mixture was stirred at 20-25 °C for 3 h. The mixture was poured into ice water (3 L) at 5-10 °C with stirring. After 30 min at 20-25° C., the solid was filtered and the wet cake was triturated with water (1.5 L) for 30 min at 20-25° C. The solid was filtered, washed with water (200 mL) and dried in a vacuum oven at 50-55° C. to give 6-(6-(methylthio)pyridin-2-yl)-N2,N4-bis((R)-1,1,1-trifluoropropan-2-yl)-1,3,5-triazine-2,4-diamine as an off-white solid (100.8 g, 98% yield). 1 H NMR: (400 MHz, DMSO-d6) δ 8.41-7.81 (m, 4H), 7.45-7.42 (m, 1H), 5.12-4.91 (m, 2H), 2.59 (s, 3H), 1.34 (d, J = 6.1 Hz, 6H). 13 C-NMR: (101 MHz, DMSO-d6) δ 170.65, 170.24, 166.65, 166.38, 160.58, 160.15, 154.20(d, J=14.4 Hz), 137.77, 127.99, 125.19, 122.87, 122.34, 119.89, 119.76 (d, J=21.9 Hz), 47.44, 47.13, 13.89 (d, J=9.6 Hz), 13.53, 13.23. LC-MS(ESI):m / z 427[M+H] +

[0187] Example 2 Preparation of 6-(6-(methylsulfinyl)pyridin-2-yl)-N2,N4-bis((R)-1,1,1-trifluoropropan-2-yl)-1,3,5-triazine-2,4-diamine (compound 2) [ka] MeOH (48 mL) and 6-(6-(methylthio)pyridin-2-yl)-N2,N4-bis((R)-1,1,1-trifluoropropan-2-yl)-1,3,5-triazine-2,4-diamine (4.0 g, 9.38 mmol) were charged to a 250 mL flask at 10-20° C. The reaction mixture was stirred for 10 min to obtain a clear solution. To the reaction solution, an aqueous solution of oxone (4.6 g, 7.48 mmol, water 40 mL) was added dropwise over 30 min at 5-15° C. The resulting mixture was stirred at 20-30° C. for 2 h. The mixture was then cooled to 10° C. and quenched with water (100 mL). The reaction mixture was then extracted with DCM (1×100 mL). The aqueous phase was extracted with DCM (20 mL). The combined organic phase was cooled to 5-10 °C and an aqueous solution of Na2SO3 (1.18 g, 9.38 mmol, water 20 mL) was added [addition is exothermic]. The phases were separated and the organic phase was washed with water (1 × 40 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo at 40-45 °C to give the crude product as an oil. The residue was purified by silica gel chromatography (eluent: n-heptane / EtOAc-5:1 to 1:1). The combined pure fractions were concentrated to dryness under vacuum to give the desired product as a white solid. The product was further recrystallized by dissolving in methanol (30 mL) at 40-45 °C followed by addition of water (60 mL) over a period of 30 min at 20-30 °C. The resulting suspension was stirred at 20-30 °C for an additional 0.5 h, then filtered, washed with water (10 mL) and dried in a vacuum oven at 50-55 °C to give 6-(6-(methylsulfinyl)pyridin-2-yl)-N2,N4-bis((R)-1,1,1-trifluoropropan-2-yl)-1,3,5-triazine-2,4-diamine as a white solid (2.2 g, 53% yield). 1 H-NMR (400 MHz, DMSO-d6) δ 8.57-8.41 (m, 2H), 8.35-8.22 (m, 2H), 8.05 (dd, J = 26.2, 7.4 Hz, 1H), 5.13-4.88 (m, 2H), 2.82 (s, 3H), 1.34 (d, J = 6.8 Hz, 6H). 13 C-NMR (101 MHz, DMSO-d6) δ 169.86, 169.51, 166.67, 166.37, 166.32, 166.11, 154.56 (d, J =9.7 Hz),140.05 (d, J = 8.1Hz), 130.61, 127.93, 126.80, 125.33, 125.22, 124.98, 124.64, 121.02 (d, J = 16.8 Hz), 47.49, 47.12 (d, J = 13.5 Hz), 41.60 (d, J = 13.5 Hz), 13.87(d, J = 15.2 Hz). LC-MS(ESI):m / z 443[M+H] +

