WO2019099653A1 - Therapeutically active compounds and their methods of use - Google Patents

Abstract

Provided are methods of treating a cancer characterized by the presence of a mutant allele of IDH1comprising administering to a subject in need thereof a compound described here.

Description

THERAPEUTICALLY ACTIVE COMPOUNDS AND THEIR METHODS OF

USE

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application No. 62/587,131, filed November 16, 2017, the entire contents of which are hereby incorporated herein by reference.

BACKGROUND OF INVENTION

Isocitrate dehydrogenases (IDHs) 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,

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) is also known as IDH; IDP; IDCD; IDPC or 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

intraperoxisomal reductions, such as the conversion of 2, 4-dienoyl-CoAs to 3-enoyl- CoAs, as well as in peroxisomal reactions that consume 2-oxoglutarate, namely the alpha-hydroxylation of phytanic acid. The 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);

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).

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).

The inhibition of mutant IDH1 and its neoactivity is, therefore, a potential therapeutic treatment for cancer. Accordingly, there is an ongoing need for inhibitors of IDH1 mutants having alpha hydroxyl neoactivity.

Given that the effect of deuteration is unpredictable, variability in deuterium effects has also led experts to question or dismiss deuterium modification as 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. In this approach, one attempts to slow the CYP-mediated metabolism of a drug or to reduce the formation of undesirable metabolites by replacing one or more hydrogen atoms with deuterium atoms at the metabolic site of the drug. Deuterium is a safe, stable, non-radioactive isotope of hydrogen. Compared to 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.

SUMMARY OF INVENTION

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:

wherein each of Ri, R , R₂, R_2% R3-R7, Rs, Rs% R9, R9’, R10-R17 is

independently H or D, provided that at least one of Ri, Rr, R₂, R_2% R3-R7, Rs, Rs_% R9, Rg_% R10-R17 is D. The compound of Formula (I) inhibits mutant IDH1, particularly mutant IDH1 having alpha hydroxyl neoactivity. Also described herein are pharmaceutical compositions comprising a compound of Formula (I) or a

pharmaceutically acceptable salt or hydrate thereof.

DETAILED DESCRIPTION OF THE INVENTION

The details of construction and the arrangement of components set forth in the following description or illustrated in the drawings are not meant to be limiting.

Other embodiments and different ways to practice the invention are expressly included. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of“including,” “comprising,” or“having,”“containing”,“involving”, and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. Definitions:

As used herein, 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.

As used herein, the terms“inhibit” or“prevent” include both complete and partial inhibition and prevention. An inhibitor may completely or partially inhibit.

The term“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.

As used herein, an amount of a compound effective to treat a disorder, or a “therapeutically effective amount” 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.

As used herein, 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. Compounds

Provided is a compound having Formula (I):

or a pharmaceutically acceptable salt or hydrate thereof, wherein each of Ri, Rr, R₂, Rr, R₃-R₇, Rs, Rs_’, R₉, R_9’, R₁₀-R₁₇ is independently H or D, provided that at least one of Ri, Rr, R₂, Rr, R3-R7, Rs, Re R9, R9’, Rio-Rnis D.

Provided is also a compound having Formula (IA):

or a pharmaceutically acceptable salt or hydrate thereof, wherein each of Ri, Rr, R₂, Rr, R₃-R₇, Rs, R_S% R₉, R_9’, R₁₀-R₁₇ is independently H or D, provided that at least one of Ri, Rr, R₂, Rr, R₃-R₇, Rs, R_S% R₉, R_9’, and R₁₀-R₁₇ is D. In some embodiments, each of R₄-R₇, Rs, Rs% R₉, R_9’, and R₁₀-R₁₇ is H. In some embodiments, each of Ri, Rr, R₂, R_2’ is D and each of R₄-R₇, R₈, Rs_% R₉, R_9’, and R₁₀-R₁₇ is H. In other embodiments, each of Ri, Rr, R₂, R r, R₃, R₄, Rs, Rs_% R₉, R_9’, and Rio-Rn is H. In other embodiments, each of R₅, R₅, and R₇ is D and each of Ri, Rr, R₂, Rr, R₃, R₄,

R₈, R_8% R₉, R_9’, and Rio-Rn is H. In another embodiment, each of Ri, Rr, R₂, Rr, R₃- R₇, and Rn-Rn is H. In another embodiment, each of R₈, R₈ , R₉, R_9’, Rio is D and each of Ri, Rr, R₂, Rr, R₃-R₇, and Rn-Rn is H. In certain embodiments, each of Ri, Rr, R₂, Rr, R₃-R₇, R_7’, R₈, R_8’, R₉, R_9’, R_IO, and R₁₄-R₁₇ is H. In certain embodiments, each of Rn-Rn is D and each of Ri, Rr, R₂, Rr, R3-R7, R7’, Rs, Rs_% R9, R9’, Rio, and Ri₄-R_l7 is H. In further embodiments, each of Ri, Rr, R₂, Rr, R₃-R₇, R_7’, R₈, R_8% R₉, R_9’, and R₁₀-R₁₃ is H. In further embodiments, each of R₁₄-R₁₇ is D and each of Ri, Rr, R2, R2’, R3-R7, R7’, Re, Re’, R9, R9’, and R10-R13 is H.

Provided is also a compound having Formula (IB):

or a pharmaceutically acceptable salt or hydrate thereof, wherein each of Ri, Rr, R₂, Rr, R₃-R₇, R₉, 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-Ri₇ is D.

Provided is also a compound having Formula (IC):

or a pharmaceutically acceptable salt or hydrate thereof, wherein each of Ri, Rr, R₂, Rr, R₃-R₇, R₉, R_9’, R₁₁-R₁₃, and R₁₅-R₁₆ is independently H or D, provided that at least one of Ri, Rr, R₂, R₂ R₃-R₇, R₉, R_9’, R₁₁-R₁₃, and R₁₅-R₁₆ is D.

Provided is also a compound having Formula (ID):

or a pharmaceutically acceptable salt or hydrate thereof, wherein each of Ri, R , R₂, Rr, R₃, R₄, R₉, R_9’, R₁₁-R₁₃, and R₁₅-R₁₆ 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.

