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WO2019099653A1 - Composés thérapeutiquement actifs et leurs procédés d'utilisation - Google Patents

Composés thérapeutiquement actifs et leurs procédés d'utilisation Download PDF

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Publication number
WO2019099653A1
WO2019099653A1 PCT/US2018/061257 US2018061257W WO2019099653A1 WO 2019099653 A1 WO2019099653 A1 WO 2019099653A1 US 2018061257 W US2018061257 W US 2018061257W WO 2019099653 A1 WO2019099653 A1 WO 2019099653A1
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Prior art keywords
compound
pharmaceutically acceptable
hydrate
acceptable salt
crystalline form
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Zhihua Sui
Robert Zahler
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Agios Pharmaceuticals Inc
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Agios Pharmaceuticals Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06139—Dipeptides with the first amino acid being heterocyclic
    • C07K5/06165—Dipeptides with the first amino acid being heterocyclic and Pro-amino acid; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06191—Dipeptides containing heteroatoms different from O, S, or N

Definitions

  • Isocitrate dehydrogenases catalyze the oxidative decarboxylation of isocitrate to 2-oxoglutarate (i.e., a-ketoglutarate). These enzymes belong to two distinct subclasses, one of which utilizes NAD(+) as the electron acceptor and the other NADP(+). Five isocitrate dehydrogenases have been reported: three NAD(+)- dependent isocitrate dehydrogenases,
  • NADP(+)-dependent isocitrate dehydrogenases which localize to the mitochondrial matrix, and two NADP(+)-dependent isocitrate dehydrogenases, one of which is mitochondrial and the other predominantly cytosolic.
  • Each NADP(+)-dependent isozyme is a homodimer.
  • IDH1 isocitrate dehydrogenase 1 (NADP+), cytosolic
  • IDP isocitrate dehydrogenase 1
  • IDCD isocitrate dehydrogenase 1
  • PICD PICD
  • the protein encoded by this gene is the NADP(+)- dependent isocitrate dehydrogenase found in the cytoplasm and peroxisomes. It contains the PTS-l peroxisomal targeting signal sequence. The presence of this enzyme in peroxisomes suggests roles in the regeneration of NADPH for
  • cytoplasmic enzyme serves a significant role in cytoplasmic NADPH production.
  • the human IDH1 gene encodes a protein of 414 amino acids.
  • the nucleotide and amino acid sequences for human IDH1 can be found as GenBank entries
  • NM_005896.2 and NP_005887.2 respectively.
  • the nucleotide and amino acid sequences for IDH1 are also described in, e.g., Nekrutenko et al., Mol. Biol. Evol. 15: 1674-1684(1998); Geisbrecht et al, J. Biol. Chem. 274:30527-30533(1999);
  • Non-mutant e.g., wild type
  • IDH1 catalyzes the oxidative decarboxylation of isocitrate to a-ketoglutarate. It has been discovered that mutations of IDH1 present in certain cancer cells result in a new ability of the enzyme to catalyze the NADPH- dependent reduction of a-ketoglutarate to i?(-)-2-hydroxyglutarate (2HG). The production of 2HG is believed to contribute to the formation and progression of cancer (Dang, L et al, Nature 2009, 462:739-44).
  • deuterium modification is a viable drug design strategy for inhibiting adverse metabolism (see Foster, A B, Adv Drug Res 1985, 14:1-40 and Fisher, M B et al, Curr Opin Drug Discov Devel, 2006, 9: 101- 09).
  • a potentially attractive strategy for improving a drug's metabolic properties is, however, deuterium modification, especially at the metabolic sites of the drug.
  • deuterium modification especially at the metabolic sites of the drug.
  • Deuterium is a safe, stable, non-radioactive isotope of hydrogen.
  • deuterium forms slightly stronger bonds with carbon, which may impact pharmacokinetics of a drug with the potential for improved drug efficacy, safety, and/or tolerability, without affecting the biochemical potency and selectivity of the drug as compared to non- deuterated analog.
  • This application provides for compounds that should have enhanced drug efficacy, safety, and/or tolerability in which deuterium atoms are introduced at various carbon atoms.
  • Described herein are methods of treating a cancer characterized by the presence of a mutant allele of IDH1.
  • the methods comprise the step of administering to a subject in need thereof a compound of Formula I, or a pharmaceutically acceptable salt or hydrate thereof:
  • compositions comprising a compound of Formula (I) or a
  • the term“elevated levels of 2HG” means 10%, 20% 30%, 50%, 75%, 100%, 200%, 500% or more 2HG than is present in a subject that does not carry a mutant IDH1 allele.
  • the term“elevated levels of 2HG” may refer to the amount of 2HG within a cell, within a tumor, within an organ comprising a tumor, or within a bodily fluid.
  • the term“bodily fluid” includes one or more of amniotic fluid surrounding a fetus, aqueous humour, blood (e.g ., blood plasma), serum, Cerebrospinal fluid, cerumen, chyme, Cowper's fluid, female ejaculate, interstitial fluid, lymph, breast milk, mucus (e.g., nasal drainage or phlegm), pleural fluid, pus, saliva, sebum, semen, serum, sweat, tears, urine, vaginal secretion, or vomit.
  • blood e.g ., blood plasma
  • serum Cerebrospinal fluid
  • cerumen cerumen
  • chyme chyme
  • Cowper's fluid female ejaculate
  • interstitial fluid lymph
  • breast milk mucus (e.g., nasal drainage or phlegm)
  • mucus e.g., nasal drainage or phlegm
  • pleural fluid pus, saliva, sebum, semen
  • inhibitor or“prevent” include both complete and partial inhibition and prevention.
  • An inhibitor may completely or partially inhibit.
  • treat means decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a cancer (e.g., a cancer delineated herein), lessen the severity of the cancer or improve the symptoms associated with the cancer.
  • an amount of a compound effective to treat a disorder refers to an amount of the compound which is effective, upon single or multiple dose administration to a subject, in treating a cell, or in curing, alleviating, relieving or improving a subject with a disorder beyond that expected in the absence of such treatment.