[0188] Example 3 Preparation of 6-(4,6-bis(((R)-1,1,1-trifluoropropan-2-yl)amino)-1,3,5-triazin-2-yl)pyridine-2-thiol (compound 3) [ka] DMSO (100 mL) and 6-(6-chloropyridin-2-yl)-N2,N4-bis((R)-1,1,1-trifluoropropan-2-yl)-1,3,5-triazine-2,4-diamine (free base of AG-881, 10 g, 24.1 mmol) were charged to a 250 mL flask under N2 at 15-20 °C. The resulting solution was stirred at 15-20 °C for 10 min to give a clear brown solution. The reaction mixture was cooled to 5 °C and Na2S (4.2 g, 90% purity, 24.4 mmol) was added portionwise over 5 min at 5-10 °C. The reaction mixture was stirred at 20-25 °C for 16 h, poured into ice water (500 mL) and acetic acid (50 mL) was added with stirring at 5-10 °C. After stirring at 10-15 °C for 15-30 min, the solid was filtered. The wet cake was dissolved in DCM (100 mL), the phases were separated and the aqueous phase was discarded. The organic phase was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to give the crude product as a pale yellow oil. The crude product was purified by silica gel chromatography (eluent: DCM / MeOH-30:1 to 10:1). Pure fractions containing the product were combined and concentrated under reduced pressure to give the crude product as a pale yellow oil. This crude product was purified by silica gel chromatography (eluent: DCM / MeOH-30:1 to 10:1). Pure fractions containing the product were combined and concentrated under reduced pressure to give the purified product as a yellow solid. This was further dried in a vacuum oven at ambient temperature to give 6-(4,6-bis(((R)-1,1,1-trifluoropropan-2-yl)amino)-1,3,5-triazin-2-yl)pyridine-2-thiol as a yellow solid (8.0 g, 80% yield). 1 H-NMR (400 MHz, DMSO-d6) δ 12.68-11.90 (m, 1H), 8.68-7.66 (m, 2H), 7.61-7.48 (m, 3H), 5.36-4.86 (m, 2H), 1.37-1.34 (m, 6H). 13C-NMR (101 MHz, DMSO-d6) δ 179.09, 178.77, 166.12, 165.71, 165.62, 163.46, 142.85, 142.66, 137.97, 137.62, 136.76, 136.35, 127.83, 125.02, 113.60, 112.82, 47.60-47.17, 14.0 (d, J = 16.2 Hz). LC-MS(ESI):m / z 413[M+H] +

[0189] Example 4 Preparation of 6,6'-(6,6'-disulfanediylbis(pyridine-6,2-diyl))bis(N2,N4-bis((R)-1,1,1-trifluoropropan-2-yl)-1,3,5-triazine-2,4-diamine) (Compound 4) [ka] Acetonitrile (30 mL) and 6-(4,6-bis(((R)-1,1,1-trifluoropropan-2-yl)amino)-1,3,5-triazin-2-yl)pyridine-2-thiol (1.0 g, 2.43 mmol) were charged to a 100 mL flask under N2 at 15-20 °C. The reaction mixture was cooled to -20 °C and trichloroisocyanuric acid (102 mg, 0.44 mmol) was added in one portion. The resulting mixture was stirred at -20 °C for 1 h and the solid was filtered. The filtrate was poured into water (50 mL) and the resulting solution was extracted with ethyl acetate (1 x 50 mL). The organic layer was concentrated under reduced pressure to give the crude product as a pale yellow oil. The residue was purified by silica gel chromatography (eluent: DCM / MeOH-50:1 to 20:1). The pure fractions containing the product were combined and concentrated to give a pale yellow solid. This was further dried in a vacuum oven at ambient temperature to give 6,6'-(6,6'-disulfanediylbis(pyridine-6,2-diyl)bis(N2,N4-bis((R)-1,1,1-trifluoropropan-2-yl)-1,3,5-triazine-2,4-diamine) as a pale yellow solid (0.82 g, 82% yield). 1 H-NMR (400 MHz, DMSO-d6) δ 8.62 (d, J = 8.0 Hz, 1H), 8.54 (d, J = 8.0 Hz, 2H), 8.27-8.24 (m, 2H), 8.19 (d, J = 8.0 Hz, 2H), 8.07-8.02 (m, 2H), 7.87 (d, J = 8.0 Hz, 2H), 5.14-4.92 (m, 4H), 1.41-1.36 (m, 12H). 13C-NMR (101 MHz, DMSO-d6) δ 169.85, 169.59, 166.70, 166.42, 166.33, 158.67, 158.53, 158.28, 154.71, 139.72 (d, J = 12.1 Hz), 130.77, 127.96, 125.15, 122.21, 121.79, 121.46, 121.32, 121.14, 121.04, 47.78-46.88, 13.94 (d, J = 11.1 Hz). LC-MS(ESI):m / z 823[M+H] +