Provided is also a compound having Formula (IE):

or a pharmaceutically acceptable salt or hydrate thereof, wherein each of Ri, Rr, R₂, Rr, R₃, R₄, R₉, R_9’, and R₁₅-R₁₆ is independently H or D, provided that at least one of Ri, Rr, R2, R2’, R3, R4, R9, R9’, and R15-R16 is D.

Provided is also a compound having Formula (IF):

or a pharmaceutically acceptable salt or hydrate thereof, wherein each of Ri, R , R₂, Rr, R₃, R₉, R_9’, and R₁₅-R₁₆ is independently H or D, provided that at least one of Ri, Rr, R2, R2’, R₃, R9, R9’, and R15-R16 is D.

Provided is also a compound having Formula (IG):

or a pharmaceutically acceptable salt or hydrate thereof, wherein each of R₃, R₉, R_9’, and R₁₅-R₁₆ is independently H or D, provided that at least one of R₃, R₉, R_9’, and R₁₅- R_l6 is D.

In certain embodiments, exemplary compounds of Formula (I) or a

pharmaceutically acceptable salt or hydrate thereof are depicted below in Table 1.

Table 1. Exemplified Compounds of Formula (I).

In certain embodiments, the compounds of Formula (I) are of Formula (II):

or a pharmaceutically acceptable salt or hydrate thereof, wherein 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

1-17 represent the positions for the deuterium substituents. Positions 1, 2, 8, and 9 can each independently have one or two deuterium substituents. In certain embodiments, 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. In certain embodiments, 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. In certain embodiments, 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. In certain

embodiments, 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. In certain embodiments, 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. In certain embodiments, 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. In certain embodiments, the compounds of Formula (II) are listed in Table 2 below.

Table 2. Exemplified Compounds of Formula (II).

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. The term “substantially free of other stereoisomers” as used herein 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. Methods of obtaining or synthesizing an individual enantiomer or stereoisomer for a given compound are known in the art and may be applied as practicable to final compounds or to starting material or intermediates. In one embodiment, 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. For example, C may be in any isotopic form, including ¹²C, ¹³C, and ¹⁴C; O may be in any isotopic form, including ¹⁶0 and ¹⁸0; and the like. For example, 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%.

Unless otherwise indicated when a disclosed compound is named or depicted by a structure without specifying the stereochemistry and has one or more chiral centers, it is understood to represent all possible stereoisomers of the compound.

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.

The skilled artisan will recognize that some variation of natural isotopic abundance occurs in a synthesized compound depending upon the origin of chemical materials used in the synthesis. Thus, a preparation of undeuterated compound of Formula (I) contains deuterium atoms at the natural abundance. The skilled artisan would consider that the concentration of naturally abundant stable hydrogen to be small and immaterial as compared to the degree of stable isotopic substitution of compounds of this invention. See, for instance, Wada, E et al., Seikagaku, 1994, 66: 15; Gannes, L Z et al., Comp Biochem Physiol Mol Integr Physiol, 1998, 119:725.

In the compounds of this invention, any atom not specifically designated as a particular isotope is meant to represent any stable isotope of that atom. Unless otherwise stated, when a position is designated specifically as“H” or“hydrogen”, the position is understood to have hydrogen at its natural abundance isotopic composition. As used herein,“D” refers to deuterium (2H). In the compound of the invention, 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.

As used herein, the term“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.

In some embodiments, 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

incorporation), at least 6333 (95% deuterium incorporation), or at least 6600 (99% deuterium incorporation.

Compounds described herein may be prepared following procedures detailed in the examples and other analogous methods known to one skilled in the art.

Compounds produced by any of the schemes set forth below may be further modified (e.g., through the addition of substituents to rings, etc.) to produce additional compounds. The specific approaches and compounds shown herein are not intended to be limiting. The suitability of a chemical group in a compound structure for use in the synthesis of another compound is within the knowledge of one of ordinary skill in the art. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the applicable compounds are known in the art and include, for example, those described in Larock R,

Comprehensive Organic Transformations , VCH Publishers (1989); Greene, TW et ah,

Protective Groups in Organic Synthesis , 3^rd Ed., John Wiley and Sons (1999); Fieser, L et ak, Fieser and Fieser’s Reagents for Organic Synthesis , John Wiley and Sons (1994); and Paquette, L, ed., Encyclopedia of Reagents for Organic Synthesis , John Wiley and Sons (1995) and subsequent editions thereof.

Combinations of substituents and variables envisioned by this invention are only those that result in the formation of stable compounds.

It may be convenient or desirable to prepare, purify, and/or handle a 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.

If the compound is cationic, or has a functional group that may be cationic ( e.g. , -NH₂ may be -NH₃ ⁺), then a salt may be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous.

Examples of 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. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.

ETnless otherwise specified, a reference to a particular compound also includes salt forms thereof.

Compositions and routes of administration

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. In another

embodiment, 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. The term“pharmaceutically acceptable carrier or adjuvant” refers to a carrier or adjuvant that may be administered to a subject, together with a compound of 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

carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat. 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.

The pharmaceutical 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

pharmaceutically acceptable acids, bases or buffers to enhance the stability of the formulated compound or its delivery form. The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrastemal, intrathecal, intralesional and intracranial injection or infusion techniques.

The pharmaceutical 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

suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in l,3-butanediol. Among the acceptable vehicles and solvents that may be used are mannitol, water, Ringer’s solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally used as a solvent or suspending medium. For this purpose, 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

pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These 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

pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.

The pharmaceutical 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. In the case of tablets for oral use, carriers that are commonly used include lactose and corn starch.

Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried com starch. When aqueous suspensions and/or emulsions are administered orally, 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.

The pharmaceutical 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. Such materials 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. For application topically to the skin, 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. Alternatively, the pharmaceutical

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.

The pharmaceutical 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.

When the 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,

intramuscularly, or subcutaneously; or orally, buccally, nasally, transmucosally, topically, in an ophthalmic preparation, or by inhalation, with a dosage ranging from about 0.5 to about 100 mg/kg of body weight, alternatively dosages between 1 mg and 1000 mg/dose, every 4 to 120 hours, or according to the requirements of the particular drug. The methods herein contemplate administration of an effective amount of compound or compound composition to achieve the desired or stated effect.

