WO2017140758A1

WO2017140758A1 - Derivatives of 2-amino-4-(2-oxazolidinon-3-yl)-pyrimidine fused with a five-membered heteroaromatic ring in 5,6-position which are useful for the treatment of various cancers

Info

Publication number: WO2017140758A1
Application number: PCT/EP2017/053464
Authority: WO
Inventors: Duncan HAY; Clive Mccarthy; Rose CHAPPELL
Original assignee: Debiopharm International S.A.; Evotec International Gmbh
Priority date: 2016-02-19
Filing date: 2017-02-16
Publication date: 2017-08-24

Abstract

The present invention relates to compounds of general Formula (I), uses of the compound of general Formula (I) for use in the treatment or prophylaxis of a disorder of the human or animal body, and pharmaceutical compositions comprising a therapeutically effective amount of the compounds of general Formula (I) as active ingredients.

Description

DERIVATIVES OF 2-AMINO-4-(2-OXAZOLIDINON-3-YL)-PYRIMIDINE

FUSED WITH A FIVE-MEMBERED HETEROAROMATIC

RING IN 5,6-POSITION WHICH ARE USEFUL FOR THE

TREATMENT OF VARIOUS CANCERS

Background of the Invention

Isocitrate dehydrogenases (IDH) represent a family of

cellular enzymes catalyzing NADP⁺ / NAD⁺ dependent oxidative decarboxylation of isocitrate, whereby a-ketoglutarate is produced and CO2 and NADPH / NADH are liberated.

Human IDHs exist in several forms; the isoforms IDH1

(cytosolic and peroxisomal) and IDH2 (mitochondrial) catalyze the reaction outside the context of the citric acid cycle and use NADP⁺ as a cofactor. Mutant forms of IDH often exhibit a neomorphic activity as a result of a "gain of function" mutation, whereby α-ketoglutarate is reduced to 2- hydroxyglutarate (2-HG) (P.S. Ward et al . , Cancer Cell 2010, 17, 225) . Regular cells have 2-HG levels, while cells and tumors with mutated IDH1 or IDH2 (referred herein as mIDHI and mIDH2) show increased 2-HG levels (See S. Gross et al . , J. Exp. Med. 2010, 207(2), 339). The dysregulation caused by the altered levels of 2-HG and α-ketoglutarate in cells bearing mIDHI and/or mIDH2 may contribute to an alteration in a number of cellular activities, e.g. signaling and gene expression, and give raise to increased angiogenesis in human cancers. Accordingly, elevated levels of 2-HG are highly associated with tumorigenesis (J.R. Prensner et al . , Nature Med. 2011, 17, 291-293) .

Malignant gliomas, including primary and secondary

glioblastomas, are among the most lethal and also the most prevalent of brain tumors. Genomic analyses of glioma genomes have revealed recurrent mutations of IDH in up to 70% of grade II-IV gliomas, in about 10% of acute myeloid leukemia (AML) cases, and in several other cancer types. In particular, IDH1 R132 and IDH2 R172 mutations have been shown to have neomorphic activity (reducing -ketoglutarate to 2- HG) , as disclosed by L. Dang et al . , Nature 2009, 462 and in WO 2011/123618 Al and WO 2015/003641 Al .

Mutant IDH1 and IDH2 have been further identified in multiple cancer types, in particular brain cancers, such as glioma, glioblastoma multiforme, paraganglioma, and supratentorial primordial neuroectodermal tumors (pNET) ; leukemia, such as acute myeloid leukemia (AML) , myelodysplasia syndrome, and chronic myelogenous leukemia (CML) , skin cancer such as melanoma, prostate cancer, thyroid cancer, colon cancer, lung cancer, sarcoma, including central chondrosarcoma, central and periosteal chondroma, and fibrosarcoma.

To date, only a few inhibitors of mlDHl and/or mIDH2 have been identified.

WO 2015/003355 A2 and WO 2015/003641 Al disclose compounds based on a 2, 4-diamino-l, 3, 5-triazine core as inhibitors of the neomorphic activity of mIDH2 R140Q and IDH2 R172K.

WO 2012/171506 Al discloses compounds based on a pyridine or a pyrimidine core fused to a saturated 6-membered ring as inhibitors of the neomorphic activity of mlDHl R132H.

WO 2013/046136 Al, WO 2014/141104 Al, WO 2014/141153 Al and WO 2014/147586 Al disclose inhibitors of the neomorphic activity of mlDHl R132H.

There is however still a need for small molecule inhibitors of mutant IDH proteins having a neomorphic activity, in particular for dual mIDHl/mIDH2 inhibitors, as well as for selective mlDHl and/or selective mIDH2 inhibitors, for use in the treatment of diseases and disorders associated with these mutant proteins. IDH related compounds are also described in WO 2015/017821 A2, WO 2015/003640 Al, WO 2015/003360 A2 , WO 2015/006591 Al, WO 2015/006592 Al, WO 2012/171337 Al, WO 2012/171506 Al, WO 2014/062511 Al, WO 2013/107291 Al, WO 2013/107405 Al and WO 2013/102431 Al .

Summary of the Invention

The present invention relates to a compound of general

Formula (I)

(I)

or a salt or solvate thereof, wherein n is 0 or 1 ;

R¹ is N (R¹¹ ) or CH;

R² is N or C (R¹²) ;

R³ is S or C (R¹³) ;

provided that

R¹ is CH and R² is CR¹² when R³ is S; and

R¹ is N(R^1X) when R³ is C(R¹³);

R¹¹ is a hydrogen atom or C¾;

R¹² is a hydrogen atom, a cyano group or C¾;

R¹³ is a hydrogen atom or a halogen atom;

R⁴, R⁵ are independently selected from a hydrogen atom; C¾OH; CH₂OCH₃; a Ci-6 alkyl group, wherein the Ci-6 alkyl group is unsubstituted or substituted with at least one halogen atom; or

R⁴, R⁵ may be joined together to form a C3-6 cycloalkyl group; R is an aromatic group, a heteroaromatic group or a C3-6 cycloalkyl group, wherein R⁶ is unsubstituted or substituted with one or two R^6a, which are the same or different;

R^6a is a halogen atom, a Ci-6 alkyl group, a Ci-6 alkoxy group, a S(0)₂-Ci-6 alkyl group, or R^6aa;

R^6aa is an aromatic group, a heteroaromatic group, or a heteroalicyclylalkyl group, wherein R^6aa is unsubstituted or substituted with one or two R^6b, which are the same or different ;

R^6b is a halogen atom; a Ci-6 alkyl group, which is

unsubstituted or substituted with at least one halogen atom; cyclopropyl or benzyloxycarbonyl ;

R⁷, R⁸ are independently selected from a hydrogen atom, or R^7a, or R⁷ and R⁸ may be joined together to form a C3-6 cycloalkyl group or a heteroalicyclic group;

R^7a is a Ci-6 alkyl group, an aromatic group, a heteroaromatic group, or a heteroalicyclic group, wherein R^7a is

unsubstituted or substituted with a substituent selected from a halogen atom, C¾, or OH;

R⁹, R¹⁰ are independently selected from a hydrogen atom, or R^9a, or R⁹ and R¹⁰ may be joined together to form a C3-6 cycloalkyl group or a heteroalicyclic group;

R^9a is a Ci-6 alkyl group, an aromatic group, a heteroaromatic group, or a heteroalicyclic group, wherein R^9a is

unsubstituted or substituted with a substituent selected from a halogen atom, C¾, or OH.

A further aspect of the present invention relates to the compound of general Formula (I) or a salt or solvate thereof as a medicament and may be used in the treatment of a human or animal body. In particular, the present invention relates to the compound of the general Formula (I) or a salt or a solvate thereof for use in the treatment or prophylaxis of a disease or disorder associated with a mutant isocitrate dehydrogenase having a neomorphic activity, preferably a cell proliferation disorder.

Yet a further aspect of the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of the compound of general Formula (I), or a salt or solvate thereof as active ingredient. In a particular embodiment, said pharmaceutical composition may be an oral dosage form.

Yet a further aspect of the present invention relates to the use of a compound of the general Formula (I) or a salt or a solvate thereof for the preparation of a medicament for the treatment or prophylaxis of a disease or disorder associated with a mutant isocitrate dehydrogenase having a neomorphic activity, preferably a cell proliferation disorder.

Yet a further aspect of the present invention is a method of treating, controlling, delaying or preventing in a mammalian patient in need of the treatment of one or more diseases or disorders associated with a mutant isocitrate dehydrogenase having a neomorphic activity, preferably a cell proliferation disorder, wherein the method comprises the administration to said patient a therapeutically effective amount of a compound of the present invention or a salt or a solvate thereof.

Detailed Description of the Invention

Compounds of the Present Invention

The term "substituted" means that one or more hydrogen atoms of a molecule or residue are replaced by a substituent.

Suitable substituents are further described herein. Generally -but not limited to-, "one or more substituents" means one, two or three, preferably one or two substituents and more preferably one substituent unless specifically defined herein. Generally these substituents can be the same or different .

The term "alkyl" or "alkyl group" means a straight-chain or branched hydrocarbon chain.

The term "C₁-4 alkyl" or "C₁-4 alkyl group" means an alkyl chain having 1 - 4 carbon atoms, e.g. if present at the end of a molecule: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, or e.g. -CH₂-, -CH₂-CH₂-, - CH(CH₃)-, -CH2-CH2-CH2-, -CH(C₂H₅)-, -C(CH₃)₂-, when two moieties of a molecule are linked by the alkyl group.

The term "Ci-6 alkyl" or "Ci-6 alkyl group" means an alkyl chain having 1 - 6 carbon atoms, e.g. if present at the end of a molecule: C₁-4 alkyl, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl; tert-butyl, n-pentyl, n-hexyl, or e.g. -CH₂-, -CH₂-CH₂-, -CH(CH₃)-, -CH₂-CH₂-CH₂- , -CH(C₂H₅)-, -C(CH₃)₂-, when two moieties of a molecule are linked by the alkyl group. The term "Ci_6 alkoxy group" (O-Ci-6 alkyl) means a Ci-6 alkyl group, which is attached to the rest of the molecule via oxygen atom. Examples are OCH₃ and OCH₂CH₃.

The term "aromatic group" means a carbocyclic aromatic system containing one, two or three rings wherein such rings may be attached together in a pendent manner or may be fused. The term "aromatic group" embraces aromatic substituents such as phenyl, naphthyl, and biphenyl . In a preferred embodiment, the term "aromatic group" refers to a phenyl group.

The term "C3-6 cycloalkyl" or "C3-6 cycloalkyl ring" or "C3-6 cycloalkyl group" means a cyclic alkyl chain having 3 - 6 carbon atoms, e.g. cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl . Preferably, cyloalkyl refers to cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

The term "3 to 7 membered heterocyclyl" or "3 to 7 membered heterocycle" means a ring with 3, 4, 5, 6 or 7 ring atoms that may contain up to the maximum number of double bonds (aromatic or non-aromatic ring which is fully, partially or unsaturated) wherein at least one ring atom up to 4 ring atoms are replaced by a heteroatom selected from the group consisting of sulfur (including -S(O)-, -S(0)₂-), oxygen and nitrogen (including =N(0)-) and wherein the ring is linked to the rest of the molecule via a carbon or nitrogen atom.

Examples for a 3 to 7 membered heterocycles are aziridine, azetidine, oxetane, thietane, furan, thiophene, pyrrole, pyrroline, imidazole, imidazoline, pyrazole, pyrazoline, oxazole, oxazoline, isoxazole, isoxazoline, thiazole, thiazoline, isothiazole, isothiazoline, thiadiazole,

thiadiazoline, tetrahydrofuran, tetrahydrothiophene,

pyrrolidine, imidazolidine, pyrazolidine, oxazolidine, isoxazolidine, thiazolidine, isothiazolidine,

thiadiazolidine, sulfolane, pyran, dihydropyran,

tetrahydropyran, imidazolidine, pyridine, pyridazine, pyrazine, pyrimidine, piperazine, piperidine, morpholine, tetrazole, triazole, triazolidine, tetrazolidine, diazepane, azepine or homopiperazine . The term "4 to 7 membered

heterocyclyl" or "4 to 7 membered heterocycle" is defined accordingly. The term "5 to 6 membered heterocyclyl" or "5 to 6 membered heterocycle" is defined accordingly.