[0190] Example 5 Preparation of (R)-2-amino-3-((6-(4,6-bis(((R)-1,1,1-trifluoropropan-2-yl)amino)-1,3,5-triazin-2-yl-pyridin-2-yl)thio)propanoic acid (compound 5) [ka] N,N-Dimethylformamide (5 mL), N,N-Diisopropylethylamine (1.88 g, 14.6 mmol) and 6-(4,6-bis(((R)-1,1,1-trifluoropropan-2-yl)amino)-1,3,5-triazin-2-yl)pyridine-2-thiol (1.0 g, 2.43 mmol) were charged to a 25 mL flask under N2 at 20-30 °C. A solution of L-2-amino-3-chloropropanoic acid (0.60 g, 4.86 mmol) in water (5 mL) was then added dropwise to the reaction mixture at 20-30 °C. The resulting mixture was heated to 60 °C and stirred for 20 h. After stirring at 60 °C for 20 h, the mixture was cooled to 20 °C and poured into water (30 mL). The resulting slurry was stirred for 15-30 min, then the solid was isolated by vacuum filtration and washed with water (10 mL). The solid was dried on the filter under air for 1-2 h, then dissolved in DMSO (30 mL). The product solution in DMSO was purified by preparative HPLC [column: YMCTA C18, 250×21.2 mm, 10 um; flow rate: 15 mL / min; gradient: 20% acetonitrile-80% water 0.1% TFA to 70% acetonitrile-30% water 0.1% TFA; @ 254 / 205 nm]. Pure fractions containing the product were combined and concentrated in vacuo at 45-50° C. to remove the solvent. The product was then lyophilized to give (R)-2-amino-3-((6-(4,6-bis(((R)-1,1,1-trifluoropropan-2-yl)amino)-1,3,5-triazin-2-yl)pyridin-2-yl)thio)propanoic acid as a white solid (170 mg, 98.9% / 220 nm, 14% yield). 1 H-NMR (400 MHz, DMSO-d6) δ8.37-8.03 (m, 6H), 7.88 (t, J = 8.0 Hz, 1H), 7.63 (d, J = 8.0 Hz, 1H), 5.19-4.95 (m, 2H), 3.61-3.32 (m, 3H), 1.42-1.35 (m, 6H). 13C-NMR (101 MHz, DMSO-d6) δ169.53, 169.27, 168.77, 166.31, 166.21, 166.14, 159.03 (d, J = 8.1 Hz), 153.24(d, J = 4.0 Hz), 138.70 (d, J = 7.1 Hz), 127.96, 125.35(d, J = 17.2 Hz), 120.71, 120.20, 56.44, 47.61, 33.77, 13.97. LC-MS(ESI):m / z 499[M+H] +

[0191] Example 6 Preparation of (S)-3-((6-(4,6-bis(((R)-1,1,1-trifluoropropan-2-yl)amino)-1,3,5-triazin-2-yl)pyridin-2-yl)thio)-2-methylpropanoic acid (compound 6) [ka] N,N-Dimethylformamide (5 mL), N,N-Diisopropylethylamine (3.1 g, 24.3 mmol) and 6-(4,6-bis(((R)-1,1,1-trifluoropropan-2-yl)amino)-1,3,5-triazin-2-yl)pyridine-2-thiol (1.0 g, 2.43 mmol) were charged to a 25 mL flask under N2 at 20-30 °C. A solution of L-2-amino-3-chloropropanoic acid (0.60 g, 4.86 mmol) in water (5 mL) was then added dropwise to the reaction mixture at 20-30 °C. The resulting mixture was heated to 60 °C and stirred for 20 h. After stirring at 60 °C for 20 h, the mixture was cooled to 20 °C and poured into water (30 mL). The resulting slurry was stirred for 15-30 min, then the solid was isolated by vacuum filtration and washed with water (10 mL). The solid was dried on the filter under air for 1-2 h, then dissolved in DMSO (30 mL). The product solution in DMSO was purified by preparative HPLC [column: YMCTA C18, 250×21.2 mm, 10 um; flow rate: 15 mL / min; gradient: 20% acetonitrile-80% water 0.1% TFA to 70% acetonitrile-30% water 0.1% TFA; @ 254 / 205 nm]. Pure fractions containing the product were combined and concentrated in vacuo at 45-50° C. to remove the solvent. The product was then lyophilized to give (S)-3-((6-(4,6-bis(((R)-1,1,1-trifluoropropan-2-yl)amino)-1,3,5-triazin-2-yl)pyridin-2-yl)thio)-2-methylpropanoic acid as a white solid (120 mg, 98.5% / 220 nm, 10% yield). 1 H-NMR (400 MHz, DMSO-d6) δ8.36-8.04 (m, 6H), 7.88 (t, J = 8.0 Hz, 1H), 7.63 (d, J = 8.0 Hz, 1H), 5.17-4.94 (m, 2H), 3.62-3.33 (m, 3H), 1.42-1.34 (m, 6H). 13C-NMR (101 MHz, DMSO-d6) δ169.55, 169.28, 168.50, 166.29, 166.15, 159.07, 158.99, 153.22, 138.71 (d, J = 7.1 Hz), 125.54, 125.38, 120.68, 120.17, 56.52, 47.59, 33.83, 13.99. LC-MS(ESI):m / z 499[M+H] +

[0192] Example 7 Preparation of 6-(6-(methylsulfonyl)pyridin-2-yl)-N2,N4-bis((R)-1,1,1-trifluoropropan-2-yl)-1,3,5-triazine-2,4-diamine (compound 7) [ka] DMSO (50 mL) and 6-(6-chloropyridin-2-yl)-N2,N4-bis((R)-1,1,1-trifluoropropan-2-yl)-1,3,5-triazine-2,4-diamine (free base of AG-881, 5.0 g, 12.06 mmol) were charged to a flask. The resulting mixture was stirred at 15-20° C. for 5 min to form a clear brown solution under nitrogen. The reaction mixture was cooled to 5° C. and then sodium thiomethoxide (NaSMe, 4.3 g, 60.28 mmol) was added portionwise over 20 min at 5-10° C. under nitrogen. The reaction mixture was heated to 90-95° C. and stirred at 90-95° C. for 3 h. The reaction solution was poured into ice water (200 mL) at 5-10° C. with stirring. After stirring for 30 min, the solid was filtered, and the filter cake was washed with water (50 mL) and dried in vacuum at 50 °C for 6 h to give 6-(6-(methylthio)pyridin-2-yl)-N2,N4-bis((R)-1,1,1-trifluoropropan-2-yl)-1,3,5-triazine-2,4-diamine as a pale yellow solid (4.8 g, purity 98.9%, crude yield 93%).