  • the term“subject” is intended to include human and non human animals.
  • exemplary human subjects include a human patient having a disorder, e.g., a disorder described herein or a normal subject.
  • the term“non-human animals” of the invention includes all vertebrates, e.g., non-mammals (such as chickens, amphibians, reptiles) and mammals, such as non-human primates, domesticated and/or agriculturally useful animals, e.g., sheep, dog, cat, cow, pig, etc.
  • each of Ri, Rr, R 2 , Rr, R 3 -R 7 , Rs, Rs ’ , R 9 , R 9’ , R 10 -R 17 is independently H or D, provided that at least one of Ri, Rr, R 2 , Rr, R3-R7, Rs, Re R9, R9’, Rio-Rnis D.
  • each of Ri, Rr, R 2 , Rr, R 3 -R 7 , Rs, R S % R 9 , R 9’ , R 10 -R 17 is independently H or D, provided that at least one of Ri, Rr, R 2 , Rr, R 3 -R 7 , Rs, R S % R 9 , R 9’ , and R 10 -R 17 is D.
  • each of R 4 -R 7 , Rs, Rs% R 9 , R 9’ , and R 10 -R 17 is H.
  • each of Ri, Rr, R 2 , R 2’ is D and each of R 4 -R 7 , R 8 , Rs % R 9 , R 9’ , and R 10 -R 17 is H.
  • each of Ri, Rr, R 2 , R r, R 3 , R 4 , Rs, Rs % R 9 , R 9’ , and Rio-Rn is H.
  • each of R 5 , R 5 , and R 7 is D and each of Ri, Rr, R 2 , Rr, R 3 , R 4 ,
  • R 8 , R 8% R 9 , R 9’ , and Rio-Rn is H.
  • each of Ri, Rr, R 2 , Rr, R 3 - R 7 , and Rn-Rn is H.
  • each of R 8 , R 8 , R 9 , R 9’ , Rio is D and each of Ri, Rr, R 2 , Rr, R 3 -R 7 , and Rn-Rn is H.
  • each of Ri, Rr, R 2 , Rr, R 3 -R 7 , R 7’ , R 8 , R 8’ , R 9 , R 9’ , R IO , and R 14 -R 17 is H.
  • each of Rn-Rn is D and each of Ri, Rr, R 2 , Rr, R3-R7, R7’, Rs, Rs % R9, R9’, Rio, and Ri 4 -R l7 is H.
  • each of Ri, Rr, R 2 , Rr, R 3 -R 7 , R 7’ , R 8 , R 8% R 9 , R 9’ , and R 10 -R 13 is H.
  • each of R 14 -R 17 is D and each of Ri, Rr, R2, R2’, R3-R7, R7’, Re, Re’, R9, R9’, and R10-R13 is H.
  • each of Ri, Rr, R 2 , Rr, R 3 -R 7 , R 9 , R 9’ , and Rn-Rn is independently H or D, provided that at least one of Ri, Rr, R2, R2’, R3-R7, R9, R9’, and Rn-Ri 7 is D.
  • each of Ri, Rr, R 2 , Rr, R 3 -R 7 , R 9 , R 9’ , R 11 -R 13 , and R 15 -R 16 is independently H or D, provided that at least one of Ri, Rr, R 2 , R 2 R 3 -R 7 , R 9 , R 9’ , R 11 -R 13 , and R 15 -R 16 is D.
  • each of Ri, R , R 2 , Rr, R 3 , R 4 , R 9 , R 9’ , R 11 -R 13 , and R 15 -R 16 is independently H or D, provided that at least one of Ri, Rr, R2, R2’, R3, R4, R9, R9’, R11-R13, and R15-R16 is D.
  • each of Ri, Rr, R 2 , Rr, R 3 , R 4 , R 9 , R 9’ , and R 15 -R 16 is independently H or D, provided that at least one of Ri, Rr, R2, R2’, R3, R4, R9, R9’, and R15-R16 is D.
  • each of Ri, R , R 2 , Rr, R 3 , R 9 , R 9’ , and R 15 -R 16 is independently H or D, provided that at least one of Ri, Rr, R2, R2’, R 3 , R9, R9’, and R15-R16 is D.
  • each of R 3 , R 9 , R 9’ , and R 15 -R 16 is independently H or D, provided that at least one of R 3 , R 9 , R 9’ , and R 15 - R l6 is D.
  • the compounds of Formula (I) are of Formula (II):
  • m, n, o, p, and q represent the number of deuterium substituents on the respective rings; m is 0, 1, 2, 3, 4 or 5; n is 0, 1, 2, 3, or 4; o is 0, 1, 2, or 3; p is 0, 1, 2, 3, 4, or 5; and q is 0, 1, 2, or 3, provided that at least one of m. n, o, p, and q is not zero; and the numerical labels
  • m is 0 or 4; n is 0, 1, 2, 3, or 4; o is 0, 1, 2, or 3; p is 0, 2, or 4; and q is 0, 1, 2, or 3, provided that at least one of m, n, o, p, and q is not zero.
  • m is 0; n is 0, 1, 2, 3, or 4; o is 0, 1, 2, or 3; p is 0, 2, or 4; and q is 0, 1, 2, or 3, provided that at least one of n, o, p, and q is not zero.
  • m is 4; n is 0, 1, 2, 3, or 4; o is 0, 1, 2, or 3; p is 0, 2, or 4; and q is 0, 1, 2, or 3.
  • n is 0, 1, 2, 3, or 4; o is 0, 1, 2, or 3; p is 0, 2, or 4; and q is 0, 1, 2, or 3.
  • m is 0 or 4; n is 0, 1, 2, 3, or 4; o is 0, 1, 2, or 3; p is 0; and q is 0, 1, 2, or 3, provided that at least one of m, n, o, and q is not zero.
  • m is 0 or 4; n is 0, 1, 2, 3, or 4; o is 0, 1, 2, or 3; p is 2; and q is 0, 1, 2, or 3.
  • m is 0 or 4; n is 0, 1, 2, 3, or 4; o is 0, 1, 2, or 3; p is 4; and q is 0, 1, 2, or 3.