"Saturated 4 to 7 membered heterocyclyl" or "saturated 4 to 7 membered heterocycle" means fully saturated "4 to 7 membered heterocyclyl" or "4 to 7 membered heterocycle".

"4 to 7 membered at least partly saturated heterocyclyl" or "4 to 7 membered at least partly saturated heterocycle" means an at least partly saturated "4 to 7 membered heterocyclyl" or "4 to 7 membered heterocycle".

"5 to 6 membered aromatic heterocyclyl" or "5 to 6 membered aromatic heterocycle" means a heterocycle derived from cyclopentadienyl or benzene, where at least one carbon atom is replaced by a heteroatom selected from the group

consisting of sulfur (including -S(O)-, -S(0)₂-), oxygen and nitrogen (including =N(0)-). Examples for such heterocycles are furan, thiophene, pyrrole, imidazole, pyrazole, oxazole, isoxazole, thiazole, isothiazole, thiadiazole, triazole, tetrazole, pyridine, pyrimidine, pyridazine, pyrazine, triazine .

The term "heteroaromatic group" means a 5 to 6 membered aromatic heterocyclyl or 5 to 6 membered aromatic

heterocycle . The term "heteroalicyclic group" means a 4 to 7 membered at least partly saturated heterocyclyl or 4 to 7 membered at least partly saturated heterocycle or a saturated 4 to 7 membered heterocyclyl or saturated 4 to 7 membered

heterocycle. Preferably the term "heteroalicyclic group" means a saturated 4 to 7 membered heterocyclyl or saturated 4 to 7 membered heterocycle.

The term "heteroalicyclylalkyl group" means a heteroalicyclyl group, which is attached to the rest of the molecule via a Ci-6 alkyl group.

The term "halogen" or "halogen atom" means fluoro, chloro, bromo or iodo. It is generally preferred that halogen is fluoro or chloro.

The term "cyano group" means -CN.

The term "benzyl" means Ph-C¾- (phenylmethyl ) , wherein "Ph" means phenyl .

The compounds of the present invention may also exist as several tautomeric forms. For instance, when the substituent _R9a is a pyridyl group substituted with a hydroxyl group, _R9a and the corresponding compound of the Formula (I) may exist in a corresponding pyridone form.

The compound of the present invention or a salt or solvate thereof is preferably the compound of general Formula (I) as defined in the summary of the invention or a salt or solvate thereof, wherein

(i) R¹ is NH, R² is CH, R³ is CH; or (ii) R is NH, IT is N, R^J is CH; or

(iii) R¹ is CH, R² is CH, R³ is S ; or

(iv) R¹ is NH, R² is CH, R³ is CF; or

(v) R¹ is NH, R² is CCN, R³ is CH; or

(vi) R¹ is NH, R² is CCH₃, R³ is CH; or

(vii) R¹ is NCH₃, R² is CH, R³ is CH.

The compound of the present invention or a salt or solvate thereof is preferably the compound of general Formula (I) as defined in the summary of the invention or a salt or solvate thereof, wherein one of R⁴, R⁵ is a hydrogen atom or CH₃ and the other is a hydrogen atom, CH₃, CH₂CH₃, CH₂OH, CH₂OCH₃, CH₂F or CF₃; or R⁴, R⁵ are joined together to form a cyclopropyl group .

The compound of the present invention or a salt or solvate thereof is preferably the compound of general Formula (I) as defined in the summary of the invention or a salt or solvate thereof, wherein R⁶ is an aromatic group or a heteroaromatic group and wherein R⁶ is unsubstituted or substituted with one or two R^6a, which are the same or different.

The compound of the present invention or a salt or solvate thereof is preferably the compound of general Formula (I) as defined in the summary of the invention or a salt or solvate thereof, wherein R⁶ is imidazolyl, pyridyl, thiazolyl, pyrazolyl, phenyl, oxadiazolyl, oxazolyl, cyclohexyl, thiophenyl or furanyl and wherein R⁶ is unsubstituted or substituted with one or two R^6a, which are the same or different .

The compound of the present invention or a salt or solvate thereof is preferably the compound of general Formula (I) as defined in the summary of the invention or a salt or solvate thereof, wherein R^6a is CH₃, OCH₃, F, CI, S(0)₂CH₃ or R^6aa.

The compound of the present invention or a salt or solvate thereof is preferably the compound of general Formula (I) as defined in the summary of the invention or a salt or solvate thereof, wherein R^6aa is phenyl, pyridyl, thiophenyl or piperazinylmethyl and wherein R^6aa is unsubstituted or

substituted with one or two R^6b, which are the same or different .

The compound of the present invention or a salt or solvate thereof is preferably the compound of general Formula (I) as defined in the summary of the invention or a salt or solvate thereof, wherein R^6b is CI, F, CF₃, cyclopropyl or

benzyloxycarbonyl .

The compound of the present invention or a salt or solvate thereof is preferably the compound of general Formula (I) as defined in the summary of the invention or a salt or solvate thereof, wherein one of R⁷, R⁸ is a hydrogen atom or CH₃ and the other is C¾F, a fluoroethyl, methyl, ethyl, isopropyl, tert. -butyl, 1-methyl-propyl, a hydroxyethyl , phenyl, pyridyl or methylimidazolyl ; or R⁷ and R⁸ are joined together to form a cyclopropyl, tetrahydrofuranyl or tetrahydropyranyl group.

The compound of the present invention or a salt or solvate thereof is preferably the compound of general Formula (I) as defined in the summary of the invention or a salt or solvate thereof, wherein R⁹ and R¹⁰ are hydrogen atoms, and at least one of R⁷ and R⁸ is other than a hydrogen atom. The compound of the present invention or a salt or solvate thereof is preferably the compound of general Formula (I) as defined in the summary of the invention or a salt or solvate thereof, wherein n is 1.

The compound of the present invention or a salt or solvate thereof is preferably the compound of general Formula (I) as defined in the summary of the invention or a salt or solvate thereof, wherein n is 1, R⁴ is a hydrogen atom and R⁵ is other than a hydro en atom to give Formula (la)

Finally, the compound of the present invention or a salt or solvate thereof is preferably selected from Table 1 herein or more preferably one of the following:

and is even more preferably

A further aspect of the present invention is a solvent or salt of a compound of formula (I) .

Accordingly, in case the compounds according to formula (I) contain one or more acidic or basic groups, the invention also comprises their corresponding salt, preferably

pharmaceutically acceptable salts. Thus, the compounds of the formula (I) which contain acidic groups can be used according to the invention, for example, as alkali metal salts,

alkaline earth metal salts or as ammonium salts. More precise examples of such salts include sodium salts, potassium salts, calcium salts, magnesium salts or salts with ammonia or organic amines such as, for example, ethylamine,

ethanolamine, triethanolamine or amino acids. Compounds of the formula (I) which contain one or more basic groups, i.e. groups which can be protonated, can be present and can be used according to the invention in the form of their addition salts with inorganic or organic acids. Examples for suitable acids include hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p- toluenesulfonic acid, naphthalenedisulfonic acids, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid,

sulfaminic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid, and other acids known to the person skilled in the art. If the compounds of the formula (I) simultaneously contain acidic and basic groups in the molecule, the invention also includes, in addition to the salt forms mentioned, inner salts or betaines ( zwitterions ) . The respective salts

according to the formula (I) can be obtained by customary methods which are known to the person skilled in the art like, for example by contacting these with an organic or inorganic acid or base in a solvent or dispersant, or by anion exchange or cation exchange with other salts. The present invention also includes all salts of the compounds of the formula (I) which, owing to low physiological

compatibility, are not directly suitable for use in

pharmaceuticals but which can be used, for example, as intermediates for chemical reactions or for the preparation of pharmaceutically acceptable salts.

Use of the Compounds of the Invention in Methods for Treatment

A compound of general Formula (I) according to the present invention or a salt or solvate thereof can be used in the treatment or prophylaxis of a human or animal body.

The use may be in the treatment or prophylaxis of a disorder associated with a mutant isocitrate dehydrogenase having a neomorphic activity. The disorder associated with a mutant isocitrate dehydrogenase having a neomorphic activity may be a cell proliferation disorder. The disorder may be one of the following: brain cancer, such as glioma, glioblastoma

multiforme, paraganglioma, and supratentorial primordial neuroectodermal tumors (pNET) , leukemia, such as acute myeloid leukemia (AML) , myelodysplasia syndrome, and chronic myelogenous leukemia (CML) , skin cancer such as melanoma, prostate cancer, thyroid cancer, colon cancer, lung cancer, sarcoma, including central chondrosarcoma, central and periosteal chondroma, and fibrosarcoma.

Pharmaceutical Compositions of the Invention

The present invention also provides pharmaceutical

compositions comprising the compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof, as active ingredient, and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable salt" includes an ammonium salt, a calcium salt, a magnesium salt, a potassium salt or a sodium salt of compound of Formula (I) .

The pharmaceutical compositions of the present invention may comprise the compound of Formula (I), or a pharmaceutically acceptable salt or solvate thereof in form of one or several polymorphs .

The term "composition" , as in pharmaceutical composition, is intended to encompass a product comprising the active

ingredient, and the inert ingredient ( s ) (such as

pharmaceutically acceptable excipients) that make up the pharmaceutically acceptable carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the

ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the

pharmaceutical compositions of the present invention

encompass any composition made by admixing the compound of Formula (I) and pharmaceutically acceptable excipients.

Any suitable route of administration may be employed for providing a patient, with an effective dosage of a compound of the present invention, including without limitation oral and parenteral (such as intravenous bolus or infusion, injection, intraperitoneal, subcutaneous or intramuscular administration) .

Pharmaceutical compositions of the present invention that are suitable for oral administration (oral dosage forms) may be presented in solid or liquid form. Suitable solid oral dosage forms include discrete units such as capsules, pills,

cachets, powders (such as effervescent) , granules or tablets, and the like, each containing a predetermined amount of the compound of Formula (I) as active ingredient. Suitable liquid oral dosage forms include solutions or suspensions in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion, including

elixirs, tinctures, solutions, suspensions, syrups and emulsions. Pharmaceutical compositions of the present

invention may also be in the form of sustained release formulations .

Any inert ingredient that is commonly used as a carrier or diluent may be used in the formulations of the present invention, such as for example, a gum, a starch, a sugar, a cellulosic material, an acrylate, or mixtures thereof. A preferred diluent is microcrystalline cellulose. The

compositions may further comprise a disintegrating agent (e.g., croscarmellose sodium) and a lubricant (e.g.,

magnesium stearate) , and may additionally comprise one or more additives selected from a binder, a buffer, a protease inhibitor, a surfactant, a solubilizing agent, a plasticizer, an emulsifier, a stabilizing agent, a viscosity increasing agent, a sweetener, a film forming agent, or any combination thereof .

The pharmaceutical compositions of the present invention may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation. For example, a tablet may be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be

prepared by compressing in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent. Desirably, each tablet, cachet or capsule contains from about 0.1 to 1,000 mg, particularly 0.1, 0.2, 0.5, 1.0, 5, 10, 25, 50, 75, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 175, 180, 200, 225, 250, 300, 350, 400, 450, 500, 750 and 1,000 milligrams of the active ingredient, for the

symptomatic adjustment of the dosage to the patient to be treated .

Examples

Methods of preparation of compound of Formula (I)

The following general methods described hereinafter in the schemes and in examples may be used to prepare compounds of Formula (I) using appropriate materials. If such starting materials are not commercially available, they may be

prepared by standard synthetic techniques. Methods for extraction, isolation, purification, treatment known by one skilled in the art may be applied. Moreover, by utilizing the procedure described below, one of ordinary skilled in the art can readily prepare additional compounds of the present invention claimed herein.