[0193] To a solution of MeOH (40 mL) and water (40 mL) was added 6-(6-(methylthio)pyridin-2-yl)-N2,N4-bis((R)-1,1,1-trifluoropropan-2-yl)-1,3,5-triazine-2,4-diamine (4.0 g, 9.38 mmol) in one portion. To this reaction mixture was added oxone (11.5 g, 18.8 mmol) in portions over 15 min, keeping the temperature below 20°C. The reaction mixture was stirred at 20-30°C for 2 h. The reaction mixture was cooled to 10°C, and then water (80 mL) and DCM (120 mL) were added. The resulting mixture was stirred at 10-15°C for 10 min and separated. The oran phase was washed with aqueous Na2SO3 (1.2 g in 80 mL water) at 5-10°C (checked with KI starch paper) and separated. The organic phase was washed with aqueous Na2SO3 (1.2 g in 80 mL water) at 5-10 °C (confirmed by KI starch paper) and separated. The organic phase was washed with water (100 mL × 2) and separated. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuum at 40-45 °C to give 4-5V slurry. The slurry was stirred at 10-15 °C for 30 min, filtered, washed with MTBE (20 mL), and dried in vacuum at 50 °C to give 6-(6-(methylsulfonyl)pyridin-2-yl)-N2,N4-bis((R)-1,1,1-trifluoropropan-2-yl)-1,3,5-triazine-2,4-diamine as a pale yellow solid (2.2 g, purity 98.7%, yield 51%). The mother liquor was concentrated to give 6-(6-(methylsulfonyl)pyridin-2-yl)-N2,N4-bis((R)-1,1,1-trifluoropropan-2-yl)-1,3,5-triazine-2,4-diamine as a yellow solid (ca. 1.5 g, ca. 90 a% / 220 nm). 1H-NMR (400 MHz, CDCl3) δ 8.57 (t, J = 8.0 Hz, 1 H), 8.21 (d, J = 8.0 Hz, 1 H), 8.11 (t, J = 8 Hz, 1 H), 5.68 (d, J = 12 Hz, 1 H), 5.31 (d, J = 8 Hz, 1 H), 5.11 (bs, 1 H), 4.88 (m, 1 H), 3.34 (s, 3 H), 1.43 (dd, J = 8.0, 4.0 Hz, 6 H). 13 C-NMR (100 MHz, CDCl3) δ 169.24, 166.18, 158.23, 154.38, 139.17, 127.46, 125.45 (q), 122.69, 47.71, 40.01, 14.34. LC-MS(ESI):m / z 459[M+H] +

[0194] Example 8 In humans 14 C]Oral administration of vorasidenib and [ 13 C3 15 N3]Characterization of the absorption, distribution, metabolism, and excretion of vorasidenib with simultaneous intravenous microdosing Metabolite profiling and identification of vorasidenib (AG-881) was performed after a single oral dose of 50 mg (100 μCi) of [14C]AG-881. 13 C3 15 N3]AG-881 was administered intravenously in small doses at 24 h after immunization with plasma, urine, and feces samples from five healthy subjects.

[0195] Plasma samples collected at selected time points 0-336 hours post-dose were pooled across subjects to generate area under the concentration-time curve (AUC) representative samples for 0-72 hours and 96-336 hours. Urine and fecal samples were pooled within subjects to generate individual urine and fecal pools. Plasma, urine, and fecal samples were extracted as appropriate, extracts were profiled using high performance liquid chromatography (HPLC), and metabolites were identified by liquid chromatography-mass spectroscopy (LC-MS and / or LC-MS / MS) analysis and retention time comparison with standards, when available.

[0196] Due to the low radioactivity in the samples, metabolite profiling in plasma was performed using accelerator mass spectrometry (AMS). In plasma, AG-881 increased 100% with a pooled AUC 0~72h and AUC 96~336h The most abundant radioactive peaks (P7; M458) accounted for 66.24% and 29.47% of the total radioactivity in plasma, respectively. 0~72 and AUC 96~336h They accounted for 10% and 43.92% of the total radioactivity in plasma, respectively. All other radioactive peaks accounted for less than 6% of the total radioactivity in plasma and were not identified.

[0197] The majority of the radioactivity recovered in feces was associated with unchanged AG-881 (55.5% of the administered dose), while AG-881 was not detected in urine. In comparison, metabolites in excreta accounted for approximately 18% of the dose in feces and 4% of the dose in urine. M515, M460-1, M499, M516 / M460-2, and M472 / M476 were the most abundant metabolites in feces, each accounting for approximately 2-5% of the radioactive dose. Meanwhile, M266 was the most abundant metabolite identified in urine, accounting for an average of 2.54% of the dose. The remaining radioactive components in urine and feces each accounted for less than 1% of the dose.

