Therapeutic active compounds and methods of use thereof

Abstract

This invention provides compounds useful for treating cancer and methods for treating cancer. [Solution] The following compounds, etc., or pharmaceutically acceptable salts thereof, in a purified form are provided. TIFF2026113537000079.tif33169 TIFF2026113537000080.tif39169 TIFF2026113537000081.tif34169

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit and priority of U.S. Provisional Application No. 63 / 149,075, filed on February 12, 2021, and U.S. Provisional Application No. 63 / 217,843, filed on July 2, 2021. The contents of these applications are hereby incorporated by reference in their entirety into this specification.

[0002] Background of the Invention Isocitrate dehydrogenase (IDH) catalyzes the oxidative decarboxylation of isocitrate to 2 - oxoglutarate (i.e., α - ketoglutarate). These enzymes belong to two different subclasses. One of them utilizes NAD(+) as an electron acceptor, and the other utilizes NADP(+). Five isocitrate dehydrogenases have been reported. Three are NAD(+)-dependent isocitrate dehydrogenases localized in the mitochondrial matrix, and two are NADP(+)-dependent isocitrate dehydrogenases. One of them is in the mitochondria and the other is mainly in the cytosol. Each NADP(+)-dependent isozyme is a homodimer.

[0003] IDH1 (isocitrate dehydrogenase 1 (NADP+), cytosolic) is also known as IDH; IDP; IDCD; IDPC or PICD. The protein encoded by this gene is a NADP(+)-dependent isocitrate dehydrogenase found in the cytoplasm and peroxisomes. This protein contains a PTS - 1 peroxisome targeting signal sequence. Since this enzyme is present in peroxisomes, it is suggested to play a role in peroxisomal reduction, such as the regeneration of NADPH for the conversion of 2,4 - dienoyl - CoA to 3 - enoyl - CoA and peroxisomal reactions that consume 2 - oxoglutarate, i.e., the α - hydroxylation of phytanic acid. The cytosolic enzyme plays an important role in cytoplasmic NADPH production.

[0004] The human IDH1 gene encodes a protein of 414 amino acids. The nucleotide sequence and amino acid sequence for human IDH1 can be found as GenBank entries NM_005896.2 and NP_005887.2, respectively. The nucleotide sequence and amino acid sequence 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] The non-mutated form, e.g., wild-type IDH1, for example, in the forward reaction, catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate, thereby reducing NAD + (NADP + ) to NADH (NADPH). Isocitrate + NAD + (NADP + ) → α-KG + CO2 + NADH (NADPH) + H +

[0006] It has been discovered that a mutation in IDH1 present in certain cancer cells confers a new ability to an enzyme that catalyzes the NADPH-dependent reduction of α-ketoglutarate to R(-)-2-hydroxyglutarate (2HG). The production of R(-)-2-hydroxyglutarate (2HG) is thought to contribute to cancer formation and progression (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 strongly associate or interact with the pyruvate dehydrogenase complex. The human IDH2 gene encodes a protein of 452 amino acids. The nucleotide sequence and amino acid sequence for IDH2 can be found as GenBank entries NM_002168.2 and NP_002159.2, respectively. The nucleotide sequence and amino acid sequence for human IDH2 are also described, for example, in Huh et al., Submitted (NOV-1992) to the EMBL / GenBank / DDBJ databases and The MGC Project Team, Genome Res. 14:2121-2127 (2004).

[0008] Non-mutated forms, such as wild-type IDH2, for example, in the forward reaction, catalyze the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG), thereby reducing NAD + (NADP + ) to NADH (NADPH). Isocitrate + NAD + (NADP + ) → α-KG + CO2 + NADH (NADPH) + H +

[0009] Mutations in IDH2 present in certain cancer cells have been found to result in a novel ability of the enzyme that catalyzes the NADPH-dependent reduction of α-ketoglutarate to R(-)-2-hydroxyglutarate (2HG). R(-)-2-hydroxyglutarate (2HG) is not formed by wild-type IDH2. The production of R(-)-2-hydroxyglutarate (2HG) is thought to contribute to cancer formation and progression (Dang, L et al., Nature 2009, 462:739-44). Therefore, inhibition of mutant IDH1 and / or mutant IDH2 and their novel activity may be a potential therapeutic treatment for cancer.

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

[0011] Boracidenib (AG-881) is described in US Patent No. 9,579,324. This patent is fully incorporated herein by reference. To better understand the clinical activity of boracidenib and to provide potential novel inhibitors of mutant IDH1 / IDH2, it is necessary to identify potential bioactive boracidenib metabolites that remain in the human body after boracidenib administration.

[0012] Summary of the Invention This specification describes compounds 1 to 7 (e.g., compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, and compound 7) and their pharmaceutically acceptable salts. [ka]

[0013] A compound selected from compounds 1 to 7, or a pharmaceutical salt thereof, or one of the embodiments described herein, inhibits at least one of mutant IDH1 or mutant IDH2. This specification also describes pharmaceutical compositions comprising a compound selected from compounds 1 to 7 or a pharmaceutical salt thereof, and methods of using such compositions to treat cancer characterized by the presence of at least one of mutant IDH1 or mutant IDH2.

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

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

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

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

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

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

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

[0021] In one embodiment, the present invention provides a pharmaceutical composition comprising one or more compounds selected from compounds 1 to 7 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.

[0022] In one embodiment, the pharmaceutical composition, [ka] or a pharmaceutically acceptable salt thereof.

[0023] In one embodiment, the pharmaceutical composition, [ka] or a pharmaceutically acceptable salt thereof.

[0024] In one embodiment, the pharmaceutical composition, [ka] or a pharmaceutically acceptable salt thereof.

[0025] In one embodiment, the pharmaceutical composition, [ka] or a pharmaceutically acceptable salt thereof.

[0026] In one embodiment, the pharmaceutical composition, [ka] or a pharmaceutically acceptable salt thereof.

[0027] In one embodiment, the pharmaceutical composition, [ka] or a pharmaceutically acceptable salt thereof.

[0028] In one embodiment, the pharmaceutical composition, [ka] or a pharmaceutically acceptable salt thereof.

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

[0030] In one embodiment, the method involves using a therapeutically effective amount of purified [ka] or administering a pharmaceutically acceptable salt thereof to the patient.

[0031] In one embodiment, the method involves using a therapeutically effective amount of purified [ka] or administering a pharmaceutically acceptable salt thereof to the patient.

[0032] In one embodiment, the method involves using a therapeutically effective amount of purified [ka] or administering a pharmaceutically acceptable salt thereof to the patient.

[0033] In one embodiment, the method involves using a therapeutically effective amount of purified [ka] or administering a pharmaceutically acceptable salt thereof to the patient.

[0034] In one embodiment, the method involves using a therapeutically effective amount of purified [ka] or administering a pharmaceutically acceptable salt thereof to the patient.

[0035] In one embodiment, the method involves using a therapeutically effective amount of purified [ka] or administering a pharmaceutically acceptable salt thereof to the patient.

[0036] In one embodiment, the method involves using a therapeutically effective amount of purified [ka] or administering a pharmaceutically acceptable salt thereof to the patient.

[0037] In one embodiment, the present invention provides a method for treating cancer, comprising administering to a patient in need of treatment a composition comprising a therapeutically effective amount of one or more compounds selected from purified compounds 1 to 7 or a pharmaceutically acceptable salt thereof, wherein the cancer is characterized by the presence of at least one mutation selected from IDH1 mutations or IDH2 mutations.

[0038] In one embodiment of the method 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 lymphoma (NHL) in the patient.

[0039] In one embodiment of the method 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 is provided for treating a glioma in a subject requiring treatment of the glioma, comprising administering to the subject one or more compounds selected from purified compounds 1 to 7 or a pharmaceutically acceptable salt thereof in a therapeutically effective amount.

[0041] In one embodiment, a method for treating gliomas involves using a therapeutically effective amount of purified [ka] This includes administering a pharmaceutically acceptable salt thereof to a subject requiring treatment for a glioma.

[0042] In one embodiment, a method for treating gliomas involves using a therapeutically effective amount of purified [ka] This includes administering a pharmaceutically acceptable salt thereof to a subject requiring treatment for a glioma.

[0043] In one embodiment, a method for treating gliomas involves using a therapeutically effective amount of purified [ka] This includes administering a pharmaceutically acceptable salt thereof to a subject requiring treatment for a glioma.

[0044] In one embodiment, a method for treating gliomas involves using a therapeutically effective amount of purified [ka] This includes administering a pharmaceutically acceptable salt thereof to a subject requiring treatment for a glioma.

[0045] In one embodiment, a method for treating gliomas involves using a therapeutically effective amount of purified [ka] This includes administering a pharmaceutically acceptable salt thereof to a subject requiring treatment for a glioma.

[0046] In one embodiment, a method for treating gliomas involves using a therapeutically effective amount of purified [ka] This includes administering a pharmaceutically acceptable salt thereof to a subject requiring treatment for a glioma.

[0047] In one embodiment, a method for treating gliomas involves using a therapeutically effective amount of purified [ka] This includes administering a pharmaceutically acceptable salt thereof to a subject requiring treatment for a glioma.

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

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

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

[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 pharmaceutically acceptable salt thereof administered to the patient is 1 to 5000 mg / day. In certain embodiments, the amount of the compound or a pharmaceutically acceptable salt thereof administered to the patient is 1 to 2000 mg / day. In certain embodiments, the amount of the compound or a pharmaceutically acceptable salt thereof administered to the patient is 1 to 1000 mg / day. In some embodiments, the amount of the compound or a pharmaceutically acceptable salt thereof administered to the patient is 1 to 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 additional therapeutic means. In certain embodiments, the additional therapeutic means is selected from radiation, surgical resection, anticancer agents, antiepileptic agents, anticonvulsants, and antiemetics.

[0056] In some embodiments, the anticancer agent is selected from chemotherapy with cytotoxic or cytostatic agents, targeted agents, antibody therapy, immunotherapy, and hormone therapy. [Brief explanation of the drawing]

[0057] [Figure 1] Figure 1 shows several proposed theoretical in vivo conversion pathways for AG-881 (ivosidenib) in humans.

[0058] Detailed explanation Details of the structure and arrangement of components described in the following description or shown in the drawings are not intended to be limiting. Other embodiments and different methods for carrying out the subject matter of this application are expressly included. The language and terminology used herein are also for illustrative purposes only and should not be considered limiting. The use of “including,” “comprising,” “having,” “containing,” “involving,” and variations thereof herein means to include the items listed below and their equivalents, as well as additional items.

[0059] definition The term "body fluids" includes amniotic fluid, aqueous humor, blood (e.g., whole blood or plasma), cerebrospinal fluid, earwax, atherosclerotic fluid, Cowper's fluid, feces, female ejaculate, interstitial fluid, lymph, breast milk, mucus (e.g., nasal discharge or sputum), 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, as well as prevention. Inhibitors may also completely or partially inhibit their intended target.