Where typical or preferred experimental conditions are given (i.e. solvent, temperature, reaction time, stoichiometry of reagents, etc.), other experimental conditions may also be used unless otherwise stated. Compounds of the general

Formula (I) might be synthesised by several processes using both solid and/or solution phase chemistry protocols. Examples of synthetic pathways for the preparation of compounds of general Formula (I) are described here below. Optimal reaction conditions may vary with particular

reactants or solvent, but such conditions can be determined by the person skilled in the art using routine optimization procedures. Compound numbers refer to the numbering in Table 1 below. A synthetic scheme for the preparation of Compound 12 is shown below. The exemplary synthetic procedures can be easily modified for the preparation of further compounds of the present invention by substituting the corresponding intermediates as described in detail below.

Scheme 1: Synthesis of Compound 12 (for reaction conditions details, see the experimental procedures below) .

Abbreviations

aq Aqueous Brine Saturated solution of sodium chloride in water

DIPEA N, -Diisopropylethylamine

DMF N, -Dimethylformamide

EtOAc Ethyl acetate

EtOH Ethanol

Et0₂ Diethyl ether

FCC Flash column chromatography

g Gram (s)

h Hour (s)

HPLC High-performance liquid chromatography

IPC In process check

LDA Lithium diisopropylamide

MeCN Acetonitrile

MeOH Methanol

Min (s) Minute (s)

mg Milligram (s)

ml Millilitre (s)

mm Millimetre (s)

mM Millimolar

aHC03 Sodium hydrogen carbonate

NaOH Sodium hydroxide

a₂S₂03 Sodium thiosulfate

NH₃ Ammonia

NH₄CI Ammonium chloride

NMR Nuclear Magnetic Resonance

RT Room temperature

SCX Cation-exchange columns with

propylsulfonic acid-tailed silica

THF Tetrahydrofuran

TBME Methyl tert-butyl ether

t_R Time of retention

μΐ Microlitre (s) ym Micrometre (s)

Analytical LCMS conditions are as follows :

System 1: ACIDIC IPC 2 min METHOD: Linear gradient 5-100 % solvent B in 1.5 mins + 0.1 mins 100 % solvent B, flow rate lml /min. Column Supelco Ascentis Express Part No. 53802-U (30 X 2.1 mm, 1.7 ym) . Solvent A = 0.1 % Formic acid in water, Solvent B = 0.1 % Formic acid in Acetonitrile .

System 2: ACIDIC FINAL 7 min METHOD: Linear gradient 5-100 % solvent B in 5.3 mins + 0.5 mins 100 % solvent B, flow rate 0.6ml/min. Column Phenomenex Kinetix-XB C18 Part No. 00D- 4498-AN (100 X 2.1 mm, 1.7 ym) . Solvent A = 0.1 % Formic acid in water, Solvent B = 0.1 % Formic acid in Acetonitrile.

Synthesis of methyl (4S) -3- [ (4-methoxyphenyl) methyl] -2-oxo-l , 3- oxazolidine-4-carboxylate : INTERMEDIATE 1 3

Sodium hydride (60%, 8.77g, 219 mmol) was added to a cooled (-10 °C) solution of methyl (4S) -2-oxo-l, 3-oxazolidine-4- carboxylate (30 g, 208.8 mmol) in dry DMF (1500 ml) under nitrogen and the mixture stirred at -10 °C for 5 min. 1- (chloromethyl) -4-methoxybenzene (31.9 ml, 230 mmol) was added and the mixture stirred at -10 °C to 0 °C over 1 hr and at RT for 2 hr . The mixture was poured into an ice cold mixture of EtOAc (1 1) and 0.5M citric acid (100 ml) in water (1 1) in 1 portion. The organic layer was separated and the aqueous re- extracted with EtOAc (500 ml) . The combined organic extracts were washed with water (4 x 1 1), sat brine (1 1), dried over sodium sulfate and concentrated in vacuo to give the crude product as a pale yellow oil which was purified by

chromatography on silica eluting with hept3.ΠΘ 5% to 50% EtOAc to give methyl (4S) -3- [ (4-methoxyphenyl) methyl] -2-oxo-l, 3- oxazolidine-4-carboxylate (18.51 g) as a yellow oil.

! H NMR (500 MHz, Chloroform-d) δ 7.18 (d, J = 8.6 Hz, 2H) , 6.86 (d, J = 8.7 Hz, 2H) , 4.82 (d, J = 14.8 Hz, 1H) , 4.41 - 4.35 (m, 1H) , 4.32 (dd, J = 9.0, 5.1 Hz, 1H) , 4.17 (d, J = 14.8 Ηζ,ΙΗ), 4.08 (dd, J = 9.5, 5.1 Hz, 1H) , 3.80 (s, 3H) , 3.75 (s, 3H) .

LCMS MH+ 265.9 RT 1.00 System 1 Synthesis of (4R) -4- (hydroxymethyl) -3- [ (4- methoxyphenyl) methyl] -1 , 3-oxazolidin-2-one : INTERMEDIATE 2

aBH4 (365 mg, 9.65 mmol) was added to a cooled (ice bath) solution of methyl (45) -3- [ (4-methoxyphenyl) methyl] -2-oxo- 1 , 3-oxazolidine-4-carboxylate (2.45 g, 8.77 mmol) in EtOH (50 ml) . The reaction mixture was allowed to warm to RT then stirred at RT . The reaction mixture was cooled in ice then acetone (5 ml) added to quench excess borohydride. The reaction mixture was allowed to warm to RT then concentrated in vacuo. The resultant mixture was partitioned between EtOAc (50 ml) and 1 M aq. HC1 (50 ml) . The phases were separated then the aqueous phase was extracted with EtOAc (50 ml) . The combined organic phases were washed with water (50 ml) and brine (50 ml) then dried over Na2SC>4 and evaporated to an amber gum which crystallised on standing to yield the crude product as an amber solid. Purification by flash column chromatography on a silica column (50 g) eluting with

EtOAc : heptane, increasing the gradient linearly from 50:50 to 100:0 afforded a colourless gum which crystallised on

standing to yield ( 4R) -4- (hydroxymethyl ) -3- [( 4- methoxyphenyl ) methyl ] -1 , 3-oxazolidin-2-one (1.45 g) as a white solid . ¹R NMR (500 MHz, Chloroform-d) δ 7.28 - 7.22 (m, 2H) , 6.90 - 6.85 (m, 2H) , 4.58 (d, J = 15.1 Hz, 1H) , 4.34 - 4.20 (m, 3H) , 3.80 (s, 3H) , 3.76 - 3.67 (m, 2H) , 3.57 - 3.49 (m, 1H)

LCMS MH+ 238.0 RT 0.85 System 1

Synthesis of (4S) -4- (fluoromethyl) -3- [ (4-methoxyphenyl) methyl] - l,3-oxazolidin-2-one: INTERMEDIATE 3

PBSF (3.3 ml, 19 mmol) was added drop-wise to a cooled (0 °C, ice bath) solution of (4R) -4- (hydroxymethyl) -3- [ (4- methoxyphenyl) methyl] -1, 3-oxazolidin-2-one (1.45 g, 6.11 mmol) and triethylamine (7.7 ml, 55 mmol) in MeCN (30 ml) . The reaction solution was stirred at 0 °C for 5 min then TREAT-HF (3.0 ml, 19 mmol) was added drop-wise. The resultant solution was left to stir at 0 °C for one hour then the reaction mixture was partitioned between EtOAc (60 ml) and water (60 ml) . The phases were separated and the aqueous phase was extracted with EtOAc (60 ml) . The combined organic phases were washed with brine (60 ml) then dried over Na2SC>4 and evaporated to give the crude product which was purified by flash column chromatography on a silica column (50 g) . The column was eluted with EtOAc : heptane, increasing the gradient linearly from 0:100 to 70:30 over 10 column volumes. The desired fractions were combined and evaporated to yield the required product as colourless oil (887 mg) ¹R NMR (500 MHz, Chloroform-d) δ 7.23 (d, J = 8.6 Hz,

6.88 (d, J = 8.6 Hz, 2H) , 4.77 (d, J = 15.1 Hz, 1H) , 4

4.29 (m, 3H) , 4.17 (d, J = 15.1 Hz, 1H) , 4.11 (dd, J =

6.0 Hz, 1H) , 3.90 - 3.79 (m, 4H) .

LCMS MH+ 239.9 RT 0.98 System 1

Synthesis of (4S) -4- (fluoromethyl) -1 , 3-oxazolidin-2-one :

INTERMEDIATE 4

A solution of (4S) -4- (fluoromethyl) -3- [ (4- methoxyphenyl ) methyl ] -1 , 3-oxazolidin-2-one (8.91 g, 37.2 mmol) in TFA (200 ml) was heated at 65 °C for 16 hours to give an orange/brown solution. The reaction mixture was concentrated in vacuo then azeotroped with EtOAc (3 x 100 ml) to yield a green mixture. Purification by flash chromatography on a silica column (100 g) eluting with DCM: EtOAc 0 to 100% afforded the required product (4.25 g, 95%) as a green oil. Further purification by chromatography on silica eluting with heptane-25% EtOAc to 100% EtOAc to give (4S) -4- (fluoromethyl) - 1 , 3-oxazolidin-2-one (4.01g, 90%) as a pale green oil (3.76 g, 85%) .

!H NMR (250 MHz, Chloroform-d) δ 6.52 (s, 1H) , 4.57 - 4.43 (m, 2H) , 4.38 - 4.04 (m, 3H) .

Scheme 2: Synthesis of Intermediate 8 (for reaction

conditions details, see the experimental procedures below) .

Synthesis of 5- (4-chlorophenyl) -l-methyl-lH-pyrazole-3- carbaldehyde : INTERMEDIATE 5

Dimethyl sulfoxide (2.17 ml, 30.48 mmol) in dichloromethane (5 ml) was added dropwise to a cooled (-78 °C) solution of oxalyl chloride (1.08 ml, 12.8 mmol) in dichloromethane (10 ml) under nitrogen and stirred for 15 minutes. A solution of 5- (4-chlorophenyl) -l-methyl-lH-pyrazol-3-yl] methanol (2.72 g, 12.19 mmol) in 1:1 dichloromethane/THF (50ml) was added dropwise and the reaction stirred for a further 60 minutes at -78 °C. Triethylamine (8.5 ml, 60.96 mmol) in dichloromethane (10 ml) was then added slowly and the reaction was allowed to warm to ambient temperature overnight. The reaction was diluted with water (75.0 ml) and aHC03 (sat aq) (50.0 ml) along with further DCM (50.0 ml) and the organic layer was separated, dried over Na2SOz_[, filtered and concentrated in vacuo. The residue was retreated in a similar manner. Oxalyl chloride (1.62 ml, 19.4 mmol) was dissolved in

dichloromethane (10 ml) under nitrogen and cooled to -78°C then dimethyl sulfoxide (2.17 ml, 30.48 mmol) in

dichloromethane (5 ml) was added dropwise and stirred for 15 minutes. The recovered residue was dissolved in

tetrahydrofuran (25.0 ml) and added slowly to the reaction washing in with DCM (25.0 ml) then the reaction was stirred for 1.5 hr at -78 °C before the slow addition of

triethylamine (8.5 ml, 60.96 mmol) in dichloromethane (10 ml) . After a further 10 minutes the reaction was allowed to warm to 0 °C and stirred for 30 mins . Reaction diluted with DCM (50.0 ml), water (75.0 ml) and NaHC03 (sat aq) (50.0 ml) then the organic layer was separated, dried over Na2SOz_[, filtered and concentrated in vacuo. The residue was treated with TBME (30.0 ml) and the resultant suspension was

filtered, washing with further TBME and dried in vacuo to give 1.637g of 5- ( 4-chlorophenyl ) -l-methyl-lH-pyrazole-3- carbaldehyde as a pale yellow powdery solid.