[0198] Overall, from the data presented, [ 14C]AG-881 was shown to undergo moderate metabolism following a single oral dose of 50 mg (100 μCi) and to be eliminated in humans by a combination of metabolism and excretion of the unchanged parent compound. Metabolism of AG-881 involved conjugation with glutathione (GSH) via oxidation and replacement of the chlorine with a chloropyridine moiety. Subsequent biotransformation of the GSH intermediate resulted in the removal of both glutamic acid and glycine to form cysteinyl conjugates (M515 and M499). The cysteinyl conjugates were further transformed by a series of biotransformation reactions, including oxidation, S-dealkylation, S-methylation, S-oxidation, S-acetylation, and N-dealkylation to form multiple metabolites.

[0199] A summary of the observed metabolites is shown in Table 2.

[0200] [Table 2]

[0201] Table 3 contains a summary of the protonated molecular ions and characteristic product ions for AG-881 and the identified metabolites.

[0202] [Table 3] JPEG2024507752000047.jpg213161

[0203] Proposed (theoretical) biotransformation pathways leading to the observed metabolites are shown in FIG.

[0204] Example 9 Enzyme assays In vitro assay for IDH1m (R132H or R132C) inhibitors The experimental procedures that can be used to obtain the data in columns 2 and 4 of Table 4 and column 2 of Table 5 are described below.

[0205] In the primary reaction, reduction of α-KG acid to 2HG is accompanied by the simultaneous oxidation of NADPH to NADP. The amount of NADPH remaining at the end of the reaction time is measured in a secondary diaphorase / resazurin reaction in which NADPH is consumed in a 1:1 molar ratio with the conversion of resazurin to the highly fluorescent resorufin. Uninhibited reactions show weak fluorescence at the end of the assay, whereas reactions in which consumption of NADPH by R132H IDH1 has been inhibited with a small molecule show strong fluorescence.

[0206] The primary reaction was carried out in a volume of 50 μL of 1× buffer (150 mM NaCl, 20 mM Tris7.5, 10 mM MgCl2, 0.05% (w / v) bovine serum albumin) containing 0.25 ug / mL (2.7 nM) IDH1wt / IDH1R132H heterodimer, 0.3 mM alpha-ketoglutarate, 4 μM NADPH, and either 300 μM NADP (saturated) or 30 μM NADP (unsaturated), and 1 uL of 50× compound in DMSO. The compound, enzyme and coenzyme mixture is preincubated at room temperature for 1 hour before adding α-ketoglutarate. To carry out the secondary reaction, 10 uL of 1× buffer containing 36 μg / mL diaphorase and 30 mM resazurin is added to the primary reaction and incubated for an additional 5 minutes at 25°C. Fluorescence is read on a Spectramax plate reader with Ex544 Em590. Compounds or compound dilutions are prepared in 100% DMSO concentration and diluted 1:50 in the final reaction. IDH1wt / IDH1R132C is assayed under similar conditions except that 1x buffer is 50mM K2HPO4 pH 6.5; 10mM MgCl2; 10% glycerol; 0.03% (w / v) bovine serum albumin with final concentrations of 0.4ug / mL (4.3nM) IDH1wt / IDH1R132C heterodimer, 0.02mM α-ketoglutarate, 4uM NADPH, and 300μM NADP (saturating) or 30μM NADP (non-saturating). IC50 is determined.

[0207] IDH1 or IDH2 wild type (wt) and mutant heterodimers are expressed and purified by methods known in the art. For example, IDH1wt / R132m heterodimers are expressed and purified as follows: IDH1wt-his and IDH1R132C-flag are co-expressed in sf9 insect cells. Cells (25 g) are resuspended in 250 mL of 50 mM Tirs, 500 mM NaCl pH 7.4 with stirring at 4° C. Cells are disrupted by passing four times through a M-Y110 Microfluidics set at 500 psi, then centrifuged at 22,000 rcf for 20 minutes at 4° C. The supernatant is collected and purified with HisTrap FF500 equilibrated with 50 mM Tirs, 500 mM NaCl pH 7.4. * Load onto a 1 ml column (GE) at 15 cm / h. Host cell contamination is removed by washing the column to baseline with equilibration buffer, followed by equilibration buffer containing 20 mM imidazole and 60 mM imidazole. Elute IDH1wt-his homodimer and IDH1wt-his / IDH1R132C-flag with equilibration buffer containing 250 mM imidazole. Pool fractions eluted with 250 mM imidazole and load onto a column pre-packed with ANTI-FLAG® M2 Affinity Gel (Sigma) at 15 cm / h, equilibrate the column with 50 mM Tris, 500 mM NaCl pH 7.4. After washing with equilibration buffer, elute IDH1wt-his / IDH1R132C-flag heterodimer with equilibration buffer containing flag peptide (0.2 mg / mL). Aliquots of IDH1wt-his / IDH1R132C-flag are snap frozen in liquid N2 and stored at -80° C. The same conditions are used for purification of IDH1wt-his / IDH1R132H-flag.