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

[0062] As used herein, the term “daily dose” refers to the total amount of the therapeutic agent administered over any 24-hour period and is used interchangeably with “amount / day.” For example, “daily dose of 100 mg” or “100 mg / day dose” or “100 mg / day amount” refers to administering a total of 100 mg of the therapeutic agent to a patient over any 24-hour period. The daily dose may be administered once daily (i.e., QD or once every day or every 24 hours) or divided into multiple doses to be administered at various times over a 24-hour period (e.g., BID or twice daily or every 12 hours; TD or three times daily or every 8 hours; QID or four times daily or every 6 hours, etc.). Each dose (e.g., daily dose or divided dose) may 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). For example, a daily dose of 1000 mg (i.e., 1000 mg / day) administered twice daily (BID) can be administered in two dosage forms (e.g., capsules or tablets), each containing 250 mg of the therapeutic agent (e.g., a compound selected from purified compounds 1-7 or a pharmaceutically acceptable salt thereof) every 12 hours.

[0063] As used herein, an effective amount of a compound to treat a disorder, or a "therapeutically effective amount," means an amount of the compound that, with a single or multiple administration to a subject, is effective in treating cells or healing, alleviating, reducing, or improving a subject with a disorder in a greater extent than would be expected without 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) or normal subjects with a disorder, e.g., a disorder as described herein. 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). In one aspect of the present invention, the term “non-human animal” 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, cattle, pigs, etc.

[0065] As used herein, the terms “purified,” “in a purified form,” or “in an isolated and purified form” in relation to a compound refer to the physical state of the compound after it has been physically separated from a synthesis process (e.g., reaction mixture), a natural source or body fluid, or a combination thereof, and / or subjected to a purification process or procedure. The “one or more purification processes” mentioned herein are either described herein or are well known to those skilled 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 well known to those skilled in the art. In some embodiments, the purified compound does not need to be physically separated from the reaction mixture or body fluid before purification; for example, the reaction mixture or body fluid is subjected to a purification process, and the desired compound is isolated in a purified form after the completion of the purification process. In further embodiments, the compound may be subjected to multiple purification processes as applicable. For example, the purification techniques disclosed herein yield isolated and purified forms of the target compounds 1-7. Such isolation and purification techniques are expected to yield compound purities of approximately 90 wt% or higher (for example, purities exceeding 90 wt%, 95 wt%, 97 wt%, 98 wt%, or 99 wt%).

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

[0067] Compounds 1-7 are metabolites that have been observed in one or more bodily fluids, or that have been proposed to be obtained when borasidenib (AG-881) is orally administered to humans, or that are newly synthesized. 14 C] Single oral administration of AG-881 and [ 13 C3 15 Example 8 describes a study to profile and identify borasidenib and its metabolites in selected plasma, urine, and fecal samples collected from human subjects after simultaneous microintravenous administration of N3]AG-881.

[0068] Compounds 1 to 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 to 7 are described in Examples 1 to 7.

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

[0070] Compound 1 is formed by treating borasidenib with thiomethoxide (e.g., sodium thiomethoxide) in a suitable organic solvent (e.g., a polar aprotic organic solvent, e.g., dimethyl sulfoxide). This reaction can be carried out at a temperature of 0 to 30°C and, if applicable, under an inert atmosphere (e.g., a nitrogen atmosphere).

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

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

[0073] Compound 3 can be obtained by dissolving borasidenib (AG-881) in a suitable organic solvent (e.g., a polar aprotic organic solvent, e.g., dimethyl sulfoxide) and treating it with a sulfide reagent (e.g., sodium sulfide). 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 2-amino-3-chloropropanoic acid of appropriate high optical purity in the presence of a base (e.g., an organic amine base, e.g., N,N-diisopropylethylamine) in an organic solvent (e.g., a polar aprotic organic solvent, e.g., N,N-dimethylformamide). The reaction can be carried out at a temperature of 20°C to 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 borasidenib (AG-881) by the method generally shown in Scheme 3. [ka]

[0075] Compound 1 is formed by treating borasidenib with thiomethoxide (e.g., sodium thiomethoxide) in a suitable organic solvent (e.g., a polar aprotic organic solvent, e.g., dimethyl sulfoxide). This reaction can be carried out at a temperature of 0 to 30°C and, if applicable, under an inert atmosphere (e.g., a nitrogen atmosphere).

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

[0077] Compounds 1-7 contain one or more chiral centers and may therefore result in, isolate, or synthesize, single enantiomers or individual stereoisomers that are substantially free from racemates, racemic mixtures, scaremic mixtures, diastereomer mixtures, and other possible enantiomers or stereoisomers. As used herein, the term "substantially free from other stereoisomers" means a preparation in which the compound having the selected stereochemistry at one or more selected stereocenters is concentrated by at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%. The term "concentrated" means that at least a specified proportion of the preparation is a compound having the 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 the final compound or starting materials or intermediates.

[0078] In certain embodiments, compounds 1 to 7 are concentrated in one or more structures having selected stereochemistry at one or more carbon atoms. For example, the compounds are concentrated in certain stereoisomers by at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.

[0079] Furthermore, compounds 1 to 7 can also be prepared by one or more isotopic substitutions. For example, H is... 1 H, 2 H (D or deuterium) and 3 It can be any isotopic form containing H (T or tritium), and C is 11 C, 12 C, 13 C and 14 It can be any isotopic form containing C, and N is 13 N, 14 N and 15 It can be any isotopic form containing N, and O is, 15 O, 16 O and 18 It can be any isotopic form containing O, and F is, 18 For example, a compound can be any isotopic form containing F. For instance, a compound is concentrated to a specific isotopic form of H, C, N, O and / or F by at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%. A specific isotope-labeled compound (e.g., 3 H and 14 Compounds labeled with 1C are useful for tissue distribution assays of compounds and / or substrates. Tritium isotopes (i.e., 3 H) and carbon-14 isotopes (i.e., 14 C) is particularly preferred because it is easy to prepare and detect. Furthermore, heavier isotopes, such as deuterium (i.e., 2Substitution with H) can provide certain therapeutic advantages resulting from higher metabolic stability (e.g., extended half-life in vivo or reduced required dose), and is therefore preferable in some situations. Isotope-labeled compounds can generally be prepared by methods similar to those disclosed in the examples, by substituting a suitable isotope-labeling reagent for a non-isotope-labeling reagent.

[0080] It may be convenient or desirable to prepare, purify, and / or handle the corresponding salt of the active compound, for example, a pharmaceutically acceptable salt. Examples of pharmaceutically 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 be anionic or may have an anionic functional group (e.g., -COOH, -COO - If it has (which may be the case), the salt can form with a suitable cation. Examples of suitable inorganic cations are alkali metal ions, for example, Na + and K + , alkaline earth cations, for example, Ca 2+ and Mg 2+ In addition, other cations, for example, Al 3+ This includes, but is not limited to, the following: A suitable example of an organic cation is the ammonium ion (i.e., NH4). + ) and substituted ammonium ions (e.g., NH3R + NH2R2 + NHR3 + NR4 +This includes, but is not limited to, ) . Some suitable examples of substituted ammonium ions are derived from ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. A common example of a quaternary ammonium ion is N(CH3)4 + That is the case.

[0082] A compound is cationic or may be cationic (e.g., -NH2, -NH3) + If it has (which may be the case), the salt can 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, nitrite, phosphoric acid, and phosphorous acid.

[0083] 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, camphor sulfonic 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. Suitable examples of high-molecular-weight organic anions include, but are not limited to, those derived from the following high-molecular-weight acids: tannic acid and carboxymethylcellulose.

[0084] Accordingly, the compounds provided herein include the compounds themselves and, where applicable, their salts, hydrates, and prodrugs. 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 biopenetration into a given biological compartment (e.g., blood, lymphatic system, central nervous system), improve oral availability, improve solubility to enable 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 pharmaceutically acceptable derivatives, which, when administered to a subject, can provide an active compound. Calcium phosphate and sodium phosphate of each of compounds 1-7 are expressly included herein where applicable. Amino acid (e.g., valine) esters of each of compounds 1-7 are expressly included herein where applicable.

[0085] Composition and route of administration The compounds used in the methods described herein may be incorporated into a pharmaceutically acceptable composition together with a pharmaceutically acceptable carrier or adjuvant before being administered to the target. In one embodiment, such a pharmaceutically acceptable composition further comprises an additional therapeutic agent in an amount effective to achieve the modulation of the disease or disease symptoms, including those described herein.

[0086] The term "pharmaceutically acceptable carrier or adjuvant" refers to a carrier or adjuvant that can be administered to a subject together with a compound according to one embodiment of the present invention, without destroying its pharmacological activity, and which is non-toxic when administered in a dose sufficient to deliver a therapeutic amount of the compound.

[0087] pharmaceutically acceptable carriers, adjuvants, and media that can be used in a pharmaceutical composition according to 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 polymer 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, polyacrylate, wax, polyethylene-polyoxypropylene-block polymer, polyethylene glycol, and lanolinic tallow. Furthermore, cyclodextrins, such as α-, β-, and γ-cyclodextrins, or chemically modified derivatives, such as hydroxyalkylcyclodextrins including 2- and 3-hydroxypropyl-β-cyclodextrin, or other solubilized derivatives, can also be advantageously used to enhance the delivery of compounds in the formulations described herein.

[0088] A pharmaceutical composition according to one aspect of the present invention may be administered orally, parenterally, or by inhalation spray topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir, preferably by oral administration or injection. A pharmaceutical composition according to one aspect of the present invention may contain any conventional non-toxic, pharmaceutically acceptable carrier, adjuvant, or medium. Optionally, the pH of the formulation may be adjusted with a pharmaceutically acceptable acid, base, or buffer to enhance the stability of the compound or its delivery form. As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intra-synovial, intrasternal, intrathecal, and intracranial injection or infusion techniques.

[0089] The pharmaceutical composition may be in the form of a sterile injectable formulation, for example, a sterile, injectable aqueous or oily suspension. This suspension may be formulated according to techniques known in the art using a suitable dispersant or wetting agent (e.g., Tween 80) and a suspending agent. The sterile injectable formulation may also be a sterile, injectable solution or suspension in a non-toxic, parenterally acceptable diluent or solvent, for example, in a solution in 1,3-butanediol. Among acceptable media and solvents, mannitol, water, Ringer's solution, and isotonic sodium chloride solutions can be used. In addition, sterile non-volatile oils have been conventionally used as solvents or suspension media. For this purpose, any sterile volatile oil, including synthetic monoglycerides or diglycerides, can be used. Fatty acids, such as oleic acid and its glyceride derivatives, are useful in the preparation of injectable formulations, and pharmaceutically acceptable natural oils, such as olive oil or castor oil, especially polyoxyethylated versions, are also useful. Furthermore, these oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, or carboxymethylcellulose, or similar dispersants commonly used in the formulation of pharmaceutically 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 the manufacture of pharmaceutically acceptable solids, liquids, or other dosage forms, may also be used for formulation purposes.

[0090] In certain embodiments, one of compounds 1 to 7 or a pharmaceutically acceptable salt thereof is used in a composition comprising one of the compounds described herein (e.g., one or more of compounds 1 to 7) or a pharmaceutically acceptable salt thereof, and one or more polymers as part of a solid dispersant (e.g., an amorphous solid dispersant). In some embodiments, the solid dispersant comprises a compound selected from compounds 1 to 7 or a pharmaceutically acceptable salt thereof, and one or more polymers. In some embodiments, the solid dispersant comprises one of compounds 1 to 7 or a pharmaceutically acceptable salt thereof, one or more polymers, and one or more surfactants. In some embodiments, the solid dispersant comprises one of compounds 1 to 7 or a pharmaceutically acceptable salt thereof, and one polymer. In some embodiments, the solid dispersant comprises one of compounds 1 to 7 or a pharmaceutically acceptable salt thereof, one polymer, and a surfactant.