  • the compounds of Formula (II) are listed in Table 2 below.
  • the compounds of this invention may contain one or more asymmetric centers and thus occur as racemates, racemic mixtures, scalemic mixtures, and diastereomeric mixtures, as well as single enantiomers or individual stereoisomers that are substantially free from another possible enantiomer or stereoisomer.
  • substantially free of other stereoisomers means a preparation enriched in a compound having a selected stereochemistry at one or more selected stereocenters by at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.
  • the term“enriched” means that at least the designated percentage of a preparation is the compound having a selected stereochemistry at one or more selected stereocenters.
  • the compound is enriched in a specific stereoisomer by at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.
  • the compounds of Formulae (I), (IA), (IB), (IC), (ID), (IE), (IF), (IG), and (II) may also comprise one or more isotopic substitutions.
  • C may be in any isotopic form, including 12 C, 13 C, and 14 C;
  • O may be in any isotopic form, including 16 0 and 18 0; and the like.
  • the compound is enriched in a specific isotopic form of C and/or O by at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.
  • the compounds of this invention may also be represented in multiple tautomeric forms, in such instances, the invention expressly includes all tautomeric forms of the compounds described herein, even though only a single tautomeric form may be represented (e.g., alkylation of a ring system may result in alkylation at multiple sites, the invention expressly includes all such reaction products). All such isomeric forms of such compounds are expressly included in the present invention.
  • any atom not specifically designated as a particular isotope is meant to represent any stable isotope of that atom.
  • a position is designated specifically as“H” or“hydrogen”, the position is understood to have hydrogen at its natural abundance isotopic composition.
  • “D” refers to deuterium (2H).
  • the positions designated specifically as“D” or“deuterium” shall be understood to have an isotopic enrichment factor for each designated deuterium atom of at least 3000. Based on the natural abundance of deuterium (about 0.015%), an isotopic enrichment factor of at least 3000 corresponds to at least 45% deuterium incorporation.
  • isotopic enrichment factor refers to the ratio between the isotopic abundance of a given isotope at a designated position of a compound and the natural abundance of that isotope.
  • the skilled artisan would understand how to prepare compounds with varying degrees of isotopic enrichment at a particular hydrogen atom. For example, the artisan could use various ratios of mixtures of compounds in which, at the hydogen of interest, one compound is fully deuterated and the other compound fully hydrogenated (of course, with deuterium being present at its natural abundance). Any level of desired enrichment can, therefore, be prepared by a skilled artisan.
  • the positions designated specifically as“D” or “deuterium” in the compound of the invention shall be understood to have an isotopic enrichment factor for each designated deuterium atom of at least 3500 (52.5% deuterium incorporation), at least 4000 (60% deuterium incorporation), at least 4500 (67.5% deuterium incorporation), at least 5000 (75% deuterium incorporation), at least 5500 (82.5% deuterium incorporation), at least 6000 (90% deuterium
  • a corresponding salt of the active compound for example, a pharmaceutically acceptable salt.
  • a pharmaceutically acceptable salt examples are discussed in Berge et al ., 1977, "Pharmaceutically Acceptable Salts.” J. Pharm. Sci. Vol. 66, pp. 1-19.
  • a salt may be formed with a suitable anion.
  • suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous.
  • Suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucoheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, and valeric.
  • a reference to a particular compound also includes salt forms thereof.
  • compositions and routes of administration are provided.
  • the compounds utilized in the methods described herein may be formulated together with a pharmaceutically acceptable carrier or adjuvant into pharmaceutically acceptable compositions prior to be administered to a subject.
  • a pharmaceutically acceptable carrier or adjuvant into pharmaceutically acceptable compositions prior to be administered to a subject.
  • such pharmaceutically acceptable compositions further comprise additional therapeutic agents in amounts effective for achieving a modulation of disease or disease symptoms, including those described herein.
  • pharmaceutically acceptable carrier or adjuvant refers to a carrier or adjuvant that may be administered to a subject, together with a compound of this invention, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound.
  • Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d- a -tocopherol poly ethyleneglycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, di sodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium
  • Cyclodextrins such as a-, b-, and g- cyclodextrin, or chemically modified derivatives such as hydroxyalkylcyclodextrins, including 2- and 3-hydroxypropyl-P-cyclodextrins, or other solubilized derivatives may also be advantageously used to enhance delivery of compounds of the Formulae described herein.
  • compositions of this invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir, preferably by oral administration or administration by injection.
  • the pharmaceutical compositions of this invention may contain any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or vehicles. In some cases, the pH of the formulation may be adjusted with
  • parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrastemal, intrathecal, intralesional and intracranial injection or infusion techniques.
  • compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension.
  • This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in l,3-butanediol.
  • a non-toxic parenterally acceptable diluent or solvent for example, as a solution in l,3-butanediol.
  • acceptable vehicles and solvents that may be used are mannitol, water, Ringer’s solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally used as a solvent or suspending medium.
  • any bland fixed oil may be used including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural
  • oils such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms such as emulsions and or suspensions.
  • Other commonly used surfactants such as Tweens or Spans and/or other similar emulsifying agents or bioavailability enhancers that are commonly used in the manufacture of
  • compositions may also be used for the purposes of formulation.
  • compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions.
  • carriers that are commonly used include lactose and corn starch.
  • Lubricating agents such as magnesium stearate, are also typically added.
  • useful diluents include lactose and dried com starch.
  • the active ingredient may be suspended or dissolved in an oily phase is combined with emulsifying and/or suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.
  • compositions of this invention may also be administered in the form of suppositories for rectal administration.
  • These compositions can be prepared by mixing a compound of this invention with a suitable non-irritating excipient that is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components.
  • suitable non-irritating excipient include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.
  • Topical administration of the pharmaceutical compositions of this invention is useful when the desired treatment involves areas or organs readily accessible by topical application.
  • the pharmaceutical composition should be formulated with a suitable ointment containing the active components suspended or dissolved in a carrier.
  • Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water.
  • the pharmaceutical compositions of this invention include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water.