[0208] In vitro assay for IDH1m (R132H or R132C) inhibitors The experimental procedures that can be used to obtain the data in columns 3 and 6 of Table 4 are described below.

[0209] Test compounds are prepared as 10 mM stocks in DMSO and diluted to 50x final concentration in DMSO for a 50 μl reaction mixture. IDH enzyme activity, which converts alpha-ketoglutarate to 2-hydroxyglutarate, is measured using an NADPH depletion assay, in which the remaining coenzyme is measured at the end of the reaction by adding a catalytic excess of diaphorase and resazurin to generate a fluorescent signal proportional to the amount of NADPH remaining. IDH1-R132 homodimeric enzyme is diluted to 0.125 μg / ml in 40 μl of assay buffer (150 mM NaCl, 20 mM Tris-Cl pH 7.5, 10 mM MgCl2, 0.05% BSA, 2 mM b-mercaptoethanol), 1 μl of test compound dilution in DMSO is added, and the mixture is incubated at room temperature for 60 minutes. The reaction is initiated by adding 10 μl of substrate mix (20 μl NADPH, 5 mM alpha-ketoglutarate in assay buffer) and the mixture is incubated for 90 minutes at room temperature. The reaction is terminated by adding 25 μl of detection buffer (36 μg / mL diaphorase, 30 mM resazurin in 1× assay buffer), incubated for 1 minute, and then read on a SpectraMax plate reader at Ex544 / Em590.

[0210] Compounds are assayed for their activity against IDH1R132C following the same assay as above with the following modifications: Assay buffer is 50 mM potassium phosphate pH 6.5, 40 mM sodium carbonate, 5 mM MgCl2, 10% glycerol, 2 mM b-mercaptoethanol and 0.03% BSA. Concentrations of NADPH and α-ketoglutarate in substrate buffer are 20 μM and 1 mM, respectively.

[0211] In vitro assay for IDH1m (R132H or R132C) inhibitors The experimental procedures that can be used to obtain the data in columns 3 and 5 of Table 5 are described below.

[0212] Test compounds are prepared as 10 mM stocks in DMSO and diluted to 50x final concentration in DMSO for a 50 μl reaction mixture. IDH enzyme activity, which converts alpha-ketoglutarate to 2-hydroxyglutarate, is measured using an NADPH depletion assay, in which the remaining coenzyme is measured at the end of the reaction, where a catalytic excess of diaphorase and resazurin is added to generate a fluorescent signal proportional to the amount of NADPH remaining. IDH1-R132 homodimeric enzyme is diluted to 0.125 μg / ml in 40 μl of assay buffer (150 mM NaCl, 20 mM Tris-Cl pH 7.5, 10 mM MgCl2, 0.05% BSA, 2 mM b-mercaptoethanol) containing 5 μM NADPH and 37.5 μM NADP, 1 μl of test compound dilution in DMSO is added, and the mixture is incubated at room temperature for 60 minutes. The reaction is initiated by adding 10 μl of substrate mix (20 μl NADPH, 5 mM alpha-ketoglutarate in assay buffer) and the mixture is incubated for 60 minutes at room temperature. The reaction is terminated by adding 25 μl of detection buffer (36 μg / mL diaphorase, 30 mM resazurin in 1× assay buffer), incubated for 1 minute, and then read on a SpectraMax plate reader at Ex544 / Em590.

[0213] Compounds are assayed for their activity against IDH1R132C following the same assay as above with the following modifications: IDH1-R132 homodimeric enzyme is diluted to 0.1875 μg / ml in 40 μl of assay buffer (50 mM potassium phosphate pH 6.5, 40 mM sodium carbonate, 5 mM MgCl2, 10% glycerol, 2 mM b-mercaptoethanol and 0.03% BSA) containing 5 uM NADPH and 28.75 uM NADP. The concentration of alpha-ketoglutarate in substrate buffer is 1 mM.

[0214] In vitro assay for IDH2mR140Q inhibitors The experimental procedures used to obtain the data in column 7 of Table 4 are described below.

[0215] Compounds are assayed for IDH2R140Q inhibitory activity by a coenzyme depletion assay. Compounds are pre-incubated with the enzyme, and then the reaction is initiated by the addition of NADPH and α-KG and allowed to proceed for 60 minutes under conditions previously demonstrated to be linear with respect to the time that both coenzyme and substrate are consumed. The reaction is terminated by the addition of a second enzyme, diaphorase, and the corresponding substrate, resazurin. Diaphorase reduces resazurin to highly fluorescent resorufin and simultaneously oxidizes NADPH to NADP. Both terminate the IDH2 reaction by depleting the available coenzyme pool, facilitating quantification of the amount of coenzyme remaining after a specific period of time by quantitatively generating an easily detected fluorescent dye.

[0216] Specifically, 1 μl of a 100× compound dilution series is added to each of 12 wells of a 384-well plate, followed by 40 μl of buffer (50 mM potassium phosphate (K2HPO4) pH 7.5, 150 mM NaCl, 10 mM MgCl2, 10% glycerol, 0.05% bovine serum albumin, 2 mM beta-mercaptoethanol) containing 0.25 μg / ml IDH2R140Q protein. The test compound is then incubated with the enzyme for 1 hour at room temperature. The IDH2 reaction is then initiated by adding 10 μl of substrate mix containing 4 μM NADPH and 1.6 mM α-KG in the above-mentioned buffer. After a further 16 h incubation at room temperature, the reaction is stopped and the remaining NADPH is measured by converting resazurin to resorufin by adding 25 μl of StopMix (36 μg / ml diaphorase enzyme and 60 μM resazurin in buffer). After 1 min incubation, the plate is read in a plate reader at Ex544 / Em590.

[0217] To measure the inhibitory potency of compounds against IDH2R140Q in an assay format similar to that described above, a similar procedure is followed except that the final test concentrations were 0.25 μg / mL IDH2R140Q protein, 4 μM NADPH and 1.6 mM α-KG.

[0218] To measure the inhibitory potency of compounds against IDH2R140Q in a high-throughput screening format, a similar procedure is performed, except that 0.25 μg / mL IDH2R140Q protein is utilized in the pre-incubation step and the reaction is initiated by the addition of 4 μM NADPH and 8 μM α-KG.

[0219] In vitro assay for IDH2mR140Q inhibitors The experimental procedures used to obtain the data in column 6 of Table 5 are described below.

[0220] Compounds are assayed for IDH2R140Q inhibitory activity by a coenzyme depletion assay. Compounds are preincubated with the enzyme and coenzyme, and the reaction is then initiated by the addition of α-KG and allowed to proceed for 60 minutes under conditions previously proven to be linear. The reaction is terminated by the addition of a second enzyme, diaphorase, and the corresponding substrate, resazurin. Diaphorase reduces resazurin to highly fluorescent resorufin and simultaneously oxidizes NADPH to NADP. Both terminate the IDH2 reaction by depleting the available coenzyme pool, facilitating quantification of the amount of coenzyme remaining after a specific period of time by quantitatively generating an easily detected fluorescent dye.

[0221] Specifically, 1 μl of 50× compound dilution series is added to each of 12 wells of a 384-well plate, followed by 40 μl of buffer (50 mM potassium phosphate (K2HPO4) pH 7.5, 150 mM NaCl, 10 mM MgCl2, 10% glycerol, 0.05% bovine serum albumin, 2 mM beta-mercaptoethanol) containing 0.39 μg / ml IDH2R140Q protein, 5 uM NADPH, and 750 uM NADP. The test compound is then incubated with the enzyme and coenzyme at room temperature for 16 hours. The IDH2 reaction is then initiated by adding 10 μl of substrate mix containing 8 mM α-KG (final concentration 1.6 mM) in the above-mentioned buffer. After a further 1 hour incubation at room temperature, the reaction is stopped and the remaining NADPH is measured by converting resazurin to resorufin by adding 25 μl of StopMix (36 μg / ml diaphorase enzyme and 60 μM resazurin in buffer). After 1 minute incubation, the plate is read in a plate reader at Ex544 / Em590.

[0222] Data from the R132H enzyme assay, R132C enzyme assay, R140Q enzyme assay, R132C cell-based assay and R140Q cell-based assay described above or similar data for various compounds according to one embodiment of the disclosure are presented in Tables 4 and 5 below.

[0223] [Table 4]

[0224] [Table 5]

[0225] Having thus described several aspects of several embodiments, it should be understood that various changes, modifications, and improvements will readily occur to those skilled in the art. Such changes, modifications, and improvements are intended to be part of this disclosure and to be within the spirit and scope of the invention. Accordingly, the foregoing description and drawings are by way of example only.

Claims

1. In purified form, 【Chemistry 1】 or a pharma- ceutically acceptable salt thereof.

2. The compound is in purified form. 【Chemistry 2】 or a pharma- ceutically acceptable salt thereof.

3. The compound is in purified form. 【Chemistry 3】 or a pharma- ceutically acceptable salt thereof.

4. The compound is in purified form. 【Chemistry 4】 or a pharma- ceutically acceptable salt thereof.

5. The compound is in purified form. 【Chemistry 5】 or a pharma- ceutically acceptable salt thereof.

6. The compound is in purified form. 【Chemistry 6】 or a pharma- ceutically acceptable salt thereof.

7. The compound is in purified form. 【Chemistry 7】 or a pharma- ceutically acceptable salt thereof.

8. The compound is in purified form. 【Chemistry 8】 or a pharma- ceutically acceptable salt thereof.

9. The compound according to any one of claims 1 to 8 or a pharma- ceutically acceptable salt thereof. Pharmaceutical compositions.

10. The compound is in purified form. 【Chemistry 9】 or a pharma- ceutically acceptable salt thereof.

11. The compound is in purified form. 【Chemistry 10】 or a pharma- ceutically acceptable salt thereof.

12. The compound is in purified form. 【Chemistry 11】 or a pharma- ceutically acceptable salt thereof.

13. The compound is in purified form. 【Chemistry 12】 or a pharma- ceutically acceptable salt thereof.

14. The compound is in purified form. 【Chemistry 13】 or a pharma- ceutically acceptable salt thereof.

15. The compound is in purified form. 【Chemistry 14】 or a pharma- ceutically acceptable salt thereof.

16. The compound is in purified form. 【Chemistry 15】 or a pharma- ceutically acceptable salt thereof.

17. 17. The pharmaceutical composition according to any one of claims 9 to 16 for treating cancer, wherein said cancer is characterized by the presence of at least one mutation selected from IDH1 mutations or IDH2 mutations.

18. The compound is in purified form. 【Chemistry 16】 or a pharma- ceutically acceptable salt thereof.

19. The compound is in purified form. 【Chemistry 17】 or a pharma- ceutically acceptable salt thereof.

20. The compound is in purified form. 【Chemistry 18】 or a pharma- ceutically acceptable salt thereof.

21. The compound is in purified form. 【Chemistry 19】 or a pharma- ceutically acceptable salt thereof.

22. The compound is in purified form. 【Chemistry 20】 or a pharma- ceutically acceptable salt thereof.

23. The compound is in purified form. 【Chemistry 21】 or a pharma- ceutically acceptable salt thereof.

24. The compound is in purified form. 【Chemical 22】 or a pharma- ceutically acceptable salt thereof.

25. 25. The pharmaceutical composition of any one of claims 17 to 24, wherein the cancer is selected from glioma (including low-grade glioma), glioblastoma (including secondary glioblastoma), grade II or III astrocytoma, grade II or III oligodendroglioma, acute myeloid leukemia (AML), sarcoma, melanoma, non-small cell lung cancer (NSCLC), cholangiocarcinoma, chondrosarcoma, myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN), colon cancer, and angioimmunoblastic non-Hodgkin's lymphoma (NHL).

26. 26. The pharmaceutical composition of claim 25, wherein the cancer is glioma.

27. 27. The pharmaceutical composition of claim 26, wherein the glioma is a low-grade glioma or a high-grade glioma.

28. The pharmaceutical composition according to any one of claims 9 to 16, for treating glioma.

29. The compound is in purified form. 【Chemistry 23】 or a pharma- ceutically acceptable salt thereof.

30. The compound is in purified form. 【Chemistry 24】 or a pharma- ceutically acceptable salt thereof.

31. The compound is in purified form. 【Chemistry 25】 or a pharma- ceutically acceptable salt thereof.

32. The compound is in purified form. 【Chemistry 26】 or a pharma- ceutically acceptable salt thereof.

33. The compound is in purified form. 【Chemistry 27】 or a pharma- ceutically acceptable salt thereof.

34. The compound is in purified form. 【Chemistry 28】 or a pharma- ceutically acceptable salt thereof.

35. The compound is in purified form. 【Chemical 29】 or a pharma- ceutically acceptable salt thereof.

36. The pharmaceutical composition according to any one of claims 28 to 35, wherein said glioma is characterized by the presence of at least one mutation selected from an IDH1 mutation and an IDH2 mutation.

37. The pharmaceutical composition of any one of claims 17 to 27 and 36, wherein the mutation is an IDH1 mutation.

38. 38. The pharmaceutical composition of claim 37, wherein the IDH1 mutation is an R132X mutation.

39. 39. The pharmaceutical composition of claim 38, wherein the IDH1 mutation is a R132H or R132C mutation.

40. The pharmaceutical composition of any one of claims 17 to 27 and 36, wherein the mutation is an IDH2 mutation.

41. 41. The pharmaceutical composition of claim 40, wherein the IDH2 mutation is an R140X or an R172X mutation.

42. 42. The pharmaceutical composition of claim 41, wherein the IDH2 mutation is an R140Q, R140W or R140L mutation.

43. 42. The pharmaceutical composition of claim 41 , wherein the IDH2 mutation is a R172K or R172G mutation.

44. 44. The pharmaceutical composition of any one of claims 9 to 43, wherein the compound or a pharma- ceutically acceptable salt thereof is administered to a patient at about 1 to 5000 mg / day.

45. 45. The pharmaceutical composition of claim 44, wherein the compound or a pharma- ceutically acceptable salt thereof is administered to a patient at about 1 to 1000 mg / day.

46. 45. The pharmaceutical composition of claim 44, wherein the compound or a pharma- ceutically acceptable salt thereof is administered to a patient at about 1 to 500 mg / day.

47. A pharmaceutical composition according to any one of claims 9 to 46 for oral administration.

48. A pharmaceutical composition according to any one of claims 9 to 47 for use in combination with an additional therapeutic procedure.

49. 49. The pharmaceutical composition of claim 48, wherein the additional therapeutic procedure is selected from radiation, surgical resection, anti-cancer medication, anti-epileptic medication, anti-convulsant medication, and anti-emetic medication.

50. 50. The pharmaceutical composition of claim 49, wherein the anti-cancer chemotherapy is selected from cytotoxic or cytostatic chemotherapy, targeted drug therapy, antibody therapy, immunotherapy and hormonal therapy.

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