[0091] In some embodiments, at least a portion of the active ingredients in the solid dispersant (e.g., one of compounds 1 to 7 or a pharmaceutically acceptable salt thereof) 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 dispersant is substantially free of crystalline compounds.

[0092] In some embodiments, the composition is an amorphous solid (e.g., spray-dried) dispersant comprising one of compounds 1 to 7 or a pharmaceutically acceptable salt thereof and a polymer. The amorphous solid dispersant 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 1% of crystalline compounds (substantially free of, for example, crystalline compounds selected from compounds 1 to 7 or pharmaceutically acceptable salts thereof).

[0093] Examples of polymers in solid dispersants include 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)); ethylcellulose or cellulose acetate phthalate); polyvinylpyrrolidone (PVP); polyethylene glycol (PEG); polyvinyl alcohol (PVA); polyvinyl esters. For example, this includes polyvinyl acetate phthalate (PVAP); acrylates, for example, polymethacrylate (e.g., Eudragit.RTM.E); cyclodextrins (e.g., beta-cyclodextrin); poly(D,L-lactide) (PLA), poly(D,L-lactide, co-glycolidic acid) (PLGA), and their copolymers and derivatives, for example, polyvinylpyrrolidone-vinyl acetate (PVP-VA), polyvinylcaprolactam-polyvinyl and acetate-polyethylene glycol copolymer, methyl acrylate / methacrylate copolymer; Soluplus; copovidone, and mixtures thereof.

[0094] In some embodiments, the solid dispersant comprises at least one water-soluble polymer. In some embodiments, the solid dispersant 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 another embodiment, the polymer is cyclodextrin. In some embodiments, the solid dispersant 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 salts, 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 crosslinked polymer, such as polyvinylpyrrolidone (e.g., crospovidone). In yet another embodiment, the polymer is polyvinylpyrrolidone (PVP).

[0099] In some embodiments, the compound (e.g., a compound selected from compounds 1 to 7) or a pharmaceutically acceptable salt thereof is present in the solid dispersant in an amount of about 10% w / w to 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 to 7) or a pharmaceutically acceptable salt thereof is present in the solid dispersant in an amount of about 10% w / w to about 80% w / w, for example, 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, for example, 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 dispersant also includes a surfactant or an inert pharmaceutically acceptable substance. Examples of surfactants in solid dispersants include sodium lauryl sulfate (SLS), vitamin E or its derivatives (e.g., vitamin E TPGS), sodium docusate, 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 dispersant in an amount of about 0.1% w / w to about 10% w / w, for example, 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 total weight of the active ingredient (e.g., a compound selected from compounds 1 to 7 or a pharmaceutically acceptable salt thereof), polymer and surfactant is 100%.

[0101] In some embodiments, solid dispersants can be prepared according to 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, rotational evaporation, freeze-drying (i.e., lyophilization), vacuum drying, melt solidification, and melt extrusion. One embodiment of the present disclosure includes a solid dispersant obtained by spray drying. In one embodiment, the product obtained by spray drying is dried to remove the solvent or solvent mixture.

[0102] Preparations disclosed herein, for example, pharmaceutical compositions, can be obtained by spray-drying a mixture comprising a compound selected from compounds 1 to 7 or a pharmaceutically acceptable salt thereof, one or more polymers, and a suitable solvent or solvent mixture. Spray-drying includes, for example, atomization of a liquid mixture containing a solid and a solvent or solvent mixture, and removal of the solvent or solvent mixture. The solvent or solvent mixture may also include a non-volatile solvent, such as glacial acetic acid. Atomization can be performed, for example, through a two-fluid nozzle, 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, spray drying is fluidized spray drying (FSD).

[0104] In a particular embodiment, a method for preparing a solid dispersant of a compound selected from compounds 1 to 7 or a pharmaceutically acceptable salt thereof is: a) Forming a compound (for example, a compound selected from compounds 1 to 7) or a pharmaceutically acceptable salt thereof, polymer, and solvent mixture; b) The mixture is spray-dried to form a solid dispersant containing the compound and polymer.

[0105] Post-drying and / or polishing of the wet spray-drying dispersant may be performed as needed to ensure that the residual solvent is below ICH or specified specifications.

[0106] These methods can be used to prepare the pharmaceutical compositions disclosed herein. The amounts and characteristics of the components used in these methods may be as disclosed herein or as determined by those skilled in the art.

[0107] In certain embodiments, a pharmaceutical composition comprising a solid dispersant can be prepared by the method described herein. For example, the pharmaceutical composition may include (a) a compound selected from compounds 1 to 7 or a pharmaceutically acceptable salt thereof, and (b) a solid dispersant comprising 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 administered orally in any orally acceptable dosage form, including but not limited to capsules, tablets, emulsions, and aqueous suspensions, dispersants, and liquids. 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. When aqueous suspensions and / or emulsions are administered orally, the active ingredient can be suspended or dissolved in an oily phase and combined with emulsifiers and / or suspending agents. Specific sweeteners and / or flavorings and / or colorings may be added as needed.

[0109] In some embodiments, a compound selected from compounds 1 to 7 or a pharmaceutically acceptable salt thereof is administered orally in amounts of 1 to 5000 mg / day (e.g., 1 to 1000 mg / day, 1000 to 2000 mg / day, 2000 to 3000 mg / day, 3000 to 4000 mg / day, or 4000 to 5000 mg / day). In one embodiment, the compound is administered in amounts of 1 to 1000 mg / day (e.g., 1 to 500 mg / day, 500 to 1000 mg / day). In another embodiment, the compound is administered in amounts of 1 to 500 mg / day (e.g., 1 to 100 mg / day, 100 to 200 mg / day, 200 to 300 mg / day, 300 to 400 mg / day, 400 to 500 mg / day). In one embodiment, the compound is administered in amounts of 500-1000 mg / day (e.g., 500-750 mg / day, 750-1000 mg / day). In another embodiment, the compound is administered in amounts of 1000-2000 mg / day (e.g., 1000-1500 mg / day, 1500-2000 mg / day). In yet another embodiment, the compound is administered in amounts of 2000-3000 mg / day (e.g., 2000-2500 mg / day, 2500-3000 mg / day). In yet another embodiment, the compound is administered in amounts of 3000-4000 mg / day (e.g., 3000-3500 mg / day, 3500-4000 mg / day). In one embodiment, the compound is administered in amounts 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 pharmaceutically acceptable salt thereof is administered orally in amounts 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 pharmaceutically acceptable salt thereof is administered orally in amounts 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, a compound selected from compounds 1 to 7 or a pharmaceutically acceptable salt thereof is administered orally in amounts of 1 to 500 mg / day, 20 to 400 mg / day, 40 to 200 mg / day, 50 to 150 mg / day, 75 to 125 mg / day, 85 to 115 mg / day, 90 to 110 mg / day, or about 100 mg / day. In some embodiments, a compound selected from compounds 1 to 7 or a pharmaceutically acceptable salt thereof is administered orally in amounts of 1 to 500 mg / day, 50 to 400 mg / day, 100 to 300 mg / day, 150 to 250 mg / day, 175 to 225 mg / day, 185 to 215 mg / day, 190 to 210 mg / day, or about 200 mg / day. In some embodiments, a compound selected from compounds 1 to 7 or a pharmaceutically acceptable salt thereof is administered orally in amounts of 1 to 500 mg / day, 100 to 500 mg / day, 200 to 400 mg / day, 250 to 350 mg / day, 275 to 375 mg / day, 285 to 315 mg / day, 290 to 310 mg / day, or approximately 300 mg / day. In some embodiments, the daily dose of a compound selected from compounds 1 to 7 or a pharmaceutically acceptable salt thereof is administered in a single dose (i.e., in one dosage form) or in one or more divided doses over a 24-hour period (i.e., in two or more dosage forms). In some embodiments, each daily dose or divided dose may be administered as a single dosage form or as multiple dosage forms (e.g., two or more dosage forms administered at each dose) to facilitate 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, the compounds disclosed herein (e.g., compounds selected from compounds 1 to 7) or pharmaceutically acceptable salts thereof are administered once daily in amounts 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.

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

[0112] Topical administration of a pharmaceutical composition according to one aspect of the present invention is useful when the desired treatment involves an area or organ that is easily accessible by topical application. For topical application to the skin, the pharmaceutical composition should be formulated into a suitable ointment containing the active ingredient suspended or dissolved on a carrier. Carriers for topical administration of the compound according to one aspect of the present invention include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compounds, emulsifying wax, and water. Alternatively, the pharmaceutical composition should be formulated into a suitable lotion or cream containing the active compound suspended or dissolved on a carrier containing a suitable emulsifier. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water. Furthermore, the pharmaceutical composition according to one aspect of the present invention can also be applied topically to the lower intestinal tract by rectal suppositories or suitable enema formulations. Topical 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 may be prepared according to well-known techniques in the art of pharmaceutical formulations and may be prepared as liquids in physiological saline using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons and / or other solubilizers or dispersants known in the art.

[0114] Alternatively, the compounds described herein may be administered, for example, by injection, intravenous, intra-arterial, subcutaneous, intraperitoneal, intramuscular, or subcutaneously, or orally, buccal, intranasal, transmucosal, topically, in ophthalmic preparations, or by inhalation, with doses ranging from about 0.5 to about 100 mg / kg body weight, or alternatively, every 4 to 120 hours or according to the requirements of the particular drug, ranging from 1 mg to 1000 mg / dose. The methods described herein are intended to administer an effective amount of the compound or a composition of the compound to achieve the desired or described effect. Typically, a pharmaceutical composition of one aspect of the present invention would be administered about 1 to about 6 times per day, or alternatively, as a continuous infusion. Such administration can be used as chronic or acute therapy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated and the specific mode of administration. Typical preparations will contain about 5% to about 95% active compound (w / w). Alternatively, such preparations contain approximately 20% to 80% of the active compound.

[0115] Lower or higher doses than those mentioned above may be required. The specific dose and treatment plan for any particular subject will depend on various factors, including the activity of the specific compound used, age, weight, general health status, sex, diet, administration time, excretion rate, concomitant use of drugs, severity and course of the disease, condition, or symptoms, the subject's constitution to the disease, condition, or symptoms, and the judgment of the treating physician.

[0116] Once the subject's condition has improved, a maintenance dose of a compound, composition, or combination according to one embodiment of the present invention may be administered as needed. Thereafter, the dose, frequency, or both of the administration may be reduced as a function of the symptoms to a level at which the improved condition is maintained when the symptoms have been alleviated to a desired level. However, if the symptoms of the disease recur, the subject may require long-term, intermittent treatment.

[0117] The pharmaceutical composition described above, comprising a compound selected from compounds 1 to 7, a pharmaceutically acceptable salt thereof, or a compound described in any one of the embodiments herein, may further comprise another therapeutic agent useful for treating cancer.