Typically, the pharmaceutical 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

administration. A typical preparation will contain from about 5% to about 95% active compound (w/w). Alternatively, such preparations contain from about 20% to about 80% active compound.

Lower or higher doses than those recited above may be required. Specific dosage and treatment regimens for any particular subject will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, condition or symptoms, the subject’s disposition to the disease, condition or symptoms, and the judgment of the treating physician.

Upon improvement of a subject’s condition, 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.

The pharmaceutical 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.

Methods of Use

Provided is 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. In one embodiment, 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. In one aspect of this embodiment, the mutant IDH1 has an R132X mutation. In one aspect of this embodiment, the R132X mutation is selected from R132H, R132C, R132L, R132V, R132S and R132G. In another aspect, the R132X mutation is R132H or R132C. In yet another aspect, 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.

In one embodiment, 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. In one aspect of this embodiment, the IDH1 mutation is an R132X mutation. In another aspect of this embodiment, the R132X mutation is selected from R132H, R132C, R132L, R132V, R132S and R132G. In another aspect, the R132X mutation is 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.

Without being bound by theory, applicants believe that 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. Thus, the compounds and methods of this invention are useful to treat any type of cancer that is

characterized by the presence of a mutant allele of IDH1 imparting such acitivity and in particular an IDH1 R132H or R132C mutation.

In one aspect of this embodiment, the efficacy of cancer treatment is monitored by measuring the levels of 2HG in the subject. Typically 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. Once the elevated levels are established, the level of 2HG is determined during the course of and/or following termination of treatment to establish efficacy.

In certain embodiments, 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. Similarly, a determination that 2HG levels are not elevated during the course of or following treatment is also indicative of efficacy. Typically, 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. A variety of different liquid

chromatography (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. 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). One method allows resolution of TCA metabolites: t = 0, 50% B; t = 5, 95% B; t= 7, 95% B; t= 8, 0% B, where B refers to an organic mobile phase of 100% methanol. Another method is specific for 2-hydroxyglutarate, 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 ¹³C-glutamine can be performed as described, e.g., in Munger et al. Nat Biotechnol 26, 1179-86, 2008.

In one embodiment 2HG is directly evaluated.

In another embodiment a derivative of 2HG formed in process of performing the analytic method is evaluated. By way of example such 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.

In another embodiment 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:

In one embodiment 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.

Table 3. IDH mutations associated with certain cancers.

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). In one embodiment, 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. In other embodiments the cancer is a glioma, and the glioma is a low grade glioma or a secondary high grade glioma. In other embodiments, the cancer is glioma, and the glioma is a low grade glioma (grade II), anaplastic (grade III) or glioblastoma (GBM, grade IV). In other embodiments the cancer is acute myelogenous leukemia (AML). In still other embodiments the AML is refractory and/or relapsed. In yet other embodiments the AML is newly diagnosed and/or previously untreated.

In another embodiment, 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.

In one embodiment, prior to and/or after treatment with a compound of Structural 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.

In one embodiment, 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 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.

In one embodiment, 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

measurement, sample analysis of bodily fluid, such as serum or spinal cord fluid analysis, or by analysis of surgical material, e.g, by mass-spectroscopy. Combination therapies

In some embodiments, 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. Exemplary 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.

The term“co-administering” as used herein with respect to an additional cancer therapeutic agents 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. Alternatively, the additional cancer therapeutic agent may be administered prior to, consecutively with, or following the administration of a compound of this invention. In such combination therapy treatment, both the compounds of this invention and the second therapeutic agent(s) are administered by conventional methods. The administration of a composition of this invention, comprising both a compound of the invention and a second therapeutic agent, to a subject does not preclude the separate administration of that same therapeutic agent, any other second therapeutic agent or any compound of this invention to said subject at another time during a course of treatment. The term“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.

In some embodiments, the additional cancer therapeutic agent is a

chemotherapy agent. Examples of 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). Exemplary agents include Aclarubicin, Actinomycin, Alitretinoin, Altretamine, Aminopterin, Aminolevulinic acid, Amrubicin, Amsacrine, Anagrelide, Arsenic trioxide, Asparaginase, Atrasentan, Belotecan, Bexarotene, bendamustine, Bleomycin, Bortezomib, Busulfan,

Camptothecin, Capecitabine, Carboplatin, Carboquone, Carmofur, Carmustine, Celecoxib, Chlorambucil, Chlormethine, Cisplatin, Cladribine, Clofarabine,

Crisantaspase, Cyclophosphamide, Cytarabine, Dacarbazine, Dactinomycin,

Daunorubicin, Decitabine, Demecolcine, Docetaxel, Doxorubicin, Efaproxiral, Elesclomol, Elsamitrucin, Enocitabine, Epirubicin, Estramustine, Etoglucid,

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

aminolevulinate, Mitobronitol, Mitoguazone, Mitotane, Mitomycin, Mitoxantrone, Nedaplatin, Nimustine, Oblimersen, Omacetaxine, Ortataxel, Oxaliplatin, Paclitaxel, Pegaspargase, Pemetrexed, Pentostatin, Pirarubicin, Pixantrone, Plicamycin, Porfimer sodium, Prednimustine, Procarbazine, Raltitrexed, Ranimustine, Rubitecan,

Sapacitabine, Semustine, 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.

Because some drugs work better together than alone, two or more drugs are often given at the same time. Often, two or more chemotherapy agents are used as combination chemotherapy.

In some embodiments, the additional cancer therapeutic agent is a

differentiation agent. Such 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).

In some embodiments 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,

Bevacizumab, Edrecolomab, and Gemtuzumab. Exemplary fusion proteins include Aflibercept and Denileukin diftitox. In some embodiments, the targeted therapy can be used in combination with a compound described herein, e.g., 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. An example of such therapy includes BEXXAR®.

In some embodiments, the additional cancer therapeutic agent is an

immunotherapy agent. Cancer immunotherapy refers to a diverse set of therapeutic strategies designed to induce the subject's own immune system to fight the tumor. 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. In some embodiments, the immunotherapy agents can be used in combination with a compound or composition described herein.