! H NMR (500 MHz, DMSO-d6) 9.94 (s, 1H) , 7.90 - 7.87 (m, 2H) , 7.54 (s, 1H) , 7.52 - 7.48 (m, 2H) , 4.16 (s, 3H)

LCMS MH+ 220.8 RT 1.17 System 1

Synthesis of (R) -N- [ (IE) - [5- (4-chlorophenyl) -1-methyl-lH-pyrazol-

3-yl]methylidene]-2-methylpropane-2-sulfinamide: INTERMEDIATE 6

Titanium tetraethoxide (3.11 ml, 14.84 mmol) was added to a solution of 5- (4-chlorophenyl) -l-methyl-lH-pyrazole-3- carbaldehyde (1.64 g, 7.42 mmol) and (R) -2-methylpropane-2- sulfinamide (0.99 g, 8.16 mmol) in tetrahydrofuran (25 ml) and the reaction heated to 55 °C under nitrogen for 4 hours. The reaction was cooled, diluted with EtOAc (50.0 ml) and water (30.0 ml) then stirred for 10 minutes. The biphasic suspension was filtered through celite, washing with further EtOAc then the organic layer was separated, dried over

Na2SOz_[, filtered and concentrated in vacuo to give (R) -N-

[ (IE) - [5- (4-chlorophenyl) -l-methyl-lH-pyrazol-3-yl ] methylidene] - 2-methylpropane-2-sulfinamide (2.189g) as a pale tan viscous oil which crystallised on standing.

! H NMR (500 MHz, DMSO-d6) 8.52 (s, 1H) , 7.88 - 7.84 (m, 2H) , 7.52 - 7.47 (m, 2H) , 7.47 (s, 1H) , 4.16 (s, 3H) , 1.21 (s, 9H) .

LCMS MH+ 423.9 RT 1.31 System 1

Synthesis of (R) -N- [ (IS) -1- [5- (4-chlorophenyl) -1-methyl-lH- pyrazol-3-yl] ethyl] -2-methylpropane-2-sulfinamide : INTERMEDIATE 7

Methyl magnesium bromide [3M in hexanes] (6.42 ml) was added slowly to a cooled (0 °C) solution of (R) -N- [ (IE) - [5- (4- chlorophenyl ) -l-methyl-lH-pyrazol-3-yl] methylidene] -2- methylpropane-2-sulfinamide (2.19 g) in tetrahydrofuran (35 ml) and the reaction was stirred for 30 mins . The reaction was quenched via the slow addition of water (10 ml) then the reaction was further diluted with EtOAc (50.0 ml) and water

(30.0 ml) and the organic layer separated, dried over Na2S04, filtered and concentrated in vacuo to give a viscous oil. Purification via neutral reverse phase Biotage (Water/MeCN) followed by further purification via column chromatography

[100% DCM to 20% MeOH in DCM] afforded 1.438g of a 7:3 mixture of the required product and (R) -N- [ ( 1R) -1- [ 5- ( 4- chlorophenyl ) -l-methyl-lH-pyrazol-3-yl ] ethyl] -2-methylpropane-2- sulfinamide as a clear oil.

! H NMR (500 MHz, DMSO-d6) 7.80 - 7.75 (m, 2H) , 7.43 (dt, J = 9.2, 1.9 Hz, 2H) , 6.73 (s, OH), 6.68 (s, 1H) , 5.67 (d, J = 5.7 Hz, OH), 5.48 (d, J = 7.3 Hz, 1H) , 4.57 (h, J = 6.9 Hz, 1H) , 3.84 (s, 1H) , 3.82 (s, 2H) , 1.57 (d, J = 6.8 Hz, 2H) , 1.53 (d, J = 6.8 Hz, 1H) , 1.12 (s, 6H) , 1.09 (s, 3H) .

LCMS MH+ 339.9 RT 1.13 System 1

Synthesis of (IS) -1- [5- (4-chlorophenyl) -l-methylpyrazol-3- yl] ethanamine: INTERMEDIATE 8

Hydrogen chloride [4M in 1,4-dioxane] (0.74 ml) was added to a solution of (R) -N- [ (IS) -1- [5- (4-chlorophenyl) -1-methyl-lH- pyrazol-3-yl ] ethyl ] -2-methylpropane-2-sulfinamide (500 mg, 1.47 mmol) in 1,4-dioxane (5 ml) to give a white precipitate. The reaction was agitated at ambient temperature for 60 mins to form a thick cake. The reaction was diluted with dioxane (20.0 ml) and triturated to give a mobile suspension which was filtered. The residual solid was washed with TBME and dried under vacuum to give 447mg of a mixture of (IS) -1- [5- (4- chlorophenyl ) -l-methylpyrazol-3-yl ] ethanamine hydrochloride and (1R) -1- [5- (4-chlorophenyl) -l-methylpyrazol-3-yl ] ethanamine hydrochloride as a white powdery solid.

! H NMR (500 MHz, DMSO-d6) 8.36 (s, 3H) , 7.77 (d, J = 8.5 Hz, 2H) , 7.48 (d, J = 8.5 Hz, 2H) , 6.90 (s, 1H) , 4.68 (s, 1H) , 3.92 (s, 3H) , 1.57 (d, J = 6.7 Hz, 3H)

LCMS MH+ 236.9 RT 0.81 System 1

(IS) -1- [1- (4-chlorophenyl) -lH-imidazol-4-yl] ethan-l-amine was synthesized according to a literature method

(IS) -1- [1- (4-fluorophenyl) -lH-imidazol-4-yl] ethan-l-amine was synthesized according to a literature method

(IS) -1- [2- (4-fluorophenyl) -1 , 3-thiazol-5-yl] ethan-l-amine was synthesized according to a literature method

(IS) -1- { 4-methyl-5- [2- (trifluoromethyl) pyridin-4-yl] pyridin-2- yl} ethan-l-amine was synthesized according to a literature method

Scheme 3: Synthesis of Intermediates A1-A5 (for reaction conditions details, see the experimental procedures below) . Synthesis of (4S) -3- [2-chloro-7- (4-methylbenzenesulfonyl) -7H- pyrrolo [2 , 3-d] pyrimidin-4-yl] -4- (fluoromethyl) -1 , 3-oxazolidin-2- one; Intermediate Al

2, 4-dichloro-7- ( 4-methylbenzenesulfonyl ) -7H-pyrrolo [2, 3- djpyrimidine (3200 mg, 9.35 mmol) , K3P04 (3.97 g, 18.7 mmol) ,

(4S) -4- (fluoromethyl) -1, 3-oxazolidin-2-one (1.114g, 9.35 mmol) and Xantphos Pd-G3 (221 mg, 0.23 mmol) were added to dry toluene (90 ml) that had been degassed by bubbling a stream of nitrogen through it for 5 minutes and the mixture was heated at 80 °C for 3.5 hr . The reaction mixture was taken up in EtOAc (200 ml) , washed with water (2 x 200 ml) dried over Na2SC>4 and concentrated in vacuo. The crude material was purified by flash column chromatography on a silica column

(100 g) eluting with heptane-EtOAc 0-60% to give (4S)-3-[2- chloro-7- ( 4-methylbenzenesulfonyl ) -7H-pyrrolo [ 2 , 3-d] pyrimidin-4- yl ] -4- ( fluoromethyl ) -1 , 3-oxazolidin-2-one (1.65 g, 39% ) as a white solid.

^XH NMR (500 MHz, Chloroform-d) δ 8.11 (d, J = 8.4 Hz, 2H) , 7.64 (d, J = 4.1 Hz, 1H) , 7.35 (d, J = 8.1 Hz, 2H) , 7.16 (d, J = 4.1 Hz, 1H) , 5.15 - 5.04 (m, 1H) , 4.85 (ddd, J = 47.9, 10.1, 3.9 Hz, 1H) , 4.73 - 4.56 (m, 3H) , 2.42 (s, 3H) .

LCMS MH+ 424.9 RT 1.28 System 1 Synthesis of (4R) -3- [2-chloro-7- (4-methylbenzenesulfonyl) -7H- pyrrolo [2 , 3-d] pyrimidin-4-yl] -4-phenyl-l , 3-oxazolidin-2-one ;

Intermediate A2

By proceeding in a similar manner to INTERMEDIATE Al but using ( 4R) -4-phenyl-l , 3-oxazolidin-2-one instead of (4S)-4- ( fluoromethyl ) -1, 3-oxazolidin-2-one, (4R) -3- [2-chloro-7- (4- methylbenzenesulfonyl ) -7H-pyrrolo [ 2 , 3-d] pyrimidin-4-yl ] -4- phenyl-1 , 3-oxazolidin-2-one was obtained as a white solid.

! H NMR (250 MHz, Chloroform-d) δ 8.10 (d, J = 8.5 Hz, 2H) , 7.64 (d, J = 4.0 Hz, 1H) , 7.46 - 7.24 (m, 7H) , 7.16 (d, J = 4.0 Hz, 1H) , 5.97 (dd, J = 9.0, 6.5 Hz, 1H) , 4.88 (t, J = 9.0 Hz, 1H) , 4.46 (dd, J = 9.0, 6.5 Ηζ,ΙΗ), 2.42 (s, 3H) .

LCMS MH+ 468.9 RT 1.56 System 1

Synthesis of (4S) -3- [2-chloro-7- (4-methylbenzenesulfonyl) -7H- pyrrolo [2 , 3-d] pyrimidin-4-yl] -4- (propan-2-yl) -1 , 3-oxazolidin-2- one; Intermediate A3

By proceeding in a similar manner but using (4S) -4- (propan-2- yl ) -1 , 3-oxazolidin-2-one instead of (4S) -4- (fluoromethyl) -1, 3- oxazolidin-2-one, (4S) -3- [2-chloro-7- ( 4-methylbenzenesulfonyl ) - 7H-pyrrolo [ 2 , 3-d] pyrimidin-4-yl ] -4- (propan-2-yl )-l,3-oxazolidin-2- one was obtained as an off white solid.

!H NMR (500 MHz, DMSO-d6) 8.03 (d, J = 8.4 Hz, 2H) , 7.89 (d, J = 4.1 Hz, 1H) , 7.50 (d, J = 8.2 Hz, 2H) , 7.08 (d, J = 4.1 Hz, 1H) , 4.83 (dt, J = 8.5, 4.2 Hz, 1H) , 4.52 (t, J = 8.8 Hz, 1H) , 4.42 (dd, J = 9.0, 4.5 Ηζ,ΙΗ), 2.39 (s, 3H) , 2.36 - 2.30 (m, 1H) , 0.86 (d, J = 7.0 Hz, 3H) , 0.74 (d, J = 6.9 Hz, 3H)

LCMS MH+ 434.8 RT 1.36 System 1

Synthesis of (4R) -4- [ (IR) -1- (tert-butoxy) ethyl] -3- [2-chloro-7- (4- methylbenzenesulfonyl) -7H-pyrrolo [2 , 3-d] pyrimidin-4-yl] -1 , 3- oxazolidin-2-one ; Intermediate A4

By proceeding in a similar manner but using ( 4R) -4- [ ( 1R) -1- (tert-butoxy) ethyl ] -1 , 3-oxazolidin-2-one instead of (4S)-4- (fluoromethyl) -1, 3-oxazolidin-2-one, (4R) -4- [ (1R) -1- (tert- butoxy) ethyl] -3- [2-chloro-7- ( 4-methylbenzenesulfonyl ) -7H- pyrrolo [ 2 , 3-d] pyrimidin-4-yl ] -1 , 3-oxazolidin-2-one was obtained as a colourless gum.

! H NMR (500 MHz, Chloroform-d) δ 8.12 (d, J = 8.4 Hz, 2H) , 7.61 (d, J = 4.0 Hz, 1H) , 7.39 - 7.32 (m, 3H) , 7.21 (d, J = 4.0 Hz, 1H) , 4.83 (dt, J = 9.0, 4.0 Hz, 1H) , 4.64 (dd, J = 9.0, 4.0 Hz, 1H) , 4.50 - 4.41 (m, 2H),2.42 (s, 3H) , 1.22 (s, 9H) , 1.01 (d, J = 6.5 Hz, 3H) .