[0118] If the compositions of this disclosure include a combination of the compounds of the formulations described herein and one or more additional therapeutic or prophylactic agents, both the compounds and the additional agents should be present at dose levels of about 1 to 100%, more preferably about 5 to 95%, of the doses typically administered in a monotherapy regimen. The additional agents may be administered separately from the compounds of one embodiment of the present invention as part of a multi-dose regimen. Alternatively, these agents may be part of a single dosage form and may be mixed together with the compounds of one embodiment of the present invention in a single composition.

[0119] How to use A method is provided for inhibiting the activity of mutant IDH1 and / or mutant IDH2, comprising contacting a target to which the inhibition of said activity is to be desired with a compound selected from compounds 1 to 7 or a pharmaceutically acceptable salt thereof.

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

[0121] In one embodiment, the cancer being treated is characterized by a mutant allele of IDH1, where the IDH1 mutation results in a novel 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 the R132X mutation. In another aspect of this embodiment, the R132X mutation is selected from R132H, R132C, R132L, R132V, R132S, and R132G. In yet another aspect, the R132X mutation is R132H or R132C. To determine the presence and specific nature of the mutation at amino acid 132 of IDH1 (e.g., the altered amino acid present), the cancer can be analyzed by sequencing a cell sample.

[0122] While not bound by theory, the applicant believes that mutant alleles of IDH1, particularly the R132H mutation of IDH1, which imparts a novel 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 within the body. For this reason, the compounds and methods of the present disclosure are useful for treating all types of cancer characterized by the presence of such activity-conferring mutant alleles of IDH1, particularly the IDH1R132H or R132C mutations.

[0123] In one embodiment, cancer is a tumor in which at least 30, 40, 50, 60, 70, 80, or 90% of tumor cells have IDH1 mutations, particularly IDH1R132H or R132C mutations, at the time of diagnosis or treatment.

[0124] The IDH1R132X mutation is 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, chondrosarcoma, myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPNs), colon cancer, and angioimmunoblastic non-Hodgkin lymphoma (NHL). Therefore, in one embodiment, the method described herein is used to treat gliomas (including low-grade gliomas), glioblastomas (including secondary glioblastomas), grade II and III astrocytomas, grade II and III oligodendrogliomas, acute myeloid leukemia, sarcomas, melanomas, non-small cell lung cancer (NSCLC), cholangiocarcinoma, chondrosarcoma, myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPNs), colon cancer, or angioimmunoblastic non-Hodgkin lymphoma (NHL) in patients.

[0127] In another embodiment, the methods described herein may be used to treat gliomas (including low-grade gliomas), glioblastomas (including secondary glioblastomas), grade II and III astrocytomas, grade II and III oligodendrogliomas, acute myeloid leukemia, sarcomas, melanomas, non-small cell lung cancer (NSCLC), cholangiocarcinoma (e.g., intrahepatic cholangiocarcinoma (IHCC)), chondrosarcoma, myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPNs), prostate cancer, chronic myelomonocytic leukemia (CMML), B-acute lymphoblastic leukemia (B-ALL), myelosarcoma, multiple myeloma, lymphoma, colon cancer, or angioimmunoblastic non-Hodgkin lymphoma (NHL) in patients.

[0128] In another embodiment, the method described herein is used to treat advanced hematological malignancies. In one embodiment, the advanced hematological malignancies treated are lymphomas (e.g., non-Hodgkin lymphoma (NHL)), B-cell lymphomas (e.g., Burkitt lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, progenitor B-lymphoblastic lymphoma and mantle cell lymphoma) and T-cell lymphomas (e.g., mycosis fungoides, anaplastic large cell lymphoma and progenitor T-lymphoblastic lymphoma).

[0129] Therefore, in one embodiment, the cancer is a cancer selected from any one of the cancer types listed in Table 1 or a cancer 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 target subject requiring such inhibition with one of the compounds of this disclosure (for example, a compound selected from compounds 1 to 7) or a pharmaceutically acceptable salt thereof.

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

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

[0133] While not bound by theory, the applicant believes that mutant alleles of IDH2, particularly the R140Q and / or R172K mutations of IDH2, which result in a novel 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 properties or location within the body. For this reason, compounds and methods according to one aspect of the present invention are useful for treating all types of cancer characterized by the presence of such activity-constituting mutant alleles of IDH2, particularly the IDH2R140Q and / or R172K mutations.

[0134] In one embodiment, cancer is a tumor in which at least 30, 40, 50, 60, 70, 80, or 90% of tumor cells have IDH2 mutations, particularly IDH2R140Q, R140W, or R140L and / or R172K or R172G mutations, at the time of diagnosis or treatment.

[0135] In another embodiment, one aspect of the present invention provides a method for 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 to 7) or a pharmaceutically acceptable salt thereof in an amount effective for treating the cancer. In more specific embodiments, the cancers treated are gliomas (including low-grade gliomas), glioblastomas (including secondary glioblastomas), astrocytomas of grade II and III, oligodendrogliomas of grade II and III, myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPNs), acute myeloid leukemia (AML), melanoma, chondrosarcoma, or angioimmunoblastic non-Hodgkin lymphoma (NHL).

[0136] The treatment methods described herein may further include various evaluation steps before and / or after treatment with the compounds of this disclosure (e.g., compounds selected from compounds 1 to 7) or pharmaceutically acceptable salts thereof.

[0137] In one embodiment, the Method further includes, before and / or after treatment with a compound of the Disclosure (e.g., a compound selected from compounds 1 to 7) or a pharmaceutically acceptable salt thereof, a step of evaluating the growth, size, weight, invasiveness, stage and / or other phenotype of the tumor using one or more techniques known and used by those skilled in the art.

[0138] In one embodiment, the method further includes a step of evaluating the IDH1 or IDH2 genotype of cancer before and / or after treatment with a compound of the disclosure (e.g., a compound selected from compounds 1 to 7) or a pharmaceutically acceptable salt thereof. This can be achieved by conventional methods in the art, such as DNA sequencing, immunoassay and / or evaluation of the presence, distribution or level of R(-)-2-hydroxyglutarate (2HG).

[0139] In one embodiment, before and / or after treatment with a compound of the Disclosure (e.g., a compound selected from compounds 1 to 7) or a pharmaceutically acceptable salt thereof, the Method further includes a step of determining the R(-)-2-hydroxyglutarlate (2HG) level in the subject. This can be achieved by spectroscopic analysis, e.g., magnetic resonance analysis, e.g., MRI and / or MRS measurement, analysis of a body fluid sample, e.g., serum or cerebrospinal fluid analysis, or analysis of a surgical material, e.g., mass spectrometry.

[0140] In one embodiment of this invention, the effectiveness 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 increase in the level of R(-)-2-hydroxyglutarate (2HG) (along with the confirmed IDH mutant status) is used to confirm the suitability of using a compound of the present disclosure (e.g., a compound selected from compounds 1 to 7) or a pharmaceutically acceptable salt thereof for treating cancer. Once an increase in the level is established, the level of R(-)-2-hydroxyglutarate (2HG) is determined during and / or after treatment to establish target involvement (i.e., inhibition of mutant IDH by administration of a compound of the present disclosure or a pharmaceutically acceptable salt thereof). In certain embodiments, the level of R(-)-2-hydroxyglutarate (2HG) is determined only during and / or after treatment. A decrease in R(-)-2-hydroxyglutarate (2HG) levels during and after treatment indicates target involvement. Typically, measurement of R(-)-2-hydroxyglutarate (2HG) would be used in conjunction with other well-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, improvement in the subject's general health, and changes in other biomarkers associated with the effectiveness of cancer treatment.

[0141] R(-)-2-hydroxyglutarate (2HG) can be detected in the sample by LC / MS. The sample is mixed with methanol in an 80:20 ratio and centrifuged at 3,000 rpm at 4°C for 20 minutes. The resulting supernatant can be collected and stored at -80°C before LC-MS / MS to assess the 2-hydroxyglutarate (2HG) level. Various liquid chromatography (LC) separation methods can be used. Each method can be coupled to a triple quadrupole mass spectrometer operating in multiple reaction monitoring (MRM) mode by negative electrospray ionization (ESI, -3.0kV), and the MS parameters are optimized with injected metabolite standard solutions. The metabolites can be separated by reverse-phase chromatography using 10 mM tributylamine as the ion-pairing agent in the aqueous mobile phase, following a modification of a previously reported method (Luo et al. J Chromatogr A 1147, 153-64, 2007). One method allows for 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 to 2-hydroxyglutarate (2HG) and involves a fast linear gradient of 50% to 5% B (the buffer defined above) over 5 minutes. As a column, Synergi Hydro-RP, 100 mm × 2 mm, particle size 2.1 μm (Phenomonex) can be used as described above. Metabolites can be quantified by comparing their peak area with that of a pure metabolite standard of known concentration. 13 Metabolite flux studies from C-glutamine can be conducted, 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 evaluated before treatment with a compound of the Disclosure (e.g., a compound selected from compounds 1 to 7) or a pharmaceutically acceptable salt thereof. In another embodiment, the concentration of R(-)-2-hydroxyglutarate (2HG) is evaluated after treatment with a compound of the Disclosure herein or a pharmaceutically acceptable salt thereof. In some embodiments, the evaluation of R(-)-2-hydroxyglutarate (2HG) is performed using biological fluids from a human patient. In other embodiments, the evaluation of R(-)-2-hydroxyglutarate (2HG) is performed using a biological sample from a biopsy or a tissue sample from a human patient. In some embodiments, 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 Disclosure, after treatment with a compound of the Disclosure, or both before and after treatment with a compound of the Disclosure.

[0143] In another embodiment, derivatives of R(-)-2-hydroxyglutarate (2HG) formed during the analytical process are evaluated. For example, such derivatives may be those formed in MS analysis. The 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 in MS analysis.

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

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

[0146] Also provided is a method for treating a disease selected from Maffucci syndrome and Orie's disease, characterized by the presence of a mutant allele of IDH1, the method comprising administering to a subject requiring treatment (a) a compound of the present disclosure (e.g., a compound selected from compounds 1 to 7) or a pharmaceutically acceptable salt thereof, or (b) a pharmaceutical composition comprising (a) and a pharmaceutically acceptable carrier.

[0147] Brain tumors treated by the method of the present invention In one embodiment, the method of the present invention is useful for treating brain tumors. This includes all tumors within the human skull (cranial cavity) or central spinal canal. Tumors may originate from the brain itself, but may also originate from lymphatic tissue, blood vessels, cranial nerves, the meninges of the brain, the skull, the pituitary gland, or the 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 present in other organs (metastatic tumors).

[0148] In some embodiments, brain tumors are gliomas, such as ependymoma, astrocytoma, 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, mitosis, endothelial proliferation, and necrosis. Tumors of grades I and II are called "low-grade gliomas" and either lack any of these features or have one of them, and include diffuse astrocytoma, pilocytic astrocytoma, low-grade astrocytoma, low-grade oligodendroglioma, low-grade oligodendroglioma, ganglioglioma, dysplastic neuroepithelial tumor, pleomorphic xanthoblastoma, and mixed glioma. Tumors of grade III and IV are called “high-grade gliomas” and 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 yet 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, brain tumors (e.g., gliomas (including low-grade gliomas), glioblastomas (including secondary glioblastomas), grade II or III astrocytomas, grade II or III oligodendrogliomas) are newly diagnosed. In further embodiments, brain tumors (e.g., gliomas (including low-grade gliomas), glioblastomas (including secondary glioblastomas), grade II or III astrocytomas, grade II or III oligodendrogliomas) are pre-treated by one or more therapeutic means, including surgery, radiotherapy, or one or more additional therapeutic agents. In other embodiments, brain tumors (e.g., gliomas (including low-grade gliomas), glioblastomas (including secondary glioblastomas), grade II or III astrocytomas, grade II or III oligodendrogliomas) are not treated by radiotherapy.