In some embodiments, the additional cancer therapeutic agent is a hormonal therapy agent. The growth of some cancers can be inhibited by providing or blocking certain hormones. Common examples of hormone-sensitive tumors include certain types of breast and prostate cancers. Removing or blocking estrogen or testosterone is often an important additional treatment. In certain cancers, administration of hormone agonists, such as progestogens may be therapeutically beneficial. In some embodiments, 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.

EXAMPLES

The chemical name of each compound described below is generated by

ChemBioOffice software.

DCM = dichloromethane TEA = triethylamine

DPPA = diphenylphosphoryl azide TFA = trifluoroacetic acid

DIPEA = AVV-Diisopropylethylamine TFAA = trifluoroacetic anhydride

The starting materials and/or intermediate compounds are known in the art or can be prepared in accordance with methods known to those skilled in the art as exemplified by the methods described in WO2013107291, W02007076034,

W02006067445, W02006067445, Atkinson, J. G. et ah, J. Amer. Chem. Soc. 90:498

(1968), Berkessel. A. et a/., Angew Chemie, Int Ed. 53: 11660 (2014); Angew. Chem. Int. Ed. Sun, X. et al., 52:4440 (2013), Topolovcan, N. et al., Eur. J. Org. Chem. 2868 (2015), and Pavlik, J. W. et al., J Heterocyclic Chem. 42:73(2005), all of which are incorporated by reference in their entireties herein. Methods for synthesizing partially or fully deuterated compounds of Formula (I) or (II) are also described herein.

The compounds of Formulae (I)-(II), or a compound of any one of the embodiments described herein, can be prepared from the illustrated synthetic schemes as below.

Example 1. Synthesis of compounds of Formula (II).

Compounds of Formula (II) can be synthesized from the key intermediate S7 according to Scheme 1 or Scheme 2. As shown in Scheme 1, benzaldehyde Sl is mixed with primary amine S2 in methanol, followed by treatment with

oxopyrrolidine-2-carboxylic acid S3 and isocyanatocyclobutane S4, to give an adduct S5 through a UGI reaction. Adduct S5 undergoes Buchwald reaction with

bromoisonicotinonitrile S6 to provide intermediate S7. Compound S7 can be purified by chromatography to give the compound of Formula (II).

Scheme 1.

Alternatively, 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 N¾ provides amide S10. Amide S10 is further converted to intermediate S7 by treatment of trifluoroacetic anhydride (TFAA) in the presence of pyridine. Scheme 2.

Detailed synthesis of the reagents or intermediates in Schemes 1 and 2 are discussed below.

Example 1.1. Synthesis of compound SI

Compound Sl can have 0, 1, 2, 3, or 4 deuterium substituents on the phenyl ring. When n is 0, the compound of Sl is 2-chlorobenzaldehyde commercially available from Sigma-Aldrich. When 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. According to Scheme 3(i), 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. Reduction of compound Sl-a with LiAlD4 or LiAlEL gives compound Sl-c or Sl-e respectively. Subsequent oxidation of compounds Sl-c and Sl-e provides compounds Sl-d and Sl-f respectively. According to Scheme 3(ii), compound Sl-g is commercially available from AK Scientific Product Catalog or Accela ChemBio Product List and can be reduced with LiAlD4, followed by oxidation to provide compound Sl-i. Further, compound Sl-j is commercially available from Accela ChemBio Product List. Reduction of compound Sl-j in the presence of D₂ gives compound Sl-k (see Synthesis, 2010, 217) that can be converted to compound Sl-l by treatment with Pt0₂ and formic acid, or reduction followed by hydrolysis (see Bull Korean Chem. Soc., 2010, 31, 473; Tet. Lett. 2002, 43,1395).

Scheme 3.

Synthesis of compound Sl-d (Scheme 3, route (i))

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

Na₂S₂0x), N-chlorosuccinimide and Pd(OAc)₂ to afford ethyl 2-chlorobenzoate-D4 (compound Sl-b). See Angew Chemie, Int Ed. 2013, 52, 4440. Ethyl 2- chlorobenzoate-D4 (compound Sl-b) is reduced in THF in the presence of LiAlD₄ at 0°C to 25° to afford (2-chlorophenyl-3,4,5,6-d4)methan-d2-ol (compound Sl-c) (see Eur. J. Org. Chem., 2015, p2868). (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).

Synthesis of compound Sl-f (Scheme 3, route (i))

Ethyl 2-Chlorobenzoate-D4 (compound Sl-b) is reduced in the presence of

LiAlEE at 0°C to 25° to afford (2-chlorophenyl-3,4,5,6-d4)methanol (compound Sl- e). (2-Chlorophenyl-3,4,5,6-d4)methanol (compound Sl-e) is then oxidized in the presence of pyridinum chlorochromate (PCC) at 25°C for 1-4 hours to give aldehyde Sl-f (see Eur. J. Org. Chem., 2015, p2868). Synthesis of compound Sl-i (Scheme 3, route (ii))

Compound Sl-g can be reduced in the presence of LiAlD₄ 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).

Synthesis of additional compound SI based on Scheme 3

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

Example 1.2. Synthesis of compound S2 ( ^{N H}2 ).

Compound S2 can be prepared according to Scheme 4. In particular, Scheme 4 provides two approaches to synthesize compounds S2-c and S2-k. According to Scheme 4(i), compound S2-a, commercially available from Sigma-Aldrich, can be reduced in the presence of D₂ 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). According to Scheme 4(ii), compound S2-d, commercially available from Aurora Building Blocks, can be reduced in the presence of D₂ 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

W02006067445). 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).