LCMS MH+ 492.9 RT 1.82 System 1

EXAMPLE 1 Synthesis of (4S) -3- (2-{ [ (IS) -1- [1- (4-chlorophenyl) -1H- imidazol-4-yl] ethyl] amino}-7H-pyrrolo [2 , 3-d] pyrimidin-4-yl) -4-

(fluoromethyl) -1 , 3-oxazolidin-2-one (Compound 12)

A solution of ( IS) -1- [ 1- (4-chlorophenyl) -lH-imidazol-4- yl ] ethan-l-amine (2.58 g, 11.65 mmol) , (4S) -3- [2-chloro-7- ( 4-methylbenzenesulfonyl) -7H-pyrrolo [2, 3-d] pyrimidin-4-yl ] -4- (fluoromethyl) -1, 3-oxazolidin-2-one (1.65 g, 3.88 mmol) and DIPEA (744 μΐ, 4.27 mmol) in DMSO (15 ml) was sealed in a nitrogen flushed pressure tube and heated at 110 °C for 16 hours. The reaction was allowed to cool then partitioned between water (60 ml) and EtOAc (60 ml) . The phases were separated and the aqueous phase extracted with more EtOAc (60 ml) . The combined organic phases were washed with water (4 x 60 ml) , dried over sodium sulfate and concentrated in vacuo to yield the crude ( 4S ) -3- (2- {[( IS ) -1- [ 1- ( 4-chlorophenyl ) -1H- imidazol-4-yl ] ethyl ] amino } -7- ( 4-methylbenzenesulfonyl ) -7H- pyrrolo [2, 3-d] pyrimidin-4-yl ) -4- ( fluoromethyl )-l,3-oxazolidin-2- one as an orange foam. 1M TBAF in THF (39 ml, 39 mmol) was added to a solution of the crude intermediate in THF (20 ml) under nitrogen and the mixture heated at 50 °C for 3 hr . The reaction mixture was taken up in EtOAc (100 ml) and washed with saturated NH4CI (2 xlOO ml), water (100 ml) and then dried over sodium sulfate to give the crude product as a brown gum.

Purification by C18 RP chromatography on silica eluting with water, 0.1% ammonia-15% to 100% MeCN to followed by further purification by chromatography on silica eluting with

heptane-30% to 100% EtOAc, and then DCM-1% to 5% MeOH

afforded (4S)-3-(2-{ [ ( IS ) -1- [ 1- ( 4-chlorophenyl ) -lH-imidazol-4- yl ] ethyl ] amino } -7H-pyrrolo [ 2 , 3-d] pyrimidin-4-yl ) -4-

( fluoromethyl ) -1 , 3-oxazolidin-2-one (602 mg, 31% ) as a

colourless solid. The sample was sonicated in hexane-ether 1:1 (2 x 5 ml), the supernatent removed by pipette and the sample dried in vacuo at 60 °C for 18hr. NMR shows 1% ether, 4% hexane w/w (402 mg, 95% w/w purity) . The sample was dried further in vacuo at 60C for 18 hr to give (4S) -3- (2-{ [ (IS) -1-

[ 1- (4-chlorophenyl ) -lH-imidazol-4-yl ] ethyl ] amino } -7H-pyrrolo [2,3- d] pyrimidin-4-yl ) -4- ( fluoromethyl ) -l,3-oxazolidin-2-one (373 mg, 20%) as a colourless solid.

!H NMR (500 MHz, Chloroform-d) δ 7.86 - 7.77 (m, 1H) , 7.47 - 7.39 (m, 2H) , 7.32 - 7.27 (m, 2H) , 7.15 - 7.10 (m, 1H) , 6.90 (d, J = 2.4 Hz, 1H) , 6.80 - 6.75 (m, 1H) , 5.34 - 4.38 (m, 7H) , 1.62 (d, J = 6.9 Hz, 3H) .

LCMS MH+ 455.0 RT 2.06 System 2

EXAMPLE 2: Synthesis of (4R) -3- (2-{ [ (IS) -1- [1- (4-chlorophenyl) - lH-imidazol-4-yl] ethyl] amino}-7H-pyrrolo [2 , 3-d] pyrimidin-4-yl) -4- phenyl-1 , 3-oxazolidin-2-one (Compound 5)

By proceeding in a similar manner to Example 1 but replacing (4S) -3- [2-chloro-7- (4-methylbenzenesulfonyl) -7H-pyrrolo [2, 3- d] pyrimidin-4-yl ] -4- ( fluoromethyl ) -1, 3-oxazolidin-2-one

(Intermediate Al) by (4R) -3- [2-chloro-7- (4- methylbenzenesulfonyl ) -7H-pyrrolo [ 2 , 3-d] pyrimidin-4-yl ] -4- phenyl-1 , 3-oxazolidin-2-one ; (Intermediate A2), (4R)-3-(2- { [ (IS) -1- [1- (4-chlorophenyl) -lH-imidazol-4-yl] ethyl] amino}-7H- pyrrolo [2, 3-d] pyrimidin-4-yl ) -4-phenyl-1 , 3-oxazolidin-2-one was obtained as a beige solid.

! H NMR (500 MHz, DMSO-d6) δ 11.16 (s, 1H) , 8.18 (d, J = 1.4 Hz, 1H) , 7.64 - 7.54 (m, 4H) , 7.43 - 7.37 (m, 3H) , 7.35 - 7.28 (m, 2H) , 7.27 - 7.20 (m, 1H) , 6.92 (dd, J = 3.6, 2.2 Hz, 1H) , 6.55 (dd, J = 3.6, 2.0 Ηζ,ΙΗ), 6.33 (d, J = 8.7 Hz, 1H) , 5.95 (dd, J = 8.7, 6.6 Hz, 1H) , 4.98 (p, J = 6.6 Hz, 1H) , 4.90 (t, J = 8.7 Hz, 1H) , 4.22 (dd, J = 8.4, 6.7 Hz, 1H) , 1.31 - 1.09 (m, 3H) .

LCMS MH+ 499.2 RT 2.47 System 2

Example 3: Synthesis of (4S) -3- (2-{ [ (IS) -1- [1- (4-chlorophenyl) - lH-imidazol-4-yl] ethyl] amino}-7H-pyrrolo [2 , 3-d] pyrimidin-4-yl) -4- (propan-2-yl) -1 , 3-oxazolidin-2-one (Compound 4)

(Intermediate Al) by (4S) -3- [2-chloro-7- (4- methylbenzenesulfonyl ) -7H-pyrrolo [ 2 , 3-d] pyrimidin-4-yl ] -4- (propan-2-yl ) -1 , 3-oxazolidin-2-one (Intermediate A3), (4S)-3-(2- { [ (IS) -1- [1- (4-chlorophenyl) -lH-imidazol-4-yl] ethyl] amino}-7H- pyrrolo [2, 3-d] pyrimidin-4-yl ) -4- (propan-2-yl )-l,3-oxazolidin-2- one was obtained as an off white solid beige solid.

! H NMR (500 MHz, Chloroform-d) 9.97 (br. s, 1H) , 7.81 (s, 1H) , 7.47 - 7.40 (m, 2H) , 7.29 (d, J = 8.7 Hz, 2H) , 7.14 (s, 1H) , 6.89 (d, J = 2.5 Hz, 1H) , 6.74 (d, J = 2.4 Hz, 1H) , 6.37 (br. s, 1H) , 5.31 (s, 1H) , 4.94 (dt,J = 8.9, 4.8 Hz, 1H) , 4.44 (t, J = 8.9 Hz, 1H) , 4.31 (dd, J = 8.8, 5.3 Hz, 1H) , 2.56 - 2.45 (m, 1H) , 1.63 (d, J = 6.8 Hz, 3H) , 0.93 (d, J = 7.1 Hz, 3H) , 0.87 (d, J = 6.9 Hz, 3H) LCMS MH+ 466.2 RT 2.39 System 2

Example 4: Synthesis of (4S) -3- (2-{ [ (IS) -1- [1- (4-fluorophenyl) - lH-imidazol-4-yl] ethyl] amino}-7H-pyrrolo [2 , 3-d] pyrimidin-4-yl) -4- (propan-2-yl) -1 , 3-oxazolidin-2-one (Compound 7)

By proceeding in a similar manner to Example 3 but replacing ( IS ) -1- [1- ( 4-chlorophenyl ) -lH-imidazol-4-yl ] ethan-1 -amine by ( IS ) -1- [1- ( 4-fluorophenyl ) -lH-imidazol-4-yl ] ethan-1 -amine) , (4S)-3-(2-{ [ (IS) -1- [1- (4-fluorophenyl) -lH-imidazol-4- yl ] ethyl ] amino } -7H-pyrrolo [ 2 , 3-d] pyrimidin-4-yl ) -4- (propan-2-yl ) - 1 , 3-oxazolidin-2-one was obtained as the formate salt.

! H NMR (500 MHz, Methanol-d4) δ 8.07 - 8.01 (m, 1H) , 7.54 - 7.45 (m, 2H) , 7.30 (s, 1H) , 7.26 - 7.17 (m, 2H) , 6.91 (d, J = 3.7 Hz, 1H) , 6.53 (d, J = 3.7 Hz, 1H) , 5.20 (q, J = 6.9 Hz, 1H) , 4.93 - 4.86 (m, 1H) , 4.48(t, J = 9.0 Hz, 1H) , 4.32 (dd, J = 8.9, 5.6 Hz, 1H) , 2.16 - 2.05 (m, 1H) , 1.60 (d, J = 6.9 Hz, 3H) , 0.72 (dd, J = 19.3, 6.9 Hz, 6H)

LCMS MH+ 450.3 RT 2.09 System 2

Synthesis of (4S) -3- (2- { [ (IS) -1- { 4-methyl-5- [2-

(trifluoromethyl) pyridin-4-yl] pyridin-2-yl}ethyl] amino} -7- (4- methylbenzenesulfonyl) -7H-pyrrolo [2 , 3-d] pyrimidin-4-yl) -4-

(propan-2-yl) -1 , 3-oxazolidin-2-one

(4S) -3- [2-chloro-7- (4-methylbenzenesulfonyl) -7H-pyrrolo [2, 3- d] pyrimidin-4-yl ] -4- (propan-2-yl) -1, 3-oxazolidin-2-one (150 mg, 0.34 mmol) and (IS) -1- { 4-methyl-5- [2- ( trifluoromethyl) pyridin-4-yl ] pyridin-2-yl } ethan- 1-amine (242.54 mg, 0.86 mmol) were dissolved in DMSO (1.5 ml) and the sealed reaction heated to 105°C for 18 hours. The

reaction was cooled then purified by column chromatography to give 480mg of the required product as a brown oil containing -50% residual DMSO.

LCMS MH+ 680 RT 1.37mins System 1

Example 5: Synthesis of (4S) -3- (2-{ [ (IS) -1- { 4-methyl-5- [2- (trifluoromethyl) pyridin-4-yl] pyridin-2-yl} ethyl] amino}-7H- pyrrolo [2 , 3-d] pyrimidin-4-yl) -4- (propan-2-yl) -1 , 3-oxazolidin-2-one (Compound 2)

(4S) -3- (2-{ [ (IS) -1- { 4-methyl-5- [2- (trifluoromethyl) pyridin-4- yl ] pyridin-2-yl } ethyl ] amino } -7- ( 4-methylbenzenesulfonyl ) -7H- pyrrolo [2, 3-d] pyrimidin-4-yl) -4- (propan-2-yl) -1, 3-oxazolidin- 2-one (49%, 480 mg, 0.35 mmol) was dissolved in TBAF [1M in THF] (1.5 ml) then the sealed reaction was heated at 50 °C for 18 hours. Reaction partitioned between DCM (10.0 ml) and water (5.0 ml) then the organic layer was separated, dried over Na2SOz_[, filtered and concentrated in vacuo.

The residue was purified via reverse phase Biotage to afford the required product.

! H NMR (500 MHz, DMSO-d6) 11.23 (s, 1H) , 8.84 (d, J = 5.0 Hz, 1H) , 8.42 (s, 1H) , 7.93 (s, 1H) , 7.77 (d, J = 5.0 Hz, 1H) , 7.36 (s, 1H) , 7.15 (d, J = 7.8 Hz, 1H) , 6.92 (s, 1H) , 6.39 (dd, J = 3.6, 1.9 Hz, 1H) , 5.16 -5.07 (m, 1H) , 4.71 (s, 1H) , 4.46 (s, 1H) , 4.27 (s, 1H) , 2.24 (s, 3H) , 1.50 (d, J = 7.1 Hz, 3H) , 0.82 - 0.35 (m, 6H) .

LCMS MH+ 526.2 RT 2.67 System 2

Synthesis of (4R)-4- [ (lR)-l- (tert-butoxy) ethyl] -3- (2-{ [ (IS) -1- { 4- methyl-5- [2- (trifluoromethyl) pyridin-4-yl] pyridin-2- yl}ethyl] amino}-7H-pyrrolo [2 , 3-d] pyrimidin-4-yl) -1 , 3-oxazolidin- 2-one : Intermediate Bl

By proceeding in a similar manner to Example 1 but replacing (4S) -3- [2-chloro-7- (4-methylbenzenesulfonyl) -7H-pyrrolo [2, 3- d] pyrimidin-4-yl ] -4- ( fluoromethyl ) -l,3-oxazolidin-2-one by (4R) -4- [ (1R) -1- (tert-butoxy) ethyl ] -3- [ 2-chloro-7- ( 4- methylbenzenesulfonyl ) -7H-pyrrolo [ 2 , 3-d] pyrimidin-4-yl ] -1 , 3- oxazolidin-2-one (Intermediate A4) and ( IS) -1- [ 1- (4- chlorophenyl) -lH-imidazol-4-yl] ethan-l-amine by (lS)-l-{4- methy1-5- [2- (trifluoromethyl) pyridin-4-yl ] pyridin-2-yl }ethan-l- amine , (4R) -4- [ (1R) -1- (tert-butoxy) ethyl] -3- (2-{ [ (IS) -l-{ 4- methy1-5- [2- (trifluoromethyl) pyridin-4-yl ] pyridin-2- yl } ethyl ] amino } -7H-pyrrolo [ 2 , 3-d] pyrimidin-4-yl )-l,3-oxazolidin 2-one is obtained as a pale amber gum.

! H NMR (500 MHz, Chloroform-d) δ 8.82 (d, J = 5.0 Hz, 1H) , 8.61 (s, 1H) , 8.38 (s, 1H) , 7.65 (s, 1H) , 7.45 (dd, J = 5.0, 1.0 Hz, 1H) , 6.87 (t, J = 3.5, 2.0 Hz, 1H) , 6.78 (t, J = 3.5 2.0 Hz, 1H) , 5.84 (d, J = 7.0 Ηζ,ΙΗ), 5.35 - 5.25 (m, 1H) , 4.91 (dt, J = 9.0, 4.5 Hz, 1H) , 4.61 (dd, J = 9.0, 4.5 Hz, 1H) , 4.45 (t, J = 9.0 Hz, 1H) , 4.43 - 4.35 (m, 1H) , 2.28 (s, 3H) , 1.62 (d, J = 7.0 Hz, 3H) , 1.23 (s, 9H) , 1.06 (d, J = 6. Hz, 3H)

LCMS MH+ 584.4 RT 1.26 System 2

Synthesis of (4R) -4- [ (1R) -1-hydroxyethyl] -3- [ (4- methoxyphenyl) methyl] -1 , 3-oxazolidin-2-one (described in WO 2014/141104 Al)

Trifluoroacetic acid (20 ml) was added to a solution of (4R) 4- [ (1R) -1- ( tert-butoxy) ethyl] -3- [ (4-methoxyphenyl) methyl] - 1, 3-oxazolidin-2-one (2.75 g, 8.95 mmol) in DCM (20 ml). The reaction was stirred for 10 min. The reaction mixture was concentrated in vacuo then partitioned between saturated aq. NaHC03 (20 ml) and EtOAc (20 ml) . The aqueous phase was extracted with more EtOAc (20 ml) then the organic phases were combined, dried over Na2SC>4 and evaporated to yield the product as a colourless oil which crystallised on standing to a give a white solid (2.255 g)

! H NMR (500 MHz, Chloroform-d) δ 7.26 - 7.22 (m, 2H) , 6.90 -

6.85 (m, 2H) , 4.70 (d, J = 15.0 Hz, 1H) , 4.28 (d, J = 15.0

Hz, 1H) , 4.21 (t, J = 9.0 Hz, 1H) , 4.11 (dd, J = 9.0, 6.5 Hz,

1H) , 3.96 (p, J = 6.5 Hz, 1H),3.80 (s, 3H) , 3.61 (dt, J = 9.0, 6.5 Hz, 1H) , 1.14 (d, J = 6.5 Hz, 3H) .

LCMS MH+ 251.9 RT 0.90 System 1

Synthesis of (4R) -4- [ (IS) -1-fluoroethyl] -3- [ (4- methoxyphenyl) methyl] -1 , 3-oxazolidin-2-one (described in WO

2014/141104 Al)

PBSF (4.71 ml, 26.3 mmol) was added drop-wise to a cooled (0 °C, ice bath) solution of (4R) -4- [ (1R) -1-hydroxyethyl] -3- [ (4- methoxyphenyl) methyl] -1, 3-oxazolidin-2-one (2.18 g, 8.69 mmol) and triethylamine (11 ml, 79 mmol) in MeCN (50 ml) . The reaction solution was stirred at 0 °C for 5 min then TREAT-HF (4.3 ml, 27 mmol) was added drop-wise. The resultant solution was left to stir at 0 °C. After 1.5 h at 0 °C, the reaction mixture was partitioned between EtOAc (100 ml) and water (100 ml) . The phases were separated and the organic phase washed with brine (100 ml), dried over Na2SC>4 and evaporated. The crude material was pre-adsorbed onto silica then purified by flash column chromatography on a silica column (50 g) . The column was eluted with EtOAc : heptane, increasing the gradient linearly from 10:90 to 50:50 over 10 column volumes then isocratic at 50:50 for 2 CVs to afford the required product as colourless oil which solidified on standing to an off- white solid.

! H NMR (500 MHz, Chloroform-d) δ 7.26 - 7.19 (m, 2H) , 6.90 - 6.85 (m, 2H) , 4.87 (d, J = 15.0 Hz, 1H) , 4.75 (dqd, J = 47.5, 6.5, 2.0 Hz, 1H) , 4.25 (td, J = 9.5, 1.0 Hz, 1H) , 4.13 (dd, J = 9.5, 6.0 Hz, 1H) , 4.10 (d, J= 15.0 Hz, 1H) , 3.80 (s, 3H) , 3.71 (dddd, J = 20.0, 9.5, 6.0, 2.0 Hz, 1H) , 1.29 (dd, J = 23.5, 6.5 Hz, 3H)

LCMS MH+ 253.9 RT 1.03 System 1

Synthesis of (4R) -4- [ (IS) -1-fluoroethyl] -1 , 3-oxazolidin-2-one (described in WO 2014/141104 Al)

A solution of (4R) -4- [ (IS) -1-fluoroethyl] -3- [ (4- methoxyphenyl) methyl] -1, 3-oxazolidin-2-one (1.77 g, 6.99 mmol) in TFA (35 ml) was heated at 65 °C for 4 hours. The reaction mixture was concentrated in vacuo then azeotroped with EtOAc to yield a green mixture. The crude material was purified by flash column chromatography on a silica column (50 g) , eluting with EtOAc:DCM, increasing the gradient linearly from 0:100 to 100:0. to yield the required product as a pale-green solid.

! H NMR (500 MHz, Chloroform-d) δ 6.46 (s, 1H) , 4.60 (dp, J = 47.3, 6.2 Hz, 1H) , 4.50 (t, J = 8.9 Hz, 1H) , 4.34 (dd, J = 9.1, 4.9 Hz, 1H) , 3.97 - 3.87 (m, 1H) , 1.37 (dd, J = 24.1, 6.3 Hz, 3H)

LCMS MH+ 133.9 RT 0.25 System 1

Synthesis of (4R) -3- [2-chloro-7- (4-methylbenzenesulfonyl) -7H- pyrrolo [2 , 3-d] pyrimidin-4-yl] -4- [ (IS) -1-fluoroethyl] -1 , 3- oxazolidin-2-one : Intermediate A5

By proceeding in a similar manner to INTERMEDIATE Al but using (4R) -4- [ (IS) -1-fluoroethyl] -1, 3-oxazolidin-2-one instead of (4S) -4- ( fluoromethyl ) -1, 3-oxazolidin-2-one, (4R) -3- [2-chloro- 7- ( 4-methylbenzenesulfonyl ) -7H-pyrrolo [ 2 , 3-d] pyrimidin-4-yl ] -4- [ (IS) -1-fluoroethyl] -1, 3-oxazolidin-2-one was obtained as a pale yellow foam.

1H NMR (500 MHz, Chloroform-d) δ 8.11 (d, J = 8.4 Hz, 2H) ,

7.64 (d, J = 4.1 Hz, 1H) , 7.34 (d, J = 8.3 Hz, 2H) , 7.17 (d, J = 4.1 Hz, 1H) , 5.28 - 5.10 (m, 1H) , 4.95 (dddd, J = 25.1, 9.1, 5.3, 1.5 Hz, 1H) , 4.62 (dd, J= 8.8, 5.3 Hz, 1H) , 4.55 (td, J = 9.0, 1.1 Hz, 1H) , 2.42 (s, 3H) , 1.38 (dd, J = 23.2, 6.6 Hz, 3H) . LCMS MH+ 438.9 RT 1.30 System 1

Example 6: Synthesis of (4R) -4- [ (1R) -1-hydroxyethyl] -3- (2- { [ (IS) -1- { 4-methyl-5- [2- (trifluoromethyl) pyridin-4-yl] pyridin-2- yl}ethyl] amino}-7H-pyrrolo [2 , 3-d] pyrimidin-4-yl) -1 , 3-oxazolidin- 2-one (Compound 9)

Trifluoroacetic acid (0.5 ml) was added to a solution of (4R) -4- [ (1R) -1- (tert-butoxy) ethyl] -3- (2-{ [ (IS) -1- { -methyl-5- [2- (trifluoromethyl ) pyridin-4 -yl ] pyridin-2 -yl } ethyl ] amino } - 7H-pyrrolo [2, 3-d] pyrimidin-4-yl )-l,3-oxazolidin-2-one (94%, 46 mg, 0.074 mmol) in DCM (0.5 ml) . The reaction solution was left to stir at RT for 1 h. The mixture was concentrated in vacuo, partitioned between DCM (2 ml) and saturated NaHCC>3 (2 ml) . The organic phase was collected using a hydrophobic frit then evaporated. The crude material was purified by basic HPLC to afford (4R) -4- [ (1R) -1-hydroxyethyl] -3- (2-{ [ (IS) -l-{ 4- methy1-5- [2- (trifluoromethyl) pyridin-4-yl ] pyridin-2- yl } ethyl ] amino } -7H-pyrrolo [ 2 , 3-d] pyrimidin-4-yl )-l,3-oxazolidin- 2-one as an off white solid.

! H NMR (500 MHz, DMSO-d6) δ 11.23 (s, 1H) , 8.84 (d, J = 5.0 Hz, 1H) , 8.42 (s, 1H) , 7.97 (s, 1H) , 7.79 (d, J = 4.9 Hz, 1H) , 7.40 (s, 1H) , 7.07 (d, J = 7.9 Hz, 1H) , 6.91 (t, J = 3.5, 2.3 Hz, 1H) , 6.42 (dd, J = 3.5, 2.0Hz, 1H) , 5.13 (q, J = 7.3 Hz, 1H) , 5.04 - 4.91 (m, 1H) , 4.91 - 4.76 (m, 1H) , 4.51 - 4.44 (m, 2H) , 4.21 - 3.86 (m, 1H) , 2.25 (s, 3H) , 1.51 (d, J = 7.0 Hz, 3H) , 0.91 - 0.61 (m, 3H)

LCMS MH+ 528.2 RT 2.24 System 2

Example 7: Synthesis of (4R) -3- (2-{ [ (IS) -1- [2- (4-chlorophenyl) - 1 , 3-thiazol-5-yl] ethyl] amino}-7H-pyrrolo [2 , 3-d] pyrimidin-4-yl) -4- [ (1R) -1-hydroxyethyl] -1 , 3-oxazolidin-2-one (Compound 3)

By proceeding in a similar manner but replacing (4R) -4- [ (1R) - 1- (tert-butoxy) ethyl] -3-(2-{[(lS) -1- { 4-methyl-5- [2-

( trifluoromethyl ) pyridin-4-yl ] pyridin-2-yl } ethyl ] amino } -7H- pyrrolo [2, 3-d] pyrimidin-4-yl) -1, 3-oxazolidin-2-one with (4R)- 4- [ (1R) -1- (tert-butoxy) ethyl] -3- (2-{ [ (IS) -1- [2- (4-chlorophenyl) - 1, 3-thiazol-5-yl] ethyl ] amino } -7H-pyrrolo [ 2 , 3-d] pyrimidin-4-yl ) - 1, 3-oxazolidin-2-one, (4R)-3-(2-{ [ (IS) -1- [2- (4-chlorophenyl) -1, 3- thiazol-5-yl ] ethyl ] amino } -7H-pyrrolo [ 2 , 3-d] pyrimidin-4-yl ) -4-

[( 1R) -1-hydroxyethyl ] -1 , 3-oxazolidin-2-one was obtained as a white solid.

! H NMR (500 MHz, DMSO-d6) δ 11.27 (s, 1H) , 7.89 - 7.83 (m, 2H) , 7.77 (s, 1H) , 7.53 - 7.48 (m, 2H) , 7.29 (d, J = 8.4 Hz, 1H) , 6.94 (dd, J = 3.5, 2.3 Hz, 1H) , 6.47 (dd, J = 3.6, 2.0 Hz, 1H) , 5.45 (p, J = 7.2 Hz, 1H),5.05 (d, J = 4.5 Hz, 1H) , 4.90 - 4.83 (m, 1H) , 4.48 (m, 2H) , 4.29 - 4.18 (m, 1H) , 1.63 (d, J = 6.9 Hz, 3H) , 0.87 (d, J = 6.0 Hz, 3H) LCMS MH+ 485.1 RT 3.03 System 2

Example 8: Synthesis of (4R) -3- (2- { [ (IS) -1- [5- (4- chlorophenyl) -1-methyl-lH-pyrazol-3-yl] ethyl] amino} -7H- pyrrolo [2 , 3-d] pyrimidin-4-yl) -4- [ (IR) -1-hydroxyethyl] -1,3- oxazolidin-2-one (Compound 8)

By proceeding in a similar manner to Example 7 but replacing

(4R)-4-[ (IR) -1- (tert-butoxy) ethyl] -3- (2-{ [ (IS) -1- [2- (4- chlorophenyl )-l,3-thiazol-5-yl] ethyl ] amino } -7H-pyrrolo [2,3- d] pyrimidin-4-yl ) -1 , 3-oxazolidin-2-one with a 7:3 mixture of

(4R)-4-[ (IR) -1- (tert-butoxy) ethyl] -3- (2-{ [ (IS) -1- [5- (4- chlorophenyl ) -l-methyl-lH-pyrazol-3-yl ] ethyl ] amino } -7H- pyrrolo [ 2 , 3-d] pyrimidin-4-yl ) -1 , 3-oxazolidin-2-one and (4R)-4-

[ (IR) -1- (tert-butoxy) ethyl] -3- (2-{ [ (IR) -1- [5- (4-chlorophenyl) -1- methyl-lH-pyrazol-3-yl ] ethyl ] amino } -7H-pyrrolo [ 2 , 3-d] pyrimidin-4- yl) -1, 3-oxazolidin-2-one, a 7:3 mixture of (4R) -3- (2-{ [ (IS) -1-

[5- (4-chlorophenyl ) -1 -methyl-lH-pyrazol-3-yl ] ethyl ] amino } -7H- pyrrolo [2, 3-d] pyrimidin-4-yl) -4- [ (IR) -1-hydroxyethyl] -1,3- oxazolidin-2-one and (4R) -3- (2-{ [ (IR) -1- [5- (4-chlorophenyl) -1- methyl-lH-pyrazol-3-yl ] ethyl ] amino } -7H-pyrrolo [ 2 , 3-d] pyrimidin-4- yl ) -4- [( IR) -1-hydroxyethyl ] -1 , 3-oxazolidin-2-one was obtained.

! H NMR (500 MHz, DMSO-d6) 11.25 (s, 1H) , 7.78 - 7.68 (m, 2H) , 7.45 - 7.36 (m, 2H) , 7.04 (d, J = 8.4 Hz, 1H) , 6.96 - 6.90 (m, 1H) , 6.65 (s, 1H) , 6.51 - 6.44 (m, 1H) , 5.34 - 5.20 (m,lH), 5.04 (d, J = 4.6 Hz, 1H) , 4.92 - 4.82 (m, 1H) , 4.52 - 4.43 (m, 2H) , 4.19 (br. s, 1H) , 3.86 (s, 3H) , 1.53 (d, J = 6.8 Hz, 3H) , 0.87 (d, J = 5.3 Hz, 3H) .

LCMS MH+ 482.2 RT 2.89 System 2

Example 9: Synthesis of (4R) -3- (2-{ [ (IS) -1- [1- (4-chlorophenyl) - lH-imidazol-4-yl] ethyl] amino}-7H-pyrrolo [2 , 3-d] pyrimidin-4-yl) -4- [ (IS) -1-fluoroethyl] -1 , 3-oxazolidin-2-one (Compound 1)

(Intermediate Al) by ( (4R) -3- [2-chloro-7- (4- methylbenzenesulfonyl ) -7H-pyrrolo [2, 3-d] pyrimidin-4-yl ] -4- [ (IS) - 1-fluoroethyl] -1, 3-oxazolidin-2-one ; ( ( 4R) -3- (2- { [ ( IS ) -1- [ 1- ( 4- chlorophenyl ) -lH-imidazol-4-yl ] ethyl ] amino } -7H-pyrrolo [2,3- d] pyrimidin-4-yl ) -4- [ (IS) -1-fluoroethyl ] -l,3-oxazolidin-2-one was obtained .

! H NMR (500 MHz, DMSO-d6) δ 11.21 (s, 1H) , 8.19 (d, J = 1.3 Hz, 1H) , 7.66 - 7.60 (m, 2H) , 7.56 - 7.50 (m, 2H) , 7.45 (s, 1H) , 6.90 (d, J = 3.6 Hz, 1H) , 6.69 (d, J = 8.0 Hz, 1H) , 6.55 (d, J = 3.6 Hz, 1H) , 5.28 - 5.01 (m, 2H) , 4.87 (ddd, J = 27.9, 8.9, 4.3 Hz, 1H) , 4.54 (t, J = 8.9 Hz, 1H) , 4.46 (dd, J = 8.8, 4.4 Hz, 1H) , 1.49 (d, J = 6.9 Hz, 3H) , 1.25 - 1.03 (m, 3H)

LCMS MH+ 470.1 RT 2.20 System 2 Example 10: Synthesis of (4R) -3- (2-{ [ (IS) -1- [1- (4-fluorophenyl) - lH-imidazol-4-yl] ethyl] amino}-7H-pyrrolo [2 , 3-d] pyrimidin-4-yl) -4- [ (IS) -1-fluoroethyl] -1 , 3-oxazolidin-2-one (Compound 10)

By proceeding in a similar manner to Example 9 but replacing ( IS ) -1- [1- ( 4-chlorophenyl ) -lH-imidazol-4-yl ] ethan-1-amine by ( IS ) -1- [1- ( 4-fluorophenyl ) -lH-imidazol-4-yl ] ethan-1-amine) , (4R)-3-(2-{ [ (IS) -1- [1- (4-fluorophenyl) -lH-imidazol-4- yl ] ethyl ] amino } -7H-pyrrolo [ 2 , 3-d] pyrimidin-4-yl )-4-[ (lS)-l- fluoroethyl ] -1 , 3-oxazolidin-2-one was obtained.

!H NMR (500 MHz, DMSO-d6) δ 11.22 (s, 1H) , 8.12 (d, J = 1.3

Hz, 1H) , 7.66 - 7.59 (m, 2H) , 7.41 (s, 1H) , 7.36 - 7.29 (m,

2H) , 6.91 (d, J = 2.0 Hz, 1H) , 6.69 (d, J = 8.0 Hz, 1H) , 6.55

(d, J = 2.6 Hz, 1H) , 5.32 - 5.02 (m, 2H) , 4.88 (ddd, J = 28.0,

9.1, 4.3 Hz, 1H) , 4.55 (t, J = 8.9 Hz, 1H) , 4.47 (dd, J =

8.8, 4.4 Hz, 1H) , 1.50 (d, J = 6.9 Hz, 3H) , 1.25 - 1.09 (m, 3H) .

LCMS MH+ 454.1 RT 1.94 System 2

Scheme 4: Synthesis of Compound 6 (for reaction conditions details, see the experimental procedures below) .

Synthesis of (4S) -3- [6-chloro-l- (oxan-2-yl) -lH-pyrazolo [3 , 4- d] pyrimidin-4-yl] -4- (propan-2-yl) -1 , 3-oxazolidin-2-one

4, 6-dichloro-l- (oxan-2-yl) -lH-pyrazolo [3, 4-d] pyrimidine (500 mg, 1.83 mmol) , (4S) -4- (propan-2-yl) -1, 3-oxazolidin-2-one (260.09 mg, 2.01 mmol), Pd2 (dba) 3 (50.29 mg, 0.05 mmol), Xantphos (31.78 mg, 0.05 mmol), K3P04 (777.19 mg, 3.66 mmol) were weighed out in an Ace pressure tube. Toluene (5 ml) was added and the mixture was "degassed" by bubbling a stream of nitrogen through it for 10 minutes. The mixture was then heated at 80 °C for 16 h. The reaction mixture was cooled to room temperature, then partitioned between DCM (10 ml) and water (10 ml) . The aqueous phase was further extracted with DCM (10 x 2ml) and the combined organic layers dried (Na2SOz_[) and concentrated. The crude material was purified by Biotage Isolera flash column chromatography (S1O2; 25 g) eluting with

10-50% EtOAc in heptane gradient to give 320 mg of required product as a mixture of diastereoisomers . LCMS MH+ 281.9 RT 1.26 System 1

Synthesis of (4S)-3-(6-{ [ (IS) -1- [1- (4-chlorophenyl) -lH-imidazol-4- yl] ethyl] amino}-!- (oxan-2-yl) -IH-pyrazolo [3 , 4-d] pyrimidin-4-yl) -4- (propan-2-yl) -1 , 3-oxazolidin-2-one

To a solution of (4S) -3- [ 6-chloro-l- (oxan-2-yl) -1H- pyrazolo [3, 4-d] pyrimidin-4-yl ] -4- (propan-2-yl) -1, 3- oxazolidin-2-one (160 mg, 0.44 mmol) in DMSO (1.5 ml) was added DIPEA (84.03 μΐ, 0.48 mmol) and ( IS) -1- [ 1- (4- chlorophenyl) -lH-imidazol-4-yl] ethan-l-amine (193.92 mg, 0. mmol) . The mixture was heated at 110 °C for 16 h. Water (5 ml) was added and the mixture extracted with DCM (3 x 5 ml) the combined organic layer was dried (Na2SOz_[) and

concentrated in vacuo. The crude material was purified by Biotage Isolera FCC (SiO2;10 g) eluting with 25-100% EtOAc in Heptane gradient to give 80 mg of required product as a mixture of diastereoisomers .