  • the pharmaceutical compositions of this invention include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water.
  • composition can be formulated with a suitable lotion or cream containing the active compound suspended or dissolved in a carrier with suitable emulsifying agents.
  • Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
  • the pharmaceutical compositions of this invention may also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable enema formulation. Topically-transdermal patches are also included in this invention.
  • compositions of this invention may be administered by nasal aerosol or inhalation.
  • Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.
  • compositions of this invention comprise a combination of a compound of the Formulae described herein and one or more additional therapeutic or prophylactic agents
  • both the compound and the additional agent should be present at dosage levels of between about 1 to 100%, and more preferably between about 5 to 95% of the dosage normally administered in a monotherapy regimen.
  • the additional agents may be administered separately, as part of a multiple dose regimen, from the compounds of this invention. Alternatively, those agents may be part of a single dosage form, mixed together with the compounds of this invention in a single composition.
  • the compounds described herein can, for example, be administered by injection, intravenously, intraarterially, subdermally, intraperitoneally,
  • the methods herein contemplate administration of an effective amount of compound or compound composition to achieve the desired or stated effect.
  • compositions of this invention will be administered from about 1 to about 6 times per day or alternatively, as a continuous infusion. Such administration can be used as a chronic or acute therapy.
  • the amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of
  • a typical preparation will contain from about 5% to about 95% active compound (w/w). Alternatively, such preparations contain from about 20% to about 80% active compound.
  • a maintenance dose of a compound, composition or combination of this invention may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained when the symptoms have been alleviated to the desired level. Subjects may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.
  • compositions described above comprising a compound of Formula (I), (IA), (IB), (IC), (ID), (IE), (IF), (IG), (II) or a compound described in any one of the embodiments herein, may further comprise another therapeutic agent useful for treating cancer.
  • a method for inhibiting a mutant IDH1 activity comprising contacting a subject in need thereof with a compound (including its tautomers and/or isotopologues) of structural Formula (I), (IA), (IB), (IC), (ID), (IE), (IF), (IG), or (II), or a compound described in any one of the embodiments herein, or a pharmaceutically acceptable salt thereof.
  • the cancer to be treated is characterized by a mutant allele of IDH1 wherein the IDH1 mutation results in a new ability of the enzyme to catalyze the NADPH-dependent reduction of a-ketoglutarate to R(-)-2- hydroxyglutarate in a subject.
  • the mutant IDH1 has an R132X mutation.
  • the R132X mutation is selected from R132H, R132C, R132L, R132V, R132S and R132G.
  • the R132X mutation is R132H or R132C.
  • the R132X mutation is R132H.
  • Also provided are methods of treating a cancer characterized by the presence of a mutant allele of IDH1 comprising the step of administering to subject in need thereof (a) a compound of Formula (I), (IA), (IB), (IC), (ID), (IE), (IF), (IG), or (II), or a compound described in any one of the embodiments herein, or a pharmaceutically acceptable salt or hydrate thereof, or (b) a pharmaceutical composition comprising (a) and a pharmaceutically acceptable carrier.
  • the cancer to be treated is characterized by a mutant allele of IDH1 wherein the IDH1 mutation results in a new ability of the enzyme to catalyze the NADPH-dependent reduction of a-ketoglutarate to f?(-)-2-hydroxyglutarate in a patient.
  • the IDH1 mutation is an R132X mutation.
  • the R132X mutation is selected from R132H, R132C, R132L, R132V, R132S and R132G.
  • the R132X mutation is R132 H or R132C.
  • a cancer can be analyzed by sequencing cell samples to determine the presence and specific nature of (e.g., the changed amino acid present at) a mutation at amino acid 132 of IDH1.
  • mutant alleles of IDH1 wherein the IDH1 mutation results in a new ability of the enzyme to catalyze the NADPH-dependent reduction of a-ketoglutarate to i?(-)-2-hydroxyglutarate, and in particular R132H mutations of IDH1, characterize a subset of all types of cancers, without regard to their cellular nature or location in the body.
  • the compounds and methods of this invention are useful to treat any type of cancer that is
  • the efficacy of cancer treatment is monitored by measuring the levels of 2HG in the subject.
  • levels of 2HG are measured prior to treatment, wherein an elevated level is indicated for the use of the compound of Formula (I), (IA), (IB), (IC), (ID), (IE), (IF), (IG), or (II), or a compound described in any one of the embodiments described herein to treat the cancer.
  • the level of 2HG is determined during the course of and/or following termination of treatment to establish efficacy.
  • the level of 2HG is only determined during the course of and/or following termination of treatment.
  • a reduction of 2HG levels during the course of treatment and following treatment is indicative of efficacy.
  • a determination that 2HG levels are not elevated during the course of or following treatment is also indicative of efficacy.
  • the these 2HG measurements will be utilized together with other well-known determinations of efficacy of cancer treatment, such as reduction in number and size of tumors and/or other cancer- associated lesions, improvement in the general health of the subject, and alterations in other biomarkers that are associated with cancer treatment efficacy.
  • 2HG can be detected in a sample by LC/MS.
  • the sample is mixed 80:20 with methanol, and centrifuged at 3,000 rpm for 20 minutes at 4 degrees Celsius.
  • the resulting supernatant can be collected and stored at -80 degrees Celsius prior to LC- MS/MS to assess 2-hydroxyglutarate levels.
  • LC separation methods can be used. Each method can be coupled by negative electrospray ionization (ESI, -3.0 kV) to triple-quadrupole mass spectrometers operating in multiple reaction monitoring (MRM) mode, with MS parameters optimized on infused metabolite standard solutions.
  • ESI, -3.0 kV negative electrospray ionization
  • MRM multiple reaction monitoring
  • Metabolites can be separated by reversed phase chromatography using 10 mM tributyl-amine as an ion pairing agent in the aqueous mobile phase, according to a variant of a previously reported method (Luo et al. J Chromatogr A 1147, 153-64, 2007).
  • Another method is specific for 2-hydroxyglutarate, running a fast linear gradient from 50% -95% B (buffers as defined above) over 5 minutes.