Scheme 4

1. (Ph0)

D₂, Pd, CD₃OD AcONa ₂P(=0)N₃, iPr₂NEt,

-BuOH, 12h, rt to reflux

18 h, rt, 1 t

atm

(i)

2. HCI, MeOH, dioxane,

pH 3, 2 h, rt

S2-a S2-b S2-c

Synthesis of compound S2-c (Scheme 4(i))

A mixture of 2,6-dichloro-5-fluoro-3-pyridinecarboxylic acid (3.00 g, 0.0143 mol), anhydrous sodium acetate (3.516 g, 0.0429 mol), and 10 % palladium-on- carbon (0.300 g) in CD₃OD (i.e., deuterated methanol) (50 mL) is deuterated (using deuterium gas) at 1 atmosphere gas pressure (balloon) for 18 hours. The reaction is vacuum filtered through a 0.45 m PTFE membrane and the filtrate is concentrated by rotary evaporation in vacuo. The residue is taken up in ethyl acetate (100 mL) and water (30 mL). The aqueous pH adjusted to 3 with 6N aqueous hydrochloric acid, and the layers separate. The organic phase is washed with water (2 x 30 mL) and brine (30 mL), then dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give compound S2-b. See W02007076034. A solution of compound S2-b (3.24 mmol) and DIEA (1.1 ml, 6.5 mmol) in tert-BuOH (16 ml) is treated with diphenylphosphoryl azide (1.4 ml, 6.5 mmol). The resulting solution is heated at 85°C for 5 h. The solution is then concentrated under reduced pressure and the crude product is dissolved in 4.0 M HC1 in 30 dioxane (20 ml). The solution is stirred at 25 °C for 12 h. The solvents are removed under reduced pressure to give the hydrochloride salt of pyridinylamine S2-c. See W02006067445.

Synthesis of compound S2-k (Scheme 4(ii))

A mixture of compound S2-d, 2,4,6-trichloropyridine-3,5-dicarboxylic acid, ( .0143 mol), anhydrous sodium acetate (3.516 g, 0.0429 mol), and 10 % palladium- on-carbon (0.300 g) in CD₃OD (i.e., deuterated methanol) (50 mL) is deuterated (using deuterium gas) at 1 atmosphere gas pressure (balloon) for 18 hours. The reaction is vacuum filtered through a 0.45 m PTFE membrane and the catalyst is thoroughly washed with methanol (25 mL). The filtrate is concentrated by rotary evaporation in vacuo. See W02007076034. The residue is taken up in ethyl acetate (100 mL) and water (30 mL), the aqueous pH adjusted to 3 with 6N aqueous hydrochloric acid, and the layers separate. The organic phase is washed with water (2 x 30 mL) and brine (30 mL), then dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give compound S2-e, which is converted to its dimethyl ester (compound S2-f) using the method described in Angew Chemie., Int. Ed., 2014, 53, pl l660. 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

diphenylphosphoryl azide (1.4 ml, 6.5 mmol). The resulting solution is heated at 85°C for 5 h. The solution is then concentrated under reduced pressure and the crude product (compound S2-h) is dissolved in 4.0 M HC1 in 30 dioxane (20 ml). The solution is stirred at 25 °C for 12 h. The solvents are removed under reduced pressure to give the hydrochloride salt of amine S2-h. See W02006067445. To a stirring solution of 1.6 mmoles methyl 5-aminonicotinate-2,4,6-d3 (compound S2-h) in 5 mL of HF-pyridine at 0°C is added 119 mg (1.72 mmol) of NaN0_2. The mixture is stirred at 0°C for 30 min. and at 50°C for 1 h. The reaction is quenched by ice and sat.

NaHCCF solution. The aqueous layer is extracted with CHCI3, dried over Na₂S04, filtered, and concentrated. See WO 2006110668. Purification by flash silica gel chromatography (1 % MeOH/CHCl₃) provides compound S2-i. Methyl 5- fluoronicotinate-2,4,6-d3 (compound S2-i) is hydrolyzed using methods known to those skilled in the art to afford 5-fluoronicotinic-2,4,6-d3 acid (compound S2-j). A solution of 5-fluoronicotinic-2,4,6-d3 acid (3.24 mmol) and DIEA (1.1 ml, 6.5 mmol) in tert-BuOH (16 ml) is treated with diphenylphosphoryl azide (1.4 ml, 6.5 mmol). The resulting solution is heated at 85°C for 5 h. The solution is then concentrated under reduced pressure and the crude product is dissolved in 4.0 M HC1 in 30 dioxane (20 ml). The solution is stirred at 25 °C for 12 h. The solvents are removed under reduced pressure to give compound S2-k. See W02006067445.

Synthesis of additional compound S2 based on Scheme 4

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.

Table 5. Additional exemplified compounds S2.

(D)P

HOoC' .C NVo

Example 1.3. Synthesis of compound S3 ( ² H ).

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 NaBD₄ or D₂ 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). As shown in Scheme 5(iii), 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 SOCl₂ 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. Chem., 1966, 31, 4121). Stirring a mixture of S3-j and renal acylase selectively hydrolyzes one of the enantiomers of S3-j to afford S3-k, which is then separated from the unhydrolyzed acyl ester of the second enantiomer (for example, via crystallization of S3-k). Hydrolysis of the ester of the second enantiomer affords S3-k\ Cyclization of compound S3-k and S3-k’ generates proline intermediates S3-1 (d-proline) and S3-f (l-proline) (see Helv. Chim. Acta., 1990, 73, 122).

Scheme 5

Additional compound 3 can be commercially obtained from LabNetwork

Compounds Product List with the following Formula:

Example 1.4. Synthesis of compound

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).

Specifically, 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. According to 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. According to route (iii), compound S4-e undergoes deprotection, formylation, and condensation to give compound S4-k. On the other hand, 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.

Scheme 6

Synthesis of compound S4-k (Scheme 6 (i) & (iii))

To a solution of an undeuterated or a partially or fully deuterated 3- oxocyclobutanecarboxylic acid (compound S4-a, 88 mmol) in dry DCM (60 mL) at 0 °C, SOCl₂ (20 mL) is added dropwise. The mixture is heated to reflux for 1.5 hours and then evaporated in vacuo. The resulting mixture is co-evaporated twice with toluene (2 x 8 mL) and the residue is dissolved in acetone (30 mL), followed by adding dropwise to a solution of NaN₃ (12 g, 185.0 mmol) in H₂0 (35 mL) at 0 °C. After addition, the mixture is stirred for another 1 hour and then quenched with ice (110 g). The resulting mixture is extracted with Et₂0 (2 xlOO mL). Combined organic layers are washed with brine, dried over anhydrous Mg₂S0₄ and concentrated to about 15 mL solution. Toluene (2 x 30 mL) is added into the residue and the mixture is co- evaporated twice to remove Et₂0 (about 30 mL solution left each time to avoid explosion). The resulting toluene solution is heated to 90 °C until the evolution of N₂ ceased. Next, 40 mL of t-BuOH is added into the reaction mixture and the resulting mixture is stirred overnight at 90 °C. The mixture is cooled and concentrated. The residue is purified by column chromatography using petroleum ether / EtOAc (V:V,

7: 1 to 5: 1) as eluent to afford S4-d.