LCMS MH+ 551.7 RT 1.16 System 1

Example 11: Synthesis of (4S) -3- (6-{ [ (IS) -1- [1- (4-chlorophenyl) - lH-imidazol-4-yl] ethyl] amino}-lH-pyrazolo [3 , 4-d] pyrimidin-4-yl) -

4- (propan-2-yl) -1 , 3-oxazolidin-2-one (Compound 6)

TFA (0.3 ml) was added to a solution of (4S) -3- ( 6- { [ (IS) -1- [ 1- ( 4-chlorophenyl ) -lH-imidazol-4-yl ] ethyl ] amino } -1- (oxan-2- yl) -lH-pyrazolo [3, 4-d] pyrimidin-4-yl) -4- (propan-2-yl) -1, 3- oxazolidin-2-one (99%, 80 mg, 0.14 mmol) in DCM (0.5 ml) and the reaction stirred at 40 °C for 3 h. The reaction mixture was concentrated in vacuo then partitioned between saturated NaHC03 (5 ml) and DCM (5 ml) . The organic phase was collected using a hydrophobic frit then evaporated to give the crude product which was purified by high pH preparative HPLC to give 46 mg of (4S) -3- ( 6- {[( IS) -1- [ 1- (4-chlorophenyl) -1H- imidazol-4-yl ] ethyl ] amino }-lH-pyrazolo [3, 4-d] pyrimidin-4-yl ) - 4 - (propan-2-yl) -1, 3-oxazolidin-2-one.

! H NMR (500 MHz, DMSO-d6) δ 12.93 (s, 1H) , 8.16 (d, J = 27.4 Hz, 2H) , 7.78 - 7.33 (m, 5H) , 7.27 (d, J = 7.9 Hz, 1H) , 5.11 (s, 1H) , 4.79 (s, 1H) , 4.47 (t, J = 8.7 Hz, 1H) , 4.38 (s, 1H) , 2.53 - 2.51 (m, 1H) , 1.52 (d, J = 6.9 Hz, 3H) , 1.10 - 0.22 (m, 6H) .

LCMS MH+ 467.1 RT 2.30 System 2 Synthesis of (4R) -4- [ (1R) -1- (tert-butoxy) ethyl] -3-{2- chlorothieno [3 , 2-d] pyrimidin-4-yl}-l , 3-oxazolidin-2-one ;

(Intermediate A6)

2, 4-dichlorothieno [3, 2-d] pyrimidine (5 g, 24.38 mmol) , (4R)- 4- [( 1R) -1- (tert-butoxy) ethyl ] -1 , 3-oxazolidin-2-one (5.48 g, 29.26 mmol) and Caesium carbonate (9.53 g, 29.26 mmol) were added to a nitrogen flushed round bottomed flask.

Acetonitrile (100 ml) was added and the reaction was heated to 60°C overnight. The reaction mixture was diluted with EtOAc (150ml) and filtered. The solvent was then removed in vacuo to give the crude product as an orange/yellow solid which was purified by column chromatography (Biotage SNAP 50g cartridge) eluting with 0-25% EtOAc in heptane to afford 2.65 g of (4R) -4- [ (1R) -1- (tert-butoxy) ethyl] -3-{2- chlorothieno[3,2-d] pyrimidin-4-yl } -1, 3-oxazolidin-2-one.

! H NMR (500 MHz, Chloroform-d) δ 8.04 (d, J = 5.6 Hz, 1H) , 7.45 (d, J = 5.6 Hz, 1H) , 4.93 (dt, J = 8.4, 4.0 Hz, 1H) , 4.72 (dd, J = 9.4, 3.7 Hz, 1H) , 4.59 - 4.54 (m, 1H) , 4.54 - 4.49 (m, 1H) , 1.26 (s, 9H) , 1.07 (d, J= 6.5 Hz, 3H) .

LCMS MH+ 355.8 RT 1.30 System 1 Synthesis of (4R)-4- [ (lR)-l- (tert-butoxy) ethyl] -3- (2-{ [ (lS)-l- [1- (4-fluorophenyl) -lH-imidazol-4-yl] ethyl] amino} thieno [3,2- d] pyrimidin-4-yl) -1 , 3-oxazolidin-2-one

(4R) -4- [ (1R) -1- (tert-butoxy) ethyl] -3- { 2-chlorothieno [ 3 , 2- d] pyrimidin-4-yl } -1 , 3-oxazolidin-2-one (150 mg, 0.42

mmol) was suspended in DMSO (2 mL) , to this DIPEA (441.74 μΐ, 2.53 mmol) and ( IS ) -1- [ 1- ( 4-fluorophenyl ) -lH-imidazol-4- yl ] ethan-l-amine hydrochloride (305.65 mg, 1.26 mmol) were added and the reaction mixture heated to 110°C for 24 hours. The reaction mixture was cooled to room temperature and retreated with ( IS ) -1- [ 1- ( 4-fluorophenyl ) -lH-imidazol-4- yl ] ethan-l-amine hydrochloride (152.82 mg, 0.63 mmol) and DIPEA (220.87 μΐ, 1.26 mmol) . The reaction mixture was heated to 110°C overnight. After cooling, the reaction mixture was diluted with EtOAc (5 ml) and water (5 ml) and the phases separated. The aqueous layer was adjusted to pH 9 and extracted with EtOAc (2 x 5 ml) . The combined organic layers were then washed with brine (5 ml) and dried over Na2S04. The solvent was removed under reduced pressure. The crude

material was purified by Reverse Phase column chromatography

(Biotage C18 12g cartridge). Elution with 0-100% MeCN (0.1% Ammonium Hydroxide) in water (0.1% Ammonium Hydroxide) afforded 172.3 mg of (4R) -4- [ (1R) -1- (tert-butoxy) ethyl] -3- (2- { [ (IS) -1- [1- (4-fluorophenyl) -lH-imidazol-4- yl ] ethyl ] amino }thieno[3,2-d] pyrimidin-4-yl ) -1, 3-oxazolidin-2- one .

LCMS MH+ 263.3 RT 1.03 System 1

Example 12: synthesis of (4R) -3- (2-{ [ (IS) -1- [1- (4-fluorophenyl) - lH-imidazol-4-yl] ethyl] amino} thieno [3 , 2-d] pyrimidin-4-yl) -4- [ (1R) -1-hydroxyethyl] -1 , 3-oxazolidin-2-one (Compound 24)

By proceeding in a similar manner to Example 7 but replacing (4R)-4-[ (1R) -1- (tert-butoxy) ethyl] -3- (2-{ [ (IS) -1- [2- (4- chlorophenyl )-l,3-thiazol-5-yl] ethyl ] amino } -7H-pyrrolo [2,3- d] pyrimidin-4-yl ) -1 , 3-oxazolidin-2-one with (4R)-4-[ ( 1R) -1- (tert- butoxy) ethyl] -3- (2-{ [ (IS) -1- [1- ( 4-fluorophenyl ) -lH-imidazol-4- yl ] ethyl ] amino } thieno [ 3 , 2-d] pyrimidin-4-yl )-l,3-oxazolidin-2- one, synthesis of ( 4R) -3- (2- {[( IS ) -1- [ 1- ( 4-fluorophenyl ) -1H- imidazol-4-yl ] ethyl ] amino } thieno [ 3 , 2-d] pyrimidin-4-yl )-4-[ (lR)-l- hydroxyethyl ] -1 , 3-oxazolidin-2-one was obtained.

! H NMR (500 MHz, DMSO-d6) δ 8.15 - 8.10 (m, 2H) , 7.68 - 7.61 (m, 2H) , 7.50 (s, 1H) , 7.38 - 7.30 (m, 2H) , 7.13 (d, J = 5.2 Hz, 1H) , 7.05 (s, 1H) , 5.22 - 5.14 (m, 1H) , 5.14 - 5.07 (m, 1H) , 4.94 (dt, J = 8.4, 4.1 Ηζ,ΙΗ), 4.57 - 4.47 (m, 2H) , 4.21 (s, 1H) , 1.52 (d, J = 6.9 Hz, 3H) , 0.86 (s, 3H) . LCMS MH+ 469.2 RT 1.77 System 2

Mutant IDH Proteins and Assays Human IDH1 R132H

Cloning

The IDH1 R132H mutant residues M1-L414 was directionally cloned via restriction sites Ndel and BamHI into pET9a vector to yield the full length protein carrying a non-cleavable C- terminal Hisg-tag.

The protein was expressed from Escherichia coli Rosetta 2 (DE3) cells transformed with the plasmid in the presence of 50 yg/ml kanamycin and after induction by 1 mM IPTG.

Protein Sequence Recombinant HsIDHl R132H carrying a non- cleavable C-terminal Hisg-tag

MSKKI SGGSVVEMQGDEMTRI IWELIKEKLIFPYVELDLHSYDLGIENRDATNDQVTKDAA EAIKKHNVGVKCATITPDEKRVEEFKLKQMWKSPNGTIR ILGGTVFREAI ICK I PRLVS GWVKPI I IGHHAYGDQYRATDFVVPGPGKVEITYTPSDGTQKVTYLVHNFEEGGGVAMGMY NQDKS IEDFAHSSFQMALSKGWPLYLSTKNTILKKYDGRFKDIFQEIYDKQYKSQFEAQKI WYEHRLIDDMVAQAMKSEGGFIWACKNYDGDVQSDSVAQGYGSLGMMTSVLVCPDGKTVEA EAAHGTVTRHYRMYQKGQETSTNPIAS IFAWTRGLAHRAKLDNNKELAFFANALEEVS IET IEAGFMTKDLAACIKGLPNVQRSDYLNTFEFMDKLGENLKIKLAQAKLSLEHHHHHHHH

Purification

On average 5 g of wet pellet weight were resuspended in 50 ml of lysis buffer (20 mM Tris/HCl pH 7.5, 500 mM NaCl, 5 mM β mercaptoethanol , 0.1% Triton X-100, 10% glycerol, 1 mM

PMSF) . Cells were lysed by sonication (40% amplitude, 2 min 30 sec, 30 sec on/off clycle) . The lysate was cleared from debris by centrifugation (45 min at 25000g) .

The IDH protein was isolated by affinity chromatography, desalting and size exclusion chromatography. Protein was loaded onto a 5 ml HisTrap Crude FF column. The column was washed with 100 ml of wash buffer (20 mM Tris/HCl pH 7.4, 500 mM NaCl, 5 mM β mercaptoethanol , 10% glycerol). The protein was eluted in a linear gradient from wash buffer to elution buffer (20 mM Tris/HCl pH 7.4, 500 mM NaCl, 5 mM β mercaptoethanol, 10% glycerol, 500 mM imidazol pH 8.0) over 20 column volumes.

Positive elution fractions were buffer exchanged using a PD- 10 column to 50 mM Tris/HCl pH 7.5, 200 mM NaCl, 10%

glycerol, 5 mM β mercaptoethanol, 2 mM MnS04. The protein was concentrated using 30 K Amicon Ultra centrifugal filters and then subjected to size exclusion chromatography on Superdex S200 16/60pg columns equilibrated in 50 mM Tris/HCl pH 7.5, 200 mM NaCl, 10% glycerol, 5 mM β mercaptoethanol, 2 mM

MnS04.

Positive fractions were combined and the protein

concentration adjusted to the required concentration using centrifugal filters. The protein was then aliquoted and after plunge-freezing in liquid nitrogen stored at -80°C.

Human IDH2 R172K

Cloning

The IDH2 R172K mutant residues M1-L414 was directionally cloned via restriction sites BamHI and Xhol into pTrilJ-HV vector to yield the full length protein carrying a non- cleavable C-terminal Hisg-tag.

This plasmid was used to produce baculovirus for infecting Sf21 insect cells. These cells were stored as BIICs

(baculovirus infected insect cells) and used to infect large scale Sf21 cell cultures. Expression was carried out at MOI-1 in Sf900 II media supplemented with 5 yg/mL Gentamicin for 72 h at 27°C. Sf21 cells were prepared at a cell density of -0.5 xlO^ cell/mL 24 hours prior to infection. Cell parameters were measured using a Cell Countess (Invitrogen) at time of infection and prior to harvest. Ideal cell density at time of infection (0 hours) is -1.2x10^ cell/mL, cell viability -99%. Set cell parameters for harvest are viability -80-90% and cell size increase >2.5ym. Usually at harvest, cell density has reached -2 xlO^ cell/mL.

Protein Sequence Recombinant HsIDH2 R172K carrying a non- cleavable C-terminal His -tag

MAGYLRVVRSLCRASGSRPAWAPAALTAPTSQEQPRRHYADKRIKVAKPVVEMDGDEMTRI IWQFIKEKLILPHVDIQLKYFDLGLPNRDQTDDQV IDSALATQKYSVAVKCA I