  • a Synergi Hydro-RP, lOOmm x 2 mm, 2.1 pm particle size (Phenomonex) can be used as the column, as described above.
  • Metabolites can be quantified by comparison of peak areas with pure metabolite standards at known concentration. Metabolite flux studies from 13 C-glutamine can be performed as described, e.g., in Munger et al. Nat Biotechnol 26, 1179-86, 2008.
  • 2HG is directly evaluated.
  • a derivative of 2HG formed in process of performing the analytic method is evaluated.
  • a derivative can be a derivative formed in MS analysis.
  • Derivatives can include a salt adduct, e.g, a Na adduct, a hydration variant, or a hydration variant which is also a salt adduct, e.g., a Na adduct, e.g., as formed in MS analysis.
  • a metabolic derivative of 2HG is evaluated.
  • examples include species that build up or are elevated, or reduced, as a result of the presence of 2HG, such as glutarate or glutamate that will be correlated to 2HG, e.g, R-2HG.
  • Exemplary 2HG derivatives include dehydrated derivatives such as the compounds provided below or a salt adduct thereof:
  • the cancer is a tumor wherein at least 30, 40, 50, 60, 70, 80 or 90% of the tumor cells carry an IDH1 mutation, and in particular an IDH1 R132H or R132C mutation, at the time of diagnosis or treatment.
  • IDH1 R132X mutations are known to occur in certain types of cancers as indicated in Table 3, below.
  • IDH1 R132H mutations have been identified in glioma, acute myelogenous leukemia, sarcoma, melanoma, non-small cell lung cancer, cholangiocarcinomas, chondrosarcoma, myelodysplastic syndromes (MDS), myeloproliferative neoplas (MPN), colon cancer, and angio-immunoblastic non-Hodgkin’s lymphoma (NHL).
  • the methods described herein are used to treat glioma, acute myelogenous leukemia, sarcoma, melanoma, non-small cell lung cancer (NSCLC) or cholangiocarcinomas, chondrosarcoma, myelodysplastic syndromes (MDS), myeloproliferative neoplas (MPN), colon cancer, or angio-immunoblastic non- Hodgkin’s lymphoma (NHL) in a patient.
  • the cancer is a glioma
  • the glioma is a low grade glioma or a secondary high grade glioma.
  • the cancer is glioma, and the glioma is a low grade glioma (grade II), anaplastic (grade III) or glioblastoma (GBM, grade IV).
  • the cancer is acute myelogenous leukemia (AML).
  • AML is refractory and/or relapsed.
  • the AML is newly diagnosed and/or previously untreated.
  • the cancer is a cancer selected from any one of the cancer types listed in Table 3, and the IDH R132X mutation is one or more of the IDH1 R132X mutations listed in Table 3 for that particular cancer type.
  • Treatment methods described herein can additionally comprise various evaluation steps prior to and/or following treatment with a compound of Formula (I), (IA), (IB), (IC), (ID), (IE), (IF), (IG), or (II), or a compound described in any one of the embodiments described herein.
  • the method further comprises the step of evaluating the growth, size, weight, invasiveness, stage and/or other phenotype of the cancer.
  • the method further comprises the step of evaluating the IDH1 genotype of the cancer. This may be achieved by ordinary methods in the art, such as DNA sequencing, immuno analysis, and/or evaluation of the presence, distribution or level of 2HG.
  • the method prior to and/or after treatment with a compound of Formula (I), (IA), (IB), (IC), (ID), (IE), (IF), (IG), or (II), or a compound described in any one of the embodiments described herein, the method further comprises the step of determining the 2HG level in the subject. This may be achieved by spectroscopic analysis, e.g., magnetic resonance-based analysis, e.g, MRI and/or MRS
  • the methods described herein comprise the additional step of co-administering to a subject in need thereof a second therapy e.g., an additional cancer therapeutic agent or an additional cancer treatment.
  • additional cancer therapeutic agents include for example, chemotherapy, targeted therapy, antibody therapies, immunotherapy, and hormonal therapy.
  • Additional cancer treatments include, for example: surgery, and radiation therapy. Examples of each of these treatments are provided below.
  • co-administering means that the additional cancer therapeutic agent may be administered together with a compound of this invention as part of a single dosage form (such as a composition of this invention comprising a compound of the invention and an second therapeutic agent as described above) or as separate, multiple dosage forms.
  • the additional cancer therapeutic agent may be administered prior to, consecutively with, or following the administration of a compound of this invention.
  • both the compounds of this invention and the second therapeutic agent(s) are administered by conventional methods.
  • composition of this invention comprising both a compound of the invention and a second therapeutic agent
  • administration of a composition of this invention does not preclude the separate administration of that same therapeutic agent, any other second therapeutic agent or any compound of this invention to said subject at another time during a course of treatment.
  • co-administering as used herein with respect to an additional cancer treatment means that the additional cancer treatment may occur prior to, consecutively with, concurrently with or following the administration of a compound of this invention.
  • the additional cancer therapeutic agent is a
  • chemotherapeutic agents used in cancer therapy include, for example, antimetabolites (e.g, folic acid, purine, and pyrimidine derivatives), alkylating agents (e.g, nitrogen mustards, nitrosoureas, platinum, alkyl sulfonates, hydrazines, triazenes, aziridines, spindle poison, cytotoxic agents, topoisomerase inhibitors and others) and hypomethylating agents (e.g., decitabine (5- aza-deoxycytidine), zebularine, isothiocyanates, azacitidine (5-azacytidine, 5-flouro- 2'-deoxycytidine, 5,6-dihydro-5-azacytidine and others).