[0150] In some embodiments, the brain tumor being 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 novel ability of 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 yet 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), malignant grade II or III astrocytoma, malignant grade II or III oligodendroglioma) have IDH1R132X mutations, e.g., R132H, R132C, R132L, R132V, R132S, or R132G mutations at the time of diagnosis or treatment. Brain tumors (e.g., glioma (including low-grade glioma), glioblastoma (including secondary glioblastoma), malignant grade II or III astrocytoma, malignant grade II or III oligodendroglioma) can be analyzed by sequencing cell samples to determine the presence and specific nature of mutations in amino acid 132 of IDH1 (e.g., the altered amino acid present).

[0151] In other embodiments, the brain tumor being 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 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 novel ability of 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 an R140X mutation. In yet another aspect of these embodiments, the R140X mutation is an R140Q mutation. In another aspect of these embodiments, the R140X mutation is the R140W mutation. In another aspect of these embodiments, the R140X mutation is the R140L mutation. In another aspect of these embodiments, mutant IDH2 has the R172X mutation. In another aspect of these embodiments, the R172X mutation is the R172K mutation. In another aspect of these embodiments, the R172X mutation is the R172G mutation. In yet another embodiment 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 IDH2R140X and / or R172X mutations, e.g., R140Q, R140W, or R140L and / or R172K or R172G mutations 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, and grade II or III oligodendrogliomas) can be analyzed by sequencing cell samples to determine the presence and specific nature of mutations (e.g., altered amino acids present) at amino acids 140 and / or 172 of IDH2.

[0152] In further embodiments, the brain tumors being 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 IDH1 and IDH2 mutations, where the IDH1 and IDH2 mutations collectively result in the accumulation of R(-)-2-hydroxyglutarate (2HG) in the patient. In one aspect of these embodiments, the accumulation of R(-)-2-hydroxyglutarate (2HG) in the patient is achieved by the IDH1 and IDH2 mutations providing a novel ability of 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 brain tumor being 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 any combination of the aforementioned IDH1 and IDH2 mutations. In yet another embodiment of these embodiments, at least 30, 40, 50, 60, 70, 80, or 90% of brain tumor cells (e.g., glioma (including low-grade glioma), glioblastoma (including secondary glioblastoma), grade II or III astrocytoma, grade II or III oligodendroglioma) have IDH1R132X mutations, e.g., R132H, R132C, R132L, R132V, R132S, or R132G mutations and IDH2R140X and / or R172X mutations, e.g., R140Q, R140W, or R140L, and / or R172K, or R172G mutations 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, and grade II or III oligodendrogliomas) can be analyzed by sequencing cell samples to determine the presence and specific nature of mutations (e.g., altered amino acids present) at amino acid 132 of IDH1 and amino acids 140 and / or 172 of IDH2.

[0153] In further embodiments, the brain tumors treated (e.g., gliomas (including low-grade gliomas), glioblastomas (including secondary glioblastomas), grade II or III astrocytomas, and grade II or III oligodendrogliomas) are characterized by the presence of an IDH1 allele that does not contain the R132X mutation and an IDH2 allele that does not contain the R140X or R172X mutation. In one aspect of these embodiments, at least 90% of the cells of the brain tumors (e.g., gliomas (including low-grade gliomas), glioblastomas (including secondary glioblastomas), grade II or III astrocytomas, and grade II or III oligodendrogliomas) do not contain mutations at amino acid 132 of IDH1 or amino acid 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, and grade II or III oligodendrogliomas) can be analyzed by sequencing cell samples to determine the presence or absence of mutations in 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 an additional step of co-administering additional therapeutic means (e.g., additional cancer treatment agents, additional treatment agents to minimize cancer symptoms or side effects of cancer treatment, or additional cancer treatments) to subjects requiring such treatment. Exemplary additional cancer treatment agents (anti-cancer agents) include, for example, chemotherapy with cytotoxic or cytostatic agents, targeted therapies (targeting agents), antibody therapies, immunotherapies, and hormone therapies. Exemplary additional treatment agents to minimize symptoms and side effects (pharmacotherapy) include, for example, antiepileptic agents, anticonvulsants, and antiemetics. Additional cancer treatments include, for example, surgery and radiotherapy. Examples of each of these treatments are provided below.

[0155] When used herein in reference to additional cancer treatments, the term “concurrent administration” means that the additional cancer treatment may be administered together with the compound of one aspect of the invention as part of a single dosage form (e.g., a composition of one aspect of the invention comprising the compound of one aspect of the invention and the second therapeutic agent described above) or as separate dosage forms. Alternatively, the additional cancer treatment may be administered prior to, consecutively with, or after the administration of the compound of one aspect of the invention. In such combination therapy treatments, both the compound of one aspect of the invention and the second therapeutic agent are administered by conventional methods. Administering a composition of one aspect of the invention, comprising both the compound of one aspect of the invention and the second therapeutic agent, to a subject does not preclude the administration of the same therapeutic agent, any other second therapeutic agent, or any compound of one aspect of the invention separately to the subject at another point in the course of treatment. When used herein in reference to additional cancer treatments, the term “concurrent administration” means that the additional cancer treatment may be performed prior to, consecutively with, simultaneously with, or after the administration of the compound of one aspect of the invention.

[0156] In some embodiments, additional cancer treatment agents are chemotherapeutic agents. Examples of chemotherapeutic agents used in cancer treatment include, for example, antimetabolites (e.g., derivatives of folic acid, purines, and pyrimidines), alkylating agents (e.g., nitrogen mustard, nitrosourea, platinum, alkyl sulfonates, hydrazines, triazenes, aziridines, spindle toxins, cytotoxic agents, topoisomerase inhibitors, etc.) and hypomethylating agents (e.g., decitabine (5-aza-deoxycytidine), zebralin, isothiocyanates, azacitidine (5-azacitidine), 5-fluoro-2'-deoxycytidine, 5,6-dihydro-5-azacitidine, etc.). Examples of drugs include acralubicin, actinomycin, alitretinoin, altretamine, aminopterin, aminolevulinic acid, amrubicin, amsacrin, anagrelide, arsenic trioxide, asparaginase, atrasentan, belotecan, bexarotene, bendamustine, bleomycin, bortezomib, busulfan, camptothecin, capecitabine, carboplatin, carbocon, carmofur, carmustine, celecoxib, chlorambucil, chlormetine, cisplatin, cladribine, clofarabine, chrysanthanspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, decitabine, demecolsin, docetaxel, doxorubicin, efapoxiral, erescromol, erusamitrusine, enocitabine, epirubicin, and estramustine. Etoglucid, etoposide, floxuridine, fludarabine, fluorouracil (5FU), fotemustine, gemcitabine, gliadel implant, hydroxycarbamide, hydroxyurea, idarubicin, ifosfamide, irinotecan, irofluben, ixabépyrone, larotaxel, leucovorin, liposomal doxorubicin, liposomal daunorubicin, ronidamine, lomustine, ru Canton, Mannosulfan, Masopropyl, Melphalan, Mercaptopurine, Mesna, Methotrexate, Methyl aminolevulinate, Mitobronitol, Mitoguazone, Mitotane, Mitomycin, Mitoxantrone, Nedaplatin, Nimustine, Oblimarcene, Omasetaxin, Ortataxel, Oxaliplatin, Paclitaxel, Pegaspargaze, Pemetrexed, Pentostatin,Pirarubicin, Pixantrone, Plicamycin, Porfimer sodium, Prednimustine, Procarbazine, Lartitrexed, Ranimustine, Rubitecan, Sapacitabine, Semustine, Citimajenceradenovec, Strataplatin, Streptozocin, Talaporfin, Tegaflu-uracil, Temoporfin, Temozolomide, Teniposide, Tesetaxel, Testaractone, Tetranitrate, Thiotepa, This includes thiazophrine, thioguanine, tipifarnib, topotecan, trabectedin, triadicone, triethylenemelamine, triplatin, tretinoin, treosulfan, trophosphamide, uramustine, barrubicin, verteporfin, vinblastine, vincristine, vindesine, vinflunin, vinorelbine, vorinostat, zolubicin, and other cytostatic or cytotoxic agents described herein.

[0157] Some drugs are more effective when used in combination than alone, so in many cases, two or more drugs are provided simultaneously. In most cases, two or more chemotherapy agents are used as combination chemotherapy.

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

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

[0160] Furthermore, targeted therapies may also include small peptides that act as "homing devices" capable of binding to receptors on the cell surface or to the affected extracellular matrix surrounding the tumor. When a radionuclide attached to these peptides (e.g., RGD) decays near the cell, the cancer cells are ultimately killed. An example of such a therapy is BEXXAR®.

[0161] In some embodiments, the additional cancer treatment 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 intravesical BCG immunotherapy for superficial bladder cancer, as well as 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, because in many cases, the donor's immune cells will attack the tumor in the 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 treatment agent is a hormone therapy agent. The growth of some cancers can be inhibited by supplying or blocking specific hormones. Common examples of hormone-sensitive tumors include certain types of breast cancer and prostate cancer. The removal or blockade of estrogen or testosterone is often an important additional treatment. In certain cancers, the administration of hormone agonists, such as progestogens, may be therapeutically beneficial. In some embodiments, hormone therapy agents can be used in combination with the compounds or compositions described herein.

[0164] Other possible additional treatment options include imatinib, gene therapy, peptide vaccines and dendritic cell vaccines, synthetic chlorotoxins, and radiolabeled drugs and antibodies.

[0165] Additional treatment options for brain cancer Additional therapeutic means used in combination with the methods for treating brain cancer described herein include therapeutic means (e.g., surgery, radiation, therapeutic agents / drugs) that are known to be useful in treating brain tumors, i.e., having a therapeutic effect against brain tumors, alleviating one or more symptoms of brain tumors, altering the progression of brain tumors, eradicating brain tumors, reducing the size of brain tumors, slowing or inhibiting the growth of brain tumors, delaying or minimizing one or more symptoms associated with brain tumors, reducing the malignancy of brain tumors or inducing quiescence of brain tumors, or alleviating or minimizing one or more side effects associated with another therapy applied or administered to treat brain tumors.

[0166] In some embodiments, the additional treatment means is surgery.

[0167] In some embodiments, the additional treatment option is radiotherapy. In some embodiments, radiotherapy is administered in accordance with the National Comprehensive Cancer Network Clinical Practice Guidelines in OncoloGy (e.g., dose and administration schedule), version 1.2016, available at nccn.org. In some embodiments, radiotherapy is administered in fractions of 1.0 to 5.0 Gy, with a cumulative dose of 20 to 100 Gy. In some embodiments, radiotherapy is performed with a cumulative dose of 20-100 Gy, 30-80 Gy, 30-60 Gy, 40-70 Gy, 40-60 Gy, 30-40 Gy, 40-50 Gy, 50-60 Gy, or 45-55 Gy in 1.0-5.0 Gy fractions, 1.5-3.0 Gy fractions, 1.5-3.0 Gy fractions, 1.5-1.0 Gy fractions, 1.0-1.5 Gy fractions, 1.0-1.5 Gy fractions, 1.5-2.0 Gy fractions, 2.5-3.0 Gy fractions, 1.8-2.0 Gy fractions, or 2.0 Gy fractions.