To a solution of an undeuterated or a partially or fully deuterated fe/7-butyl 3-oxocyclobutylcarbamate (compound S4-d, 111.07 mmol) in dry DCM (190 mL), DAST (diethylaminosulfur trifluoride) (41.0 mL, 222.14 mmol) is added dropwise at 0 °C under the atmosphere of N₂. The mixture is then allowed to warm up to r.t and stirred overnight. The resulting mixture is slowly added into a pre-cooled saturated aq. NaHCCh solution and extracted with DCM (3 x 200 mL). Combined organic layers are washed with brine, dried over anhydrous MgS0₄, and concentrated in vacuo. The residue is purified by column chromatography using petroleum ether / EtOAc (V:V, 15: 1) as eluent to afford compound S4-e. See WO06110668.

To a solution of MeOH (170 mL) and CH₃COCl (65 mL), an undeuterated or a partially or fully deuterated /cvv-butyl 3,3-difluoro-cyclobutylcarbamate (compound S4-e, 58.42 mmol) is added in one portion dropwise at 0 °C. The reaction mixture is stirred at 0 °C for 20 min, and then allowed to warm up to r.t and stirred for another 1.5 h. The reaction mixture is concentrated and dissolved in H₂0 (200 mL). The resulting mixture is extracted by Et₂0 (150 mL) and the aqueous layer is adjusted to pH=l 1 with solid Na₂C0₃ and extracted by DCM (2 x 150 mL). The combined organic layers are dried over anhydrous MgS0₄, filtered and concentrated in vacuo using a cold-water bath (<20 °C). The residue is dissolved in HCOOEt (90 mL), and transferred into a sealed pressure tube. This reaction mixture is heated to 80 °C and stirred overnight. The solvent is removed, and the residue is purified by column chromatography using petroleum ether / EtOAc (V:V, 1 : 1 to 1 :3) as eluent to afford compound S4-j .

To a solution of an undeuterated or a partially or fully deuterated A-(3,3- difluorocyclobutyl)-formamide (compound S4-j, 14.81 mmol) and PPh₃ (4.27 g, 16.29 mmol) in DCM (35 mL) are added CCl₄ (1.43 mL, 14.81 mmol) and TEA (2.06 mL, 14.81 mmol). The reaction mixture is stirred at 45 °C overnight under a N₂

atmosphere. The resulting mixture is evaporated in vacuo at 0 °C. The residue is suspended in Et₂0 (25 mL) at 0 °C for 30 min and then filtered. The filtrate is evaporated to about 5 mL at 0 °C under reduced pressure. The residue is purified by column chromatography using Et₂0 as eluent to afford compound S4-k.

Synthesis of compound S4-k (Scheme 6(ii) & (iii))

A mixture of an undeuterated or a partially or fully deuterated 3- oxocyclobutanecarboxylic acid (compound S4-a, 44 mmol), potassium carbonate (12 g, 88 mmol) and benzyl bromide (11.2 g, 66 mmol) in acetone (50 mL) is refluxed for 16 h. The solvent is then removed under reduced pressure and the residue is partitioned between ethyl acetate and water. Combined organic layers are dried over anhydrous MgS0₄, filtered and concentrated. The residue is purified with silica gel chromatography eluting with a gradient of 100% hexane to 96% hexane / EtOAc to give compound S4-f.

To a solution of an undeuterated or a partially or fully deuterated benzyl 3- oxocyclobutanecarboxylate (compound S4-f, 6.03 mmol) in DCM (35 mL) is added DAST (0.8 mL, 6.03 mmol) dropwise under nitrogen. The mixture is stirred at room temperature for 16 hours and then diluted with DCM. After successive washes with saturated sodium bicarbonate, 1N aq. hydrochloride acid, and brine, the organic layer is dried over anhydrous sodium sulfate, filtered and concentrated. The crude product is purified by silica gel chromatography with 93% hexane / EtOAc as eluent to give compound S4-g.

An undeuterated or a partially or fully deuterated Benzyl 3,3- difluorocyclobutanecarboxylate (compound S4-g, 3.72 mol) is dissolved in ethanol (40 mL), and approximately 0.02 g palladium on activated carbon is added. The mixture is stirred at room temperature for 12 hours under the atmosphere of H₂ and then filtered through a pad of Celite. The filtrates are concentrated and dried in vacuo to give compound S4-h.

An undeuterated or a partially or fully deuterated 3,3- difluorocyclobutanecarboxylic acid (compound S4-h, 27.3 mmol), DPPA (7.87 g, 27 mmol) and TEA (2.87 g, 28.4 mmol) are dissolved in /-BuOH (25 mL). The mixture is refluxed for 5 hours and then diluted with ethyl acetate (about 200 mL). The organic phase is washed twice with 5% citric acid and saturated sodium hydrogen carbonate respectively, dried over anhydrous Mg₂S0₄ and evaporated under reduced pressure. The residue is purified by silica gel chromatography with 50% hexane / EtOAc to give compound S4-e, which can be converted to compound S4-k as discussed above in Scheme 6(iii).