  • antimetabolites e.g, folic acid, purine, and pyrimidine derivatives
  • alkylating agents e.g, nitrogen mustards, nitrosoureas, platinum, alkyl sulfonates
  • agents include Aclarubicin, Actinomycin, Alitretinoin, Altretamine, Aminopterin, Aminolevulinic acid, Amrubicin, Amsacrine, Anagrelide, Arsenic trioxide, Asparaginase, Atrasentan, Belotecan, Bexarotene, bendamustine, Bleomycin, Bortezomib, Busulfan,
  • Etoposide Floxuridine, Fludarabine, Fluorouracil (5FET), Fotemustine, Gemcitabine, Gliadel implants, Hydroxycarbamide, Hydroxyurea, Idarubicin, Ifosfamide,
  • Irinotecan Irofulven, Ixabepilone, Larotaxel, Leucovorin, Liposomal doxorubicin, Liposomal daunorubicin, Lonidamine, Lomustine, Lucanthone, Mannosulfan, Masoprocol, Melphalan, Mercaptopurine, Mesna, Methotrexate, Methyl
  • Sapacitabine Semustine, Sitimagene ceradenovec, Strataplatin, Streptozocin, Talaporfm, Tegafur-uracil, Temoporfm, Temozolomide, Teniposide, Tesetaxel, Testolactone, Tetranitrate, Thiotepa, Tiazofurine, Tioguanine, Tipifamib, Topotecan, Trabectedin, Triaziquone, Triethylenemelamine, Triplatin, Tretinoin, Treosulfan, Trofosfamide, ETramustine, Valrubicin, Verteporfm, Vinblastine, Vincristine, Vindesine, Vinflunine, Vinorelbine, Vorinostat, Zorubicin, and other cytostatic or cytotoxic agents described herein.
  • two or more drugs are often given at the same time.
  • two or more chemotherapy agents are used as combination chemotherapy.
  • the additional cancer therapeutic agent is a
  • differentiation agent includes retinoids (such as all-trans-retinoic acid (ATRA), 9-cis retinoic acid, l3-cis-retinoic acid (l3-cRA) and 4-hydroxy-phenretinamide (4- HPR)); arsenic trioxide; histone deacetylase inhibitors HDACs (such as azacytidine (Vidaza) and butyrates (e.g., sodium phenylbutyrate)); hybrid polar compounds (such as hexamethylene bisacetamide ((HMBA)); vitamin D; and cytokines (such as colony- stimulating factors including G-CSF and GM-CSF, and interferons).
  • retinoids such as all-trans-retinoic acid (ATRA), 9-cis retinoic acid, l3-cis-retinoic acid (l3-cRA) and 4-hydroxy-phenretinamide (4- HPR)
  • arsenic trioxide such as azacytidine
  • the additional cancer therapeutic agent is a targeted therapy agent.
  • Targeted therapy constitutes the use of agents specific for the deregulated proteins of cancer cells.
  • Small molecule targeted therapy drugs are generally inhibitors of enzymatic domains on mutated, overexpressed, or otherwise critical proteins within the cancer cell.
  • Prominent examples are the tyrosine kinase inhibitors such as Axitinib, Bosutinib, Cediranib, dasatinib, erlotinib, imatinib, gefitinib, lapatinib, Lestaurtinib, Nilotinib, Semaxanib, Sorafenib, Sunitinib, and Vandetanib, and also cyclin-dependent kinase inhibitors such as Alvocidib and Seliciclib.
  • Monoclonal antibody therapy is another strategy in which the therapeutic agent is an antibody which specifically binds to a protein on the surface of the cancer cells. Examples include the anti-HER2/neu antibody trastuzumab (HERCEPTIN®) typically used in breast cancer, and the anti-CD20 antibody rituximab and
  • Tositumomab typically used in a variety of B-cell malignancies.
  • Other exemplary antibodies include Cetuximab, Panitumumab, Trastuzumab, Alemtuzumab,
  • the targeted therapy can be used in combination with a compound described herein, e.g., a biguanide such as metformin or phenformin, preferably phenformin.
  • a biguanide such as metformin or phenformin, preferably phenformin.
  • Targeted therapy can also involve small peptides as“homing devices” which can bind to cell surface receptors or affected extracellular matrix surrounding the tumor. Radionuclides which are attached to these peptides (e.g, RGDs) eventually kill the cancer cell if the nuclide decays in the vicinity of the cell.
  • RGDs Radionuclides which are attached to these peptides
  • An example of such therapy includes BEXXAR®.
  • the additional cancer therapeutic agent is an
  • Cancer immunotherapy refers to a diverse set of therapeutic strategies designed to induce the subject's own immune system to fight the tumor.
  • Contemporary methods for generating an immune response against tumors include intravesicular BCG immunotherapy for superficial bladder cancer, and use of interferons and other cytokines to induce an immune response in renal cell carcinoma and melanoma subjects.
  • Allogeneic hematopoietic stem cell transplantation can be considered a form of immunotherapy, since the donor’s immune cells will often attack the tumor in a graft-versus-tumor effect.
  • the immunotherapy agents can be used in combination with a compound or composition described herein.
  • the additional cancer therapeutic agent is a hormonal therapy agent.
  • the growth of some cancers can be inhibited by providing or blocking certain hormones.
  • hormone-sensitive tumors include certain types of breast and prostate cancers. Removing or blocking estrogen or testosterone is often an important additional treatment.
  • administration of hormone agonists, such as progestogens may be therapeutically beneficial.
  • the hormonal therapy agents can be used in combination with a compound or a composition described herein.
  • Other possible additional therapeutic modalities include imatinib, gene therapy, peptide and dendritic cell vaccines, synthetic chlorotoxins, and radiolabeled drugs and antibodies.
  • Adduct S5 undergoes Buchwald reaction with
  • compound S7 can be synthesized according to Scheme 2 from adduct S5.
  • Adduct S5 undergoes an aromatic substitution reaction with ethyl 2- bromoisonicotinate S8 to give intermediate S9.
  • Reduction of intermediate S9 in the presence of N3 ⁇ 4 provides amide S10.
  • Amide S10 is further converted to intermediate S7 by treatment of trifluoroacetic anhydride (TFAA) in the presence of pyridine.
  • TFAA trifluoroacetic anhydride
  • Compound Sl can have 0, 1, 2, 3, or 4 deuterium substituents on the phenyl ring.
  • the compound of Sl is 2-chlorobenzaldehyde commercially available from Sigma-Aldrich.