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

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

[0170] In some embodiments, one or more additional therapeutic agents include one or more additional cancer therapeutic agents (i.e., anticancer agents) (e.g., DNA-reactive agents, PARP inhibitors, immunotherapies (e.g., checkpoint inhibitors), PVC chemotherapy, antibody therapies (e.g., bevacizumab), gemcitabine), antiemetics, anticonvulsants, or antiepileptic agents.

[0171] In some embodiments, one or more additional therapeutic agents are additional cancer treatment agents (e.g., anticancer drugs).

[0172] In some embodiments, the additional cancer treatment agent is a DNA-reactive agent. As used herein, “DNA-reactive agent” refers to agents such as alkylating agents, crosslinking agents, and DNA intercalating agents. These interact with cellular DNA covalently or non-covalently. For example, DNA-reactive drugs include adzeresin, altretamine, bizeresin, busulfan, carboplatin, carboquan, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, estramustine, fotemustine, hepsulfame, ifosfamide, improsulfan, ilofluben, lomustine, mechloretamine, melphalan, mitozolomide, nedaplatin, oxaliplatin, piposulfan, procarbazine, semustine, streptozocin, temozolomide, thiotepa, treosulfan, diethylnitrosamine, benzo(a)pyrene, doxorubicin, and mitomycin-C. Many of these DNA-reactive drugs are useful in cancer treatment as DNA-reactive chemotherapy agents.

[0173] In some embodiments, the DNA-reactive agent is temozolomide (TMZ). In one aspect of these embodiments, TMZ is administered in accordance with the National Comprehensive Cancer Network Clinical Practice Guidelines in OncoloGy (e.g., dose and administration schedule), version 1.2016, available at nccn.org. In one aspect of these embodiments, TMZ is administered in accordance with the prescribing information for TEMODAR® (temozolomide) capsules and TEMODAR® (temozolomide) for injection. In some aspects of these embodiments, TMZ is administered at a daily dose of 100–250 mg / m², 100–150 mg / m², 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 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 at a daily dose of 125–175 mg / m², based on the patient's body surface area, for five consecutive days within a 28-day treatment cycle. In some aspects of these embodiments, TMZ is administered in combination with radiotherapy at 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 radiotherapy 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 radiotherapy at a daily dose of 70–80 mg / m² for 42 days, based on the patient's body surface area. In some aspects of these embodiments, where the brain tumor is a high-grade glioma or glioblastoma, TMZ is administered in combination with radiotherapy at a daily dose of 70–80 mg / m² for 42 days, based on the patient's body surface area.In some embodiments of these models where the brain tumor is an anaplastic astrocytoma, TMZ is administered at a daily dose of 125–175 mg / m2 based on the patient's body surface area for five consecutive days of a 28-day treatment cycle. In some embodiments of these models where the brain tumor is an anaplastic astrocytoma, TMZ is administered at a daily dose of 175–225 mg / m2 based on the patient's body surface area for five consecutive days of a 28-day treatment cycle.

[0174] In some embodiments, one or more additional cancer treatment agents (anticancer 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, lucaparib, veraparib, iniparib, talazoparib, and niraparib.

[0175] In some embodiments, one or more additional cancer treatment agents (anticancer agents) are immunotherapies, such as checkpoint inhibitors. As used herein, “checkpoint inhibitor” refers to a treatment agent that inhibits an immune checkpoint (e.g., CTLA-4, PD-1 / PD-L1, etc.) that would in some way prevent the immune system from attacking cancer cells, thereby enabling 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, one or more additional cancer treatment agents (anti-cancer agents) constitute PVC chemotherapy. As used herein, “PVC chemotherapy” refers to a chemotherapy regimen comprising the combined administration of procarbazine, lomustine (marketed under the trade name CCNU®), and vincristine (marketed under the trade name Onocovin®). Typically, vincristine is administered intravenously, while procarbazine and lomustine are administered orally. PVC chemotherapy is often administered cyclically, where each cycle comprises a single dose of vincristine and lomustine, followed by a 10-day treatment with procarbazine.

[0177] In some embodiments, one or more additional cancer treatment agents (anticancer agents) are antibodies, such as bevacizumab. Bevacizumab, marketed under the trade name Avastin®, is a recombinant humanized monoclonal antibody.

[0178] In some embodiments, one or more additional cancer treatment agents (anticancer agents) are gemcitabine. Gemcitabine, marketed under the trade name Gemzar®, is a pyrimidine nucleoside analog.

[0179] In some embodiments, one or more additional therapeutic agents are antiemetics. As used herein, “antiemetic” refers to an agent effective in reducing symptoms of vomiting and nausea. Examples of antiemetics include 5-HT3 receptor antagonists (e.g., drasetron, granisetron, ondansetron, tropisetron, palonosetron, mirtazapine, etc.), dopamine agonists (e.g., domperidone, olanzapine, droperidol, haloperidol, chlorpromazine, prochlorperazine, arizaprid, prochlorperazine, metoclopramide, etc.), NK1 receptor antagonists (e.g., aprepitant, casopitant, lorapitant, etc.), and antihistamines (e.g., cinnarizine, cyclizine, dife). This includes benzodiazepines (e.g., dimenhydrinate, doxylamine, meclizine, promethazine, hydroxyzine, etc.), cannabinoids (e.g., cannabis, dronabinol, synthetic cannabinoids, etc.), 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, ajuwine, etc.

[0180] In some embodiments, one or more additional therapeutic agents are anticonvulsants or antiepileptic agents. As used herein, “anticeps or antiepileptic agents” refers to agents effective in treating or preventing seizures, including epileptic seizures. Examples of anticonvulsants include paraaldehyde, stiripentol, phenobarbital, methylphenobarbital, barbexacron, clobazam, clonazepam, chlorazepam, diazepam, midazolam, lorazepam, nitrazepam, temazepam, nimetazepam, potassium bromide, felbamate, carbamazepine, oxcarbazepine, eslicarbazepine acetate, valproic acid, sodium valproate, divalproex sodium, vigabatrin, progavid, thiagabin, topiramate, gaba Contains pentin, pregabalin, etotoin, phenytoin, mephenytoin, fosphenytoin, paramethadione, trimethadione, etadione, beclamide, primidone, buribaracetam, etilacetam, levetiracetam, celetracetam, ethosuximide, fenximide, mesximide, acetazolamide, sultiame, metazolamide, zonisamide, lamotrigine, phenetulide, phenasemide, valbromide, valoctamide, perampanel, stiripentol, pyridoxine, etc.

[0181] Examples General experimental considerations In the following examples, reagents (chemical substances) were purchased from commercial suppliers (e.g., Alfa, Acros, Sigma Aldrich, TCI, and Shanghai Chemical Reagent Company) and used without further purification. Nuclear magnetic resonance (NMR) spectra were obtained using a Brucker AMX-400 NMR spectrometer (Brucker, Switzerland). Chemical shifts were reported at parts per million (ppm, δ) of a low magnetic field from tetramethylsilane. Mass spectra were obtained by electrospray ionization (ESI) using a Waters LCT TOF mass spectrometer (Waters, USA) or a Shimadzu LCMS-2020 mass spectrometer (Shimadzu, Japan). Microwave reactions were carried out using 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 the preparation of the compound such that it is concentrated at least about 90%, 95%, 96%, 97%, 98%, or 99% at the specified stereocenter. The chemical names of each exemplary compound listed below are generated by ChemDraw software.

[0183] List of Abbreviations general anhy. aq. Water-based min hrs time mL (milliliter) nmol (millimol) mole 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 doublet of quintets dd doublet of doublets dt doublet of triplets

[0185] Solvents and Reagents DAST Diethylaminosulfur trifluoride CHCl3 chloroform DCM dichloromethane DMF dimethylformamide Et2O diethyl ether EtOH ethanol EtOAc ethyl acetate MeOH methanol 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 hexamethyldisilazide LiHMDS lithium hexamethyldisilazide LAH Lithium Aluminum Hydrogen 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(diphenylphosphin)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(diphenylphosphin)ferrocene HATU O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium BINAP 2,2'-bis(diphenylphosphanyl)-1,1'-binaphthyl MTBE Methyl tert-butyl ether NaSMe sodium thiomethoxide

[0186] Example 1 Preparation of 6-(6-(methylthio)pyridine-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-chloropyridine-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 packed into a 1 L flask under N2 conditions at 15-20°C. The resulting solution was stirred at 15-20°C for 10 minutes to form a clear brown solution. This reaction solution was cooled to 5°C, and sodium thiomethoxide (NaSMe, 35.6 g, 0.508 mol) was added gradually over 20 minutes at 5-10°C. The reaction mixture was stirred at 20-25°C for 3 hours. While stirring, the mixture was poured into ice water (3 L) at 5-10°C. After 30 minutes at 20-25°C, the solid was filtered, and the moist cake was ground with water (1.5L) for 30 minutes at 20-25°C. This solid was filtered, washed with water (200mL), and dried in a vacuum oven at 50-55°C to give 6-(6-(methylthio)pyridine-2-yl)-N2,N4-bis((R)-1,1,1-trifluoropropan-2-yl)-1,3,5-triazine-2,4-diamine as an off-white solid (100.8g, yield 98%). 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)pyridine-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)pyridine-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 packed into a 250 mL flask at 10-20°C. The reaction mixture was stirred for 10 minutes to obtain a clear solution. To this reaction solution, an aqueous solution of Oxon (4.6 g, 7.48 mmol, 40 mL of water) was added dropwise over 30 minutes at 5-15°C. The resulting mixture was stirred at 20-30°C for 2 hours. 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, 20 mL of water) was added [the addition is exothermic]. The phases were separated, the organic phase was washed with water (1 × 40 mL), dried over anhydrous Na2SO4, filtered, and concentrated under vacuum at 40-45°C to obtain the crude product as an oily substance. The residue was purified by silica gel chromatography (eluent: n-heptane / siRNA-5:1 to 1:1). The combined pure fraction was concentrated to dryness under vacuum to give the desired product as a white solid. This product was dissolved in methanol (30 mL) at 40-45°C, and then recrystallized by adding water (60 mL) over a period of 30 minutes at 20-30°C. The resulting suspension was stirred for a further 0.5 hours at 20-30°C, then filtered, washed with water (10 mL), and dried in a vacuum oven at 50-55°C to give 6-(6-(methylsulfinyl)pyridine-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, yield 53%). 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 13C-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 = 8.1 Hz), 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)