Synthesis of compound S4-p (Scheme 6(iv))

To a solution of an undeuterated or a partially or fully deuterated fer/-butyl 3- oxocyclobutylcarbamate (compound S4-d, 10.8 mmol, 2 eq) in EtOH (20 mL) is added NaBH₄ (204 mg, 1 eq) at 0 °C. The mixture is then allowed to warm to room temperature and stirred for 30 min. The mixture is concentrated in vacuo and the residue is purified by column chromatography using petroleum ether / EtOAc (V:V, 2: 1 to pure EtOAc) as eluent to afford compound S44. To a solution of an undeuterated or a partially or fully deuterated /cvV-butyl 3 -hydroxy cyclobutyl - carbamate (compound S4-1, 5.35 mmol) in dry DCM (20 mL) at -70 °C is added DAST dropwise (lg, 0.85 mL, 1.17 eq) under the atmosphere of N_2. The mixture is then slowly warmed to room temperature and stirred overnight. The resulting mixture is washed with diluted aq. NaHC0_3. The organic layer is dried over anhydrous Mg₂S0₄ and concentrated. The residue is purified by flash chromatography using petroleum ether / EtOAc (V:V, 20: 1 to 2: 1) as eluent to afford compound S4-m, which can be converted to compound S4-p following the similar procedures as set forth in Scheme 6 (iii).

Additional compound S4 can be synthesized from commercial reagents based on Scheme 7. Both compound S4-q and S4-t are commercially available from Abacipharm Product List. According to Scheme 7(i), compound S4-q can be converted to compound S4-r in a solution of NaOD in D₂0 (see J. Amer. Chem. Soc. 1968, 90, 498). Compound S4-r can subsequently be transformed to compound S4-s using the similar procedures as compound S4-h converted to compound S4-k as shown in Scheme 6 (ii) & (iii). Moreover, compound S4-t can be treated with base and benzyl bromide to form benzyl ester S4-v. Compound S4-v can be converted to compound S4-w using the similar procedures as compound S4-f converted to compound S4-k in Scheme 6(iii) & (iii). Scheme 7

S4-t S4-u S4-v S4-w

Example 1.5. Synthesis of compound

An exemplified compound S6 is commercially available from CombiPhos Product List and has the following Formula:

Additional compound S6 can be synthesized from Scheme 8. Specifically, compound S6-a is commercially available form ACES Pharma Product List.

Compound S6-a is deuterated in the presence of D₂0 to give compound S6-b (see J. Heterocyclic Chem. 2005, 42, 73). Esterification of compound S6-b followed by selective bromination gives compound S6-d (see J. Heterocyclic Chem. 2005, 42, 73; CN102199120). The methyl ester of compound S6-d can be converted to nitrile after coupling with compound S5 as shown in Scheme 2.

Scheme 8

Specifically, 4-carboxypyridine 1 -oxide (compound S6-a) is reacted with deuterated sodium hydroxide (i.e., Na in D₂0 or NaOD) at 80°C to afford 4- carboxypyridine 1 -oxide-2, 6-d2. For example, sodium deuteroxide (prepared from sodium metal, 0.71 g, 31.0 mmol, and D₂0) ) may be reacted with 4-carboxypyridine l-oxide (21.6 mmol ) at 80 °C. After 4 hr. 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.

Acidification of the reaction mixture affords 4-carboxypyridine 1 -oxide-2, 6-d2 (compound S6-b), which is refluxed in a mixture of toluene (10 mL), methanol (10 mL), and cone sulfuric acid (2 mL) followed by azeotropic distillation. The residue is poured onto ice (10 g), made basic with aqueous sodium carbonate (10 %), extracted with dichloromethane (5 x 20 mL) and the dried extract (sodium sulfate) evaporated to provide compound S6-c. Brominating compound S6-c with phosphoryl tribromide at l00°C for 8-24 hours affords methyl 2-bromoisonicotinate-6-d (compound S6-d). See J. Heterocyclic Chem. 2005, 42, 73; CN102199120.

Example 1.6. Synthesis of compound S7 (Scheme 1)

A mixture of compound Sl (0.78 mmol), compound S2 (0.78 mmol), compound S3 (0.78 mmol), and compound S4 (0.78 mmol) is stirred in MeOH overnight and undergoes UGI reaction to give compound S5. A mixture of compound S5 (0.20 mmol), 2-bromoisonicotinonitrile (0.30 mmol), Cs₂C0₃ (129 mg, 0.39 mmol), Pd₂(dba)₃ (18 mg, 0.02 mmol) and Xant-Phos (9.4 mg, 0.02 mmol) in 1,4- dioxane (10 mL) is stirred under N₂ at 80 °C overnight. After filtration, the filtrate is concentrated in vacuo and the residue is purified by a standard method to give compound S7.

Example 1.9. Synthesis of compound S7 (Scheme 2)

Alternatively, according to Scheme 2, 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.

Specifically, a mixture of compound S5 (0.20 mmol), compound S6-d (0.30 mmol), Cs₂C0₃ (129 mg, 0.39 mmol), Pd₂(dba)₃ (18 mg, 0.02 mmol) and Xant- Phos (9.4 mg, 0.02 mmol) in l,4-dioxane (10 mL) is stirred under N₂ at 80 °C overnight. After filtration, the filtrate is concentrated in vacuo and the residue is purified by a standard method to give compound S9. A mixture of compound S9 (0.3 mmole), NH₃ (0.4-1 mmole), and MeOH are stirred overnight at 60°C, sealed tube). After filtration, the filtrate is concentrated in vacuo and the residue is purified by a standard method to give compound S10. A mixture of compound S10 (0.3 mmole), pyridine, and TFAA in THF at 0°C overnight. The reaction mixture is concentrated in vacuo and the residue is purified by a standard method to give compound S7.

Example 2.0. Synthesis of compound of Formula (II) from compound S7

(Schemes 1 & 2)

Compound S7 may be purified using standard chromatographic methods to provide the substantially pure enantiomer compound of Formula (II).

Example 2: In Vitro Assays for IDHlm (R132H or R132C) Inhibitors

A 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 MgCl₂, 0.05% BSA, 2 mM b-mercaptoethanol); 1 mΐ 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 mΐ of Substrate Mix (20 mΐ 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 mΐ 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.

Compounds are assayed for their activity against IDH1 R132C following the same assay as above with the following modifications: Assay Buffer is (50 mM potassium phosphate, pH 6.5; 40 mM sodium carbonate, 5 mM MgCl₂, 10% glycerol, 2 mM b-mercaptoethanol, and0.03% BSA). The concentration of NADPH and alpha- ketoglutarate in the Substrate Buffer is 20 mM and 1 mM, respectively.