  • n is 1, 2, 3, or 4
  • the relevant compound Sl can be synthesized according to Scheme 3.
  • Scheme 3 shows synthesis of four compounds Sl-d, Sl-f, Sl-i, and Sl-l.
  • compound Sl-a commercially available from DSK Biopharma Product List, can be selectively chlorinated using Pd catalyzed C-H chlorination as described in Angew Chemie, Int Ed. 2013, 52, 4440.
  • Ethyl benzoate-D5 (compound Sl-a) is stirred for 1-10 hours at 90°C with triflic acid (TfOH) as the solvent in the presence of a strong oxidant (such as
  • (2-Chlorophenyl-3,4,5,6-d4)methan-d2-ol (compound Sl-c) can be oxidized to provide aldehyde (compound Sl-d) in the presence of pyridinum chlorochr ornate (PCC) at 25°C for 1-4 hours (see Eur. J. Org. Chem., 2015, p2868).
  • Compound Sl-g can be reduced in the presence of LiAlD 4 at 0°C to 25° to afford (2-chlorophenyl)methan-d2-ol (compound Sl-h), which can be subsequently oxidized in methylene chloride in the presence of pyridinum chlorochromate (PCC) at 25C for 1-4 hours to afford 2-chlorobenzaldehyde-a-dl (compound Sl-i) (see Eur. J. Org. Chem., 2015, p2868).
  • PCC pyridinum chlorochromate
  • Additional compounds Sl can be prepared following the procedure as set forth above and Scheme 3. Exemplified additional compounds Sl are listed in Table 4 below. Table 4. Synthesis of additional compounds Sl based on Scheme 3
  • Scheme 4 provides two approaches to synthesize compounds S2-c and S2-k.
  • compound S2-a commercially available from Sigma-Aldrich, can be reduced in the presence of D 2 to give carboxylic acid S2-b, which can be subsequently reduced with diphenylphosphoryl azide to give pyridinylamine hydrochloride salt S2-c (see W02007076034 and W02006067445).
  • compound S2-d commercially available from Aurora Building Blocks, can be reduced in the presence of D 2 to give dicarboxylic acid S2-e, which undergoes esterification to give dicarboxylic ester S2-f (see Angew Chemie., Int. Ed., 2014, 53, pl l660).
  • Selective hydrolysis of dicarboxylic ester S2-f provides compound S2-g.
  • Reduction of the carboxylic group in compound S2-g in the presence of diphenylphosphoryl azide gives pyridinylamine S2-h (see
  • Compound S2-h can be subsequently fluorinated with HF- pyridine to generate compound S2-i (see W02006110668). Further hydrolysis of compound S2-i followed by another reduction with diphenylphosphoryl azide provides compound S2-k (see W02006067445).
  • Compound S2-f is partially hydrolyzed to 5-(methoxycarbonyl)nicotinic- 2,4,6-d3 acid (compound S2-g) in the presence of NaOH in methanolic water at room temperature for 5 minutes to up to an hour.
  • a solution of compound S2-g (3.24 mmol) and DIEA (1.1 ml, 6.5 mmol) in tert-BuOH (16 ml) is treated with
  • Additional compounds S2 can be prepared following the procedure as set forth above and Scheme 4 or purchased from commercial sources. Exemplified additional compounds S2 are listed in Table 5 below.
  • Compound S3 can be obtained from commercial sources or prepared according to Scheme 5. As shown in Scheme 5(i) and (ii), ethyl 3-chloro-3- oxopropanoate (compound S3-a) or tert-butyl methyl malonate (compound S3-d) can be reduced in the presence of NaBD 4 or D 2 to give hydroxypropanoate S3-b and S3-e respectively (see Jiang, Jinlong; Bunda, Jaime L. e-EROS Encyclopedia Reagents for Organic Synthesis; ChemCatChem. 2013, 5, 3228). Brominating compound S3-b or S3-e provides compound S3-c or S3-f (see W02010077836).
  • compound S3-g undergoes nucleophilic substitution with compound S3-c or S3-f to give compound S3-h (see Organic Syntheses, Coll. Vol. Ill, John Wiley and Sons, Inc., New York, N. Y., 1955, p 381; J. Org. Chem., 1966, 31, 4121).
  • Refluxing compound S3-h in the presence of SOCl 2 followed by protection with an acetyl group provides a compound of S3-j (see J. Org. Chem., 1966, 31, 4121).
  • Compound S3-j is treated with renal acylase to separate chiral compound S3-k from its enantiomer S3-k’ (see J. Org.
  • Additional compound 3 can be commercially obtained from LabNetwork
  • Compound S4 can be prepared according to Scheme 6. According to Scheme 6, two types of compound S4 can be prepared based on route (iii) and route (iv).
  • route (iii) provides compound S4-k with di-fluoro substituents on the cyclobutyl ring, while route (iv) generates compound S4-p with mono-fluoro substituent on the cyclobutyl ring.
  • the starting reagent S4-e of route (iii) can be synthesized from compound S4-a based on route (i) or (ii) in Scheme 6.
  • route (i) compound S4-a can be converted to acyl chloride S4-b, which reacts with sodium azide to form acyl azide S4-c. Reduction of acyl azide S4-c, followed by fluorination gives compound S4-e.
  • Compound S4-e can be alternatively synthesized based on route (ii) in Scheme 6.
  • Compound S4-a reacts with benzyl bromide to form benzyl ester S4-f, which undergoes fluorination to give compound S4-g.
  • Deprotection of compound S4-g and subsequent treatment with diphenylphosphoryl azide (DPP A) followed by Curtius rearrangement gives compound S4-e.
  • DPP A diphenylphosphoryl azide
  • Curtius rearrangement gives compound S4-e.
  • compound S4-e undergoes deprotection, formylation, and condensation to give compound S4-k.
  • compound S4-p can be synthesized from compound S4-d based on route (iv).
  • Compound S4-d can be reduced with NaBH 4. Subsequent fluorination gives compound S4-m, which undergoes deprotection, formylation, and condensation to give compound S4-p.
  • An exemplified compound S6 is commercially available from CombiPhos Product List and has the following Formula:
  • 4-carboxypyridine 1 -oxide (compound S6-a) is reacted with deuterated sodium hydroxide (i.e., Na in D 2 0 or NaOD) at 80°C to afford 4- carboxypyridine 1 -oxide-2, 6-d2.
  • deuterated sodium hydroxide i.e., Na in D 2 0 or NaOD
  • sodium deuteroxide (prepared from sodium metal, 0.71 g, 31.0 mmol, and D 2 0) ) may be reacted with 4-carboxypyridine l-oxide (21.6 mmol ) at 80 °C.
  • the cooled solution is acidified (cone hydrochloric acid) and the resulting precipitate of partially deuterated compound is subjected to a second hydrogen-deuterium exchange as above.
  • compound S5 and compound 8 undergo a Buchwald reaction to give compound S9.
  • the ethyl ester of compound S9 can be reduced to form amide S10, which can be further condensed to introduce the nitrile functional group in compound S7.
  • Compound S7 may be purified using standard chromatographic methods to provide the substantially pure enantiomer compound of Formula (II).
  • test compound is prepared as 10 mM stock in DMSO and diluted to 50X final concentration in DMSO, for a 50 pl reaction mixture.
  • IDH enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutaric acid is measured using a NADPH depletion assay. In the assay the remaining cofactor is measured at the end of the reaction with the addition of a catalytic excess of diaphorase and resazurin, to generate a fluorescent signal in proportion to the amount of NADPH remaining.
  • IDH1-R132 homodimer enzyme is diluted to 0.125 pg/ml in 40 pl of Assay
  • Buffer (l50 mM NaCl, 20 mM Tris-Cl pH 7.5, 10 mM MgCl 2 , 0.05% BSA, 2 mM b-mercaptoethanol); 1 m ⁇ of test compound dilution in DMSO is added and the mixture is incubated for 60 minutes at room temperature. The reaction is started with the addition of 10 m ⁇ of Substrate Mix (20 m ⁇ NADPH, 5 mM alpha-ketoglutarate, in Assay Buffer) and the mixture is incubated for 90 minutes at room temperature.
  • the reaction is terminated with the addition of 25 m ⁇ of Detection Buffer (36 pg/ml diaphorase, 30 mM resazurin, in IX Assay Buffer), and is incubated for 1 minute before reading on a SpectraMax platereader at Ex544/Em590.
  • Detection Buffer 36 pg/ml diaphorase, 30 mM resazurin, in IX Assay Buffer
  • Assay Buffer is (50 mM potassium phosphate, pH 6.5; 40 mM sodium carbonate, 5 mM MgCl 2 , 10% glycerol, 2 mM b-mercaptoethanol, and0.03% BSA).
  • concentration of NADPH and alpha- ketoglutarate in the Substrate Buffer is 20 mM and 1 mM, respectively.
  • Example 3 Cellular Assays for IDHlm (R132H or R132C) Inhibitors.
  • Cells (HT1080 or U87MG) are grown in T125 flasks in DMEM containing 10% FBS, lx penicillin/streptomycin and 500ug/mL G418 (present in U87MG cells only). They are harvested by trypsin and seeded into 96 well white bottom plates at a density of 5000 cell/well in 100 ul/well in DMEM with 10% FBS. No cells are placed in columns 1 and 12. Cells are incubated overnight at 37°C in 5% C0 2. The next day test compounds are made up at 2x the final concentration and lOOul are added to each cell well. The final concentration of DMSO is 0.2% and the DMSO control wells are plated in row G.
  • the plates are then placed in the incubator for 48 hours. At 48 hours, lOOul of media is removed from each well and analyzed by LC- MS for 2-HG concentrations. The cell plate is placed back in the incubator for another 24 hours. At 72 hours post compound addition, 10 mL/plate of Promega Cell Titer Glo reagent is thawed and mixed. The cell plate is removed from the incubator and allowed to equilibrate to room temperature. Then lOOul of Promega Cell Titer Glo reagent is added to each well of media. The cell plate is then placed on an orbital shaker for 10 minutes and then allowed to sit at room temperature for 20 minutes.
  • the plate is then read for luminescence with an integration time of 500ms.
  • the IC50 for inhibition of 2-HG production (concentration of test compound to reduce 2HG production by 50% compared to control) in these two cell lines for various compounds of Formula I is set forth in Table 3 above.
  • Metabolic stabilities of compounds of Formula I can be tested with the following assay and species specific liver microsomes (LM) extraction ratio (Eh) can be calculated:
  • Buffer A 1.0 L of 0.1 M monobasic Potassium Phosphate buffer containing 1.0 mM EDTA
  • Buffer B 1.0 L of 0.1 M Dibasic Potassium Phosphate buffer containing 1.0 mM EDTA
  • Buffer C 0.1 M Potassium Phosphate buffer, 1.0 mM EDTA, pH 7.4 by titrating 700 mL of buffer B with buffer A while monitoring with the pH meter.
  • 500 mM spiking solution add 10 pL of 10 mM DMSO stock solution into 190 pL CAN;
  • NADPH stock solution (6 mM) is prepared by dissolving NADPH into buffer C.

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Abstract

La présente invention porte sur des méthodes de traitement d'un cancer caractérisé par la présence d'un allèle mutant de IDH1, consistant à administrer à un sujet qui en a besoin un composé tel que présenté dans la description.
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US20170057994A1 (en) * 2013-07-25 2017-03-02 Agios Pharmaceuticals, Inc. Therapeutically active compounds and their methods of use
WO2017066571A1 (fr) * 2015-10-15 2017-04-20 Agios Pharmaceuticals, Inc. Polythérapie pour le traitement de tumeurs malignes

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Publication number Priority date Publication date Assignee Title
US20170057994A1 (en) * 2013-07-25 2017-03-02 Agios Pharmaceuticals, Inc. Therapeutically active compounds and their methods of use
WO2017066571A1 (fr) * 2015-10-15 2017-04-20 Agios Pharmaceuticals, Inc. Polythérapie pour le traitement de tumeurs malignes

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