Chemical Structure

[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-triazine-2-yl)pyridine-2-thiol (1.0 g, 2.43 mmol) were packed into 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 all at once. The resulting mixture was stirred at -20°C for 1 hour, and the solid was filtered. The filtrate was poured into water (50 mL), and the resulting solution was extracted with ethyl acetate (1 × 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 (eluate: 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 obtain 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, yield 82%). 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-triazine-2-ylpyridine-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-triazine-2-yl)pyridine-2-thiol (1.0 g, 2.43 mmol) were packed into a 25 mL flask under N2 conditions at 20-30 °C. Then, a solution of L-2-amino-3-chloropropanoic acid (0.60 g, 4.86 mmol) in water (5 mL) was added dropwise to the reaction mixture at 20-30 °C. The resulting mixture was heated to 60 °C and stirred for 20 hours. After stirring at 60 °C for 20 hours, the mixture was cooled to 20 °C and poured into water (30 mL). The obtained slurry was stirred for 15-30 minutes, then the solid was isolated by vacuum filtration and washed with water (10 mL). The solid was dried on a filter under air for 1-2 hours, and then dissolved in DMSO (30 mL). The product solution in DMSO was purified by preparation HPLC [column: YMCTA C18, 250 × 21.2 mm, 10 μm; flow rate: 15 mL / min; gradient: 20% acetonitrile - 80% water 0.1% TFA to 70% acetonitrile - 30% water 0.1% TFA; @254 / 205 nm]. The pure fractions containing the product were combined and concentrated under vacuum at 45-50°C to remove the solvent. Next, the product was freeze-dried to give (R)-2-amino-3-((6-(4,6-bis(((R)-1,1,1-trifluoropropan-2-yl)amino)-1,3,5-triazine-2-yl)pyridine-2-yl)thio)propanoic acid as a white solid (170 mg, 98.9% / 220 nm, yield 14%). 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-triazine-2-yl)pyridine-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-triazine-2-yl)pyridine-2-thiol (1.0 g, 2.43 mmol) were packed into a 25 mL flask under N2 conditions at 20-30 °C. Then, a solution of L-2-amino-3-chloropropanoic acid (0.60 g, 4.86 mmol) in water (5 mL) was added dropwise to the reaction mixture at 20-30 °C. The resulting mixture was heated to 60 °C and stirred for 20 hours. After stirring at 60 °C for 20 hours, the mixture was cooled to 20 °C and poured into water (30 mL). The obtained slurry was stirred for 15-30 minutes, then the solid was isolated by vacuum filtration and washed with water (10 mL). The solid was dried on a filter under air for 1-2 hours, and then dissolved in DMSO (30 mL). The product solution in DMSO was purified by preparation HPLC [column: YMCTA C18, 250 × 21.2 mm, 10 μm; flow rate: 15 mL / min; gradient: 20% acetonitrile - 80% water 0.1% TFA to 70% acetonitrile - 30% water 0.1% TFA; @254 / 205 nm]. The pure fractions containing the product were combined and concentrated under vacuum at 45-50°C to remove the solvent. Next, the product was freeze-dried to give (S)-3-((6-(4,6-bis(((R)-1,1,1-trifluoropropan-2-yl)amino)-1,3,5-triazine-2-yl)pyridine-2-yl)thio)-2-methylpropanoic acid as a white solid (120 mg, 98.5% / 220 nm, yield 10%). 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)pyridine-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-chloropyridine-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 packed into a flask. The resulting mixture was stirred at 15-20°C for 5 minutes to form a clear brown solution under nitrogen. This reaction mixture was cooled to 5°C, and then sodium thiomethoxide (NaSMe, 4.3 g, 60.28 mmol) was gradually added over 20 minutes under nitrogen at 5-10°C. This reaction mixture was heated to 90-95°C and stirred at 90-95°C for 3 hours. The reaction solution was poured into ice water (200 mL) at 5-10°C while stirring. After stirring for 30 minutes, the solid was filtered, the filtered cake was washed with water (50 mL), and dried in a vacuum at 50°C for 6 hours to obtain 6-(6-(methylthio)pyridine-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), 6-(6-(methylthio)pyridine-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) was added all at once. To this reaction mixture, Oxon (11.5 g, 18.8 mmol) was added gradually over 15 minutes while maintaining the temperature below 20°C. The reaction mixture was stirred at 20-30°C for 2 hours. 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 minutes and separated. The orane phase was washed with an aqueous solution of Na2SO3 (1.2 g in 80 mL of water) at 5-10°C (confirmed with KI starch paper) and separated. The organic phase was washed with an aqueous solution of Na2SO3 (1.2 g in 80 mL of water) at 5-10°C (confirmed by KI starch paper) and separated. The organic phase was washed with water (100 mL x 2) and separated. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated in vacuum at 40-45°C to obtain a 4-5V slurry. This slurry was stirred at 10-15°C for 30 minutes, filtered, washed with MTBE (20 mL), and dried in vacuum at 50°C to give 6-(6-(methylsulfonyl)pyridine-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 obtain 6-(6-(methylsulfonyl)pyridine-2-yl)-N2,N4-bis((R)-1,1,1-trifluoropropan-2-yl)-1,3,5-triazine-2,4-diamine as a yellow solid (approximately 1.5 g, approximately 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 boracidenib and [ 13 C3 15 [N3] Evaluation of the characteristics of absorption, distribution, metabolism, and excretion of borasidenib with simultaneous microintravenous administration. To profile and identify the metabolites of borasidenib (AG-881), a single oral dose of 50 mg (100 μCi) of [14C]AG-881 was administered, 13 C3 15 The study involved plasma, urine, and fecal samples collected from five healthy subjects who received simultaneous microintravenous administration of [N3]AG-881.

[0195] Plasma samples collected at selected time points from 0 to 336 hours after administration were pooled among subjects to create representative area under the concentration-time curve (AUC) samples for 0–72 hours and 96–336 hours. Urine and fecal samples were pooled for each subject to create individual urine and fecal pools. Plasma, urine, and fecal samples were extracted as appropriate, and the extracts were profiled using high-performance liquid chromatography (HPLC). Metabolites were identified by liquid chromatography-mass spectroscopy (LC-MS and / or LC-MS / MS) analysis and, where available, by comparison of retention times with reference materials.

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

[0197] The majority of the radioactivity recovered in feces was associated with unchanged AG-881 (55.5% of the dose), but AG-881 was not detected in urine. In comparison, metabolites in excrement accounted for approximately 18% of the dose in feces and approximately 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, [ 14[C]AG-881 underwent moderate metabolism after a single oral administration of 50 mg (100 μCi), and in humans, it was shown to be eliminated through a combination of metabolism and excretion of the unchanged parent compound. The metabolism of AG-881 involved conjugation with glutathione (GSH) by oxidation and chlorine substitution at the chloropyridine moiety. Subsequent biotransformation of the GSH intermediate removed both glutamate and glycine, forming cysteinyl conjugates (M515 and M499). The cysteinyl conjugates were further transformed by a series of biotransformation reactions, e.g., oxidation, S-dealkylation, S-methylation, S-oxidation, S-acetylation, and N-dealkylation, forming multiple metabolites.

[0199] Table 2 shows a summary of the metabolites observed.

[0200] [Table 2]

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

[0202] [Table 3] JPEG2026113537000047.jpg213161

[0203] Figure 1 shows the proposed (theoretical) biotransformation pathways leading to the observed metabolites.

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

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

[0206] The primary reaction was carried out in 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), along with 1 μL of 50× compound in DMSO. Before adding α-ketoglutarate, the mixture of compound, enzyme, and coenzyme was pre-incubated at room temperature for 1 hour. To carry out the secondary reaction, 10 μL of 1× buffer containing 36 μg / mL diaphorase and 30 mM rezazurin was added to the primary reaction and incubated at 25°C for a further 5 minutes. Fluorescence is read using a Spectramax plate reader with Ex544 Em590. The compound or compound dilution is prepared at 100% DMSO concentration and diluted 1:50 in the final reaction. IDH1wt / IDH1R132C is assayed under the same conditions, except that the 1× buffer is 50mM K2HPO4 pH6.5; 10mM MgCl2; 10% glycerol; 0.03% (w / v) bovine serum albumin, with a final concentration of 0.4ug / mL (4.3nM) IDH1wt / IDH1R132C heterodimer, 0.02mM α-ketoglutarate, 4uM NADPH, and 300μM NADP (saturated) or 30μM NADP (unsaturated). The 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, the IDH1wt / R132m heterodimer is expressed and purified as follows: Co-expression of IDH1wt-his and IDH1R132C-flag is performed in sf9 insect cells. Cells (25g) are resuspended in 250mL of 50mM Tirs, 500mM NaCl pH 7.4 with stirring at 4°C. Cells are lysed by passing them four times through an M-Y110 microfluidizer set to 500psi, and then centrifuged at 22,000 rcf for 20 minutes at 4°C. The supernatant is collected and Histrap FF5 is equilibrated with 50mM Tirs, 500mM NaCl pH 7.4. * Load a 1 ml column (GE) at 15 cm / h. Remove host cell contamination by washing the column to baseline with equilibration buffer, followed by equilibration buffer containing 20 mM imidazole and 60 mM imidazole. Elute the IDH1wt-his homodimer and IDH1wt-his / IDH1R132C-flag with equilibration buffer containing 250 mM imidazole. Pool the fractions eluted with 250 mM imidazole and load them at 15 cm / h onto a column pre-packed with ANTI-FLAG® M2AffinityGel (Sigma). Equilibrate the column with 50 mM Tris and 500 mM NaCl pH 7.4. After washing with equilibration buffer, elute the IDH1wt-his / IDH1R132C-flag heterodimer with equilibration buffer containing flag peptide (0.2 mg / mL). Aliquotes of IDH1wt-his / IDH1R132C-flag are flash-frozen in liquid N2 and stored at -80°C. The same conditions are used for the purification of IDH1wt-his / IDH1R132H-flag.

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

[0209] The test compound is prepared as a 10 mM stock in DMSO and diluted to 50 × final concentration in DMSO for 50 μl of the reaction mixture. The IDH enzyme activity that converts alpha-ketoglutarate to 2-hydroxyglutaric acid is measured using an NADPH depletion assay. In this assay, the remaining coenzyme is measured at the end of the reaction, when a catalytic excess of diaphorase and rezazurin is added to generate a fluorescence signal proportional to the amount of remaining NADPH. The 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 β-mercaptoethanol), 1 μl of the diluted test compound in DMSO is added, and the mixture is incubated at room temperature for 60 minutes. The reaction is initiated by adding 10 μl of the substrate mixture (20 μl of NADPH and 5 mM alpha-ketoglutarate in assay buffer) and incubating the mixture at room temperature for 90 minutes. The reaction is terminated by adding 25 μl of detection buffer (36 μg / mL diaphorase and 30 mM rezazurin in 1× assay buffer), incubating for 1 minute, and then reading the result on a SpectraMax plate reader using Ex544 / Em590.

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

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

[0212] The test compound is prepared as a 10 mM stock in DMSO and diluted to 50 × final concentration in DMSO for 50 μl of the reaction mixture. The IDH enzyme activity that converts alpha-ketoglutarate to 2-hydroxyglutaric acid is measured using an NADPH depletion assay. In this assay, the remaining coenzyme is measured at the end of the reaction, when a catalytic excess of diaphorase and rezazurin is added to generate a fluorescence signal proportional to the amount of remaining NADPH. The IDH1-R132 homodimeric enzyme is diluted to 0.125 μg / ml in 40 μl of assay buffer containing 5 μM NADPH and 37.5 μM NADP (150 mM NaCl, 20 mM Tris-Cl pH 7.5, 10 mM MgCl2, 0.05% BSA, 2 mM β-mercaptoethanol), 1 μl of the diluted test compound in DMSO is added, and the mixture is incubated at room temperature for 60 minutes. The reaction is initiated by adding 10 μl of the substrate mixture (20 μl of NADPH and 5 mM alpha-ketoglutarate in assay buffer) and incubating the mixture at room temperature for 60 minutes. The reaction is terminated by adding 25 μl of detection buffer (36 μg / mL diaphorase and 30 mM rezazurin in 1 × assay buffer), incubating for 1 minute, and then reading the result on a SpectraMax plate reader using Ex544 / Em590.

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

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

[0215] The compound is assayed for IDH2R140Q inhibitory activity by a coenzyme depletion assay. The compound is pre-incubated with the enzyme, and the reaction is then initiated by adding NADPH and α-KG, and allowed to proceed for 60 minutes under conditions previously proven to be linear in terms of the time it takes for both the coenzyme and substrate to be consumed. The reaction is terminated by adding a second enzyme, diaphorase, and the corresponding substrate, rezazulin. Diaphorase reduces rezazulin to highly fluorescent resorphine and simultaneously oxidizes NADPH to NADP. Both actions halt the IDH2 reaction by depleting the available coenzyme pool, and the amount of coenzyme remaining after a specific period can be easily quantified by quantitatively generating a readily detectable fluorescent dye.

[0216] Specifically, 1 μl of the 100× compound dilution series is added to each of the 12 wells of a 384-well plate, followed by 40 μl of buffer containing 0.25 μg / ml IDH2R140Q protein (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). The test compound is then incubated with the enzyme at room temperature for 1 hour. Subsequently, 10 μl of substrate mix containing 4 μM NADPH and 1.6 mM α-KG is added to the buffer to initiate the IDH2 reaction. After incubation at room temperature for a further 16 hours, the reaction is stopped, and the remaining NADPH is measured by adding 25 μl of StopMix (36 μg / ml diaphorase enzyme and 60 μM resazurin in buffer) to convert resazurin to resorphine. After incubation for 1 minute, the plate is placed on a plate reader and read using Ex544 / Em590.

[0217] To measure the inhibitory effect of the compound on IDH2R140Q using the same assay format as above, the same procedure was 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 efficacy of compounds against IDH2R140Q in a high-throughput screening format, 0.25 μg / mL IDH2R140Q protein was used in the pre-incubation step, and the reaction was carried out using the same method as before, except that 4 μM NADPH and 8 μM α-KG were added to initiate the reaction.

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

[0220] The compound is assayed for IDH2R140Q inhibitory activity using a coenzyme depletion assay. The compound is pre-incubated with the enzyme and coenzyme, and the reaction is then initiated by adding α-KG and allowed to proceed for 60 minutes under conditions previously proven to be linear. The reaction is terminated by adding a second enzyme, diaphorase, and its corresponding substrate, rezazulin. Diaphorase reduces rezazulin to highly fluorescent resorphine and simultaneously oxidizes NADPH to NADP. Both actions deplete the available coenzyme pool, thereby terminating the IDH2 reaction and facilitating the quantitative generation of readily detectable fluorescent dyes, which allows for the quantitative determination of the amount of coenzyme remaining after a specific period.

[0221] Specifically, 1 μl of the 50× compound dilution series is added to each of the 12 wells of a 384-well plate, followed by 40 μl of a buffer containing 0.39 μg / ml IDH2R140Q protein, 5 μM NADPH, and 750 μM NADP (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). The test compound is then incubated with the enzyme and coenzyme at room temperature for 16 hours. Subsequently, 10 μl of a substrate mix containing 8 mM α-KG (final concentration 1.6 mM) is added to the buffer to initiate the IDH2 reaction. After incubating at room temperature for another hour, the reaction is stopped, and the remaining NADPH is measured by adding 25 μl of StopMix (36 μg / ml diaphorase enzyme and 60 μM resazurin in buffer) to convert resazurin to resorphine. After incubation for 1 minute, the plate is placed on a plate reader and read using Ex544 / Em590.

[0222] For various compounds according to one aspect of this disclosure, the data from the R132H enzyme assay, R132C enzyme assay, R140Q enzyme assay, R132C cell-based assay, and R140Q cell-based assay, or similar data, are shown 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 modifications, modifications, and improvements will be readily available to those skilled in the art. Such modifications, modifications, and improvements are intended to be part of this disclosure and within the spirit and scope of the invention; therefore, the foregoing description and drawings are illustrative only.

Claims

1. The following are the purified forms: 【Chemistry 43】 A compound selected from or a pharmaceutically acceptable salt thereof.

2. The compound is in a purified form. 【Chemistry 44】 The compound according to claim 1, or a pharmaceutically acceptable salt thereof.

3. The compound is in a purified form. 【Chemistry 45】 The compound according to claim 1, or a pharmaceutically acceptable salt thereof.

4. The compound is in a purified form. 【Chemistry 46】 The compound according to claim 1, or a pharmaceutically acceptable salt thereof.

5. The compound is in a purified form. 【Chemistry 47】 The compound according to claim 1, or a pharmaceutically acceptable salt thereof.

6. The compound is in a purified form. 【Chemistry 48】 The compound according to claim 1, or a pharmaceutically acceptable salt thereof.

7. The compound is in a purified form. 【Chemistry 49】 The compound according to claim 1, or a pharmaceutically acceptable salt thereof.

8. The compound is in a purified form. [Transformation 50] The compound according to claim 1, or a pharmaceutically acceptable salt thereof.

9. A compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof, comprising one or more pharmaceutically acceptable excipients, Pharmaceutical composition.

10. The compound is 【Chemistry 51】 The pharmaceutical composition according to claim 9, or a pharmaceutically acceptable salt thereof.

11. The compound is 【Chemistry 52】 The pharmaceutical composition according to claim 9, or a pharmaceutically acceptable salt thereof.

12. The compound is 【Chemistry 53】 The pharmaceutical composition according to claim 9, or a pharmaceutically acceptable salt thereof.

13. The compound is 【Chemistry 54】 The pharmaceutical composition according to claim 9, or a pharmaceutically acceptable salt thereof.

14. The compound is 【Transformation 55】 The pharmaceutical composition according to claim 9, or a pharmaceutically acceptable salt thereof.

15. The compound is 【Transformation 56】 The pharmaceutical composition according to claim 9, or a pharmaceutically acceptable salt thereof.

16. The compound is 【Chemistry 57】 The pharmaceutical composition according to claim 9, or a pharmaceutically acceptable salt thereof.

17. A method of treating cancer, The treatment comprises administering a therapeutically effective amount of the purified compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 8 to a patient in need of treatment for cancer, wherein the cancer is characterized by the presence of at least one mutation selected from IDH1 mutations or IDH2 mutations. method.

18. The purified compound is 【Chemistry 58】 The method according to claim 17, or a pharmaceutically acceptable salt thereof.

19. The purified compound is 【Chemistry 59】 The method according to claim 17, or a pharmaceutically acceptable salt thereof.

20. The purified compound is 【Transformation 60】 The method according to claim 17, or a pharmaceutically acceptable salt thereof.

21. The purified compound is 【Chemistry 61】 The method according to claim 17, or a pharmaceutically acceptable salt thereof.

22. The purified compound is 【Transformation 62】 The method according to claim 17, or a pharmaceutically acceptable salt thereof.

23. The purified compound is 【Transformation 63】 The method according to claim 17, or a pharmaceutically acceptable salt thereof.

24. The purified compound is 【Chemistry 64】 The method according to claim 17, or a pharmaceutically acceptable salt thereof.

25. A method of treating cancer, The method comprises administering a therapeutically effective amount of the composition according to any one of claims 9 to 16 to a patient in need of treatment for cancer, wherein the cancer is characterized by the presence of at least one mutation selected from IDH1 mutations or IDH2 mutations. method.

26. The method according to any one of claims 17 to 25, wherein the cancer in the patient 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 lymphoma (NHL).

27. The method according to claim 26, wherein the cancer is a glioma.

28. The method according to claim 27, wherein the glioma is a low-grade glioma or a high-grade glioma.

29. A method for treating gliomas in patients requiring treatment for gliomas, The method comprises administering to the subject a therapeutically effective amount of the purified compound according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof. method.

30. The purified compound is 【Transformation 65】 The method according to claim 29, or a pharmaceutically acceptable salt thereof.

31. The purified compound is 【Chemical Formula 66】 The method according to claim 29, or a pharmaceutically acceptable salt thereof.

32. The purified compound is 【Transformation 67】 The method according to claim 29, or a pharmaceutically acceptable salt thereof.

33. The purified compound is 【Transformation 68】 The method according to claim 29, or a pharmaceutically acceptable salt thereof.

34. The purified compound is 【Transformation 69】 The method according to claim 29, or a pharmaceutically acceptable salt thereof.

35. The purified compound is 【Transformation 70】 The method according to claim 29, or a pharmaceutically acceptable salt thereof.

36. The purified compound is 【Chemistry 71】 The method according to claim 29, or a pharmaceutically acceptable salt thereof.

37. A method for treating gliomas, The method comprises administering a therapeutically effective amount of the composition according to any one of claims 9 to 16 to a patient requiring treatment for a glioma, wherein the glioma is characterized by the presence of at least one mutation selected from IDH1 mutations or IDH2 mutations. method.

38. The method according to any one of claims 29 to 36, wherein the glioma is characterized by the presence of at least one mutation selected from IDH1 mutations and IDH2 mutations.

39. The method according to any one of claims 17 to 28 and 37, wherein the mutation is an IDH1 mutation.

40. The method according to claim 39, wherein the IDH1 mutation is an R132X mutation.

41. The method according to claim 40, wherein the IDH1 mutation is an R132H or R132C mutation.

42. The method according to any one of claims 17 to 28 and 37, wherein the mutation is an IDH2 mutation.

43. The method according to claim 42, wherein the mutation is an R140X or R172X mutation.

44. The method according to claim 43, wherein the mutation is an R140Q, R140W, or R140L mutation.

45. The method according to claim 43, wherein the mutation is an R172K or R172G mutation.

46. The method according to any one of claims 17 to 45, wherein the amount of the purified compound or a pharmaceutically acceptable salt thereof administered to the patient is about 1 to 5000 mg / day.

47. The method according to claim 46, wherein the amount of the purified compound or a pharmaceutically acceptable salt thereof administered to the patient is about 1 to 1000 mg / day.

48. The method according to claim 46, wherein the amount of the purified compound or a pharmaceutically acceptable salt thereof administered to the patient is about 1 to 500 mg / day.

49. The method according to any one of claims 17 to 48, wherein the purified compound or composition is administered orally.

50. The method according to any one of claims 17 to 49, wherein the purified compound or composition is administered in combination with additional therapeutic means.

51. The method according to claim 50, wherein additional therapeutic means are selected from radiation, surgical resection, anticancer agents, antiepileptic agents, anticonvulsants and antiemetics.

52. The method according to claim 51, wherein the anticancer agent is selected from chemotherapy with cytotoxic agents or cytostatic agents, targeted agents, antibody therapy, immunotherapy, and hormone therapy.

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