Representative compounds of Formula I set forth in Table 1 are tested in this assay or a similar assay.

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.

Example 4: Metabolic Stabilities of Compounds of Formula I

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:

1. 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.

2. Reference compounds (Ketanserin) and test compounds spiking solution:

500 mM spiking solution: add 10 pL of 10 mM DMSO stock solution into 190 pL CAN;

1.5 pM spiking solution in microsomes (0.75 mg/mL): add 1.5 pL of 500 pM spiking solution and 18.75 pL of 20 mg/mL liver microsomes into 479.75pL of Buffer C.

3. NADPH stock solution (6 mM) is prepared by dissolving NADPH into buffer C.

4. Dispense 30 pL 1.5X compound/liver microsome solution in 96-well plate and immediately add 135 pL ACN containing IS before adding l5uL Buffer C to prepare real 0 minute samples.

5. Add 15 pL of NADPH stock solution (6 mM) to the wells designated as

Time 30, and start timing.

6. At the end of incubation (0 min), add 135 pL of ACN containing the internal standard Osalmid) to all the wells (30 min, and 0 min). Then add 15 pL of NADPH stock solution (6 mM) to the wells designated as Time 0.

7. After quenching, centrifuge the reaction mixtures at 3220g for 10 min.

8. Transfer 50 pL of the supernatant from each well into a 96-well sample plate containing 50 pL of ultra pure water (Millipore) for LC/MS analysis.

Claims

Claims

1. A compound of F ormul a (I)

or a pharmaceutically acceptable salt or hydrate or crystalline form thereof, wherein each of Ri, Rr, R₂, Rr, R₃-R₇, Rs, Rs_% R₉, R , R₁₀-R₁₇ is independently H or D, provided that at least one of the R groups is D.

2. The compound of claim 1 or a pharmaceutically acceptable salt or hydrate or crystalline form thereof having Formula (IA):

3. The compound or a pharmaceutically acceptable salt or hydrate or crystalline form thereof of any one of claims 1-2, wherein each of R₄-R₇, Rx, Rx , R₉, R_9’, and R10-R17 is H.

4. The compound or a pharmaceutically acceptable salt or hydrate or crystalline form thereof of any one of claims 1-2, wherein each of Ri, Rr, R₂, Rr, R₃, R₄, R₈, R_8’, R₉, R_9’, and R₁₀-R₁₇ is H.

5. The compound of claims 1-2 or a pharmaceutically acceptable salt or hydrate or crystalline form thereof, wherein each of Ri, Rr, R₂, R₂ , R₃-R₇, and Rn- Rn is H.

6. The compound of any one of claims 1-2 or a pharmaceutically acceptable salt or hydrate or crystalline form thereof, wherein each of Ri, Rr, R₂, Rr, R₃-R₇, R7’, Rs, Re R9, R9’, Rio, and R14-R17 is H.

7. The compound of any one of claims 1-2 or a pharmaceutically acceptable salt or hydrate or crystalline form thereof, wherein each of Ri, Rr, R₂, Rr, R₃-R₇, R7 , R₈, R_8% R9, R9’, and R10-R13 is H.

8. The compound of claim 2 or a pharmaceutically acceptable salt or hydrate or crystalline form thereof having Formula (IB):

wherein each of Ri, Rr, R₂, R r, R₃-R₇, R₉, R , and Rn-R_l7 is independently H or D, provided that at least one of the R groups is D.

9. The compound of claim 2 or a pharmaceutically acceptable salt or hydrate or crystalline form thereof having Formula (IC):

wherein each of Ri, Rr, R₂, R , R₃-R₇, R₉, Rr, R₁₁-R₁₃, and R₁₅-R₁₆ is independently H or D, provided that at least one of the R groups is D.

10. The compound of claim 2 or a pharmaceutically acceptable salt or hydrate or crystalline form thereof having Formula (ID):

wherein each of Ri, Rr, R₂, R r, R₃, R₄, R₉, R_9’, R₁₁-R₁₃, and R₁₅-R₁₆ is independently H or D, provided that at least one of the R groups is D.

11. The compound of claim 2 or a pharmaceutically acceptable salt or hydrate or crystalline form thereof having Formula (IE):

wherein each of Ri, Rr, R₂, R_2% R₃, R₄, R₉, R_9’, and R_I5-R_I6 is independently H or D, provided that at least one of the R groups is D.

12. The compound of claim 2 or a pharmaceutically acceptable salt or hydrate or crystalline form thereof having Formula (IF):

wherein each of Ri, Rr, R₂, R_2% R₃, R₉, R9_’, and R₁₅-R₁₆ is independently H or D, provided that at least one of the R groups is D.

13. The compound of claim 2 or a pharmaceutically acceptable salt or hydrate or crystalline form thereof having Formula (IG):

wherein each of R₃, R₉, R_9’, and R₁₅-R₁₆ is independently H or D, provided that at least one of the R groups is D.

14. The compound of claim 2 having Formula (II):

15.

or a pharmaceutically acceptable salt or hydrate or crystalline form thereof, wherein 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 1-17 represent the positions for the deuterium substituents.

16. A compound of claim 1 or a pharmaceutically acceptable salt or hydrate or crystalline form thereof, that is chosen from:

17. A method of treating a cancer characterized by the presence of an IDH1 mutation, , comprising the step of administering to the patient in need thereof a composition comprising the compound of any of claims 1-16 or a pharmaceutically acceptable salt or hydrate thereof.

18. The method of claim 17, wherein the IDH1 mutation is an IDH1 R132H or R132C mutation.

19. The method of claim 17, wherein the cancer is selected from glioma, acute myelogenous leukemia, melanoma, non-small cell lung cancer (NSCLC), cholangiocarcinomas, chondrosarcoma, myelodysplastic syndromes (MDS), myeloproliferative neoplasm (MPN), colon cancer in a patient.

20. The method of claim 19, further comprising administering to the patient in need thereof a second therapeutic agent useful in the treatment of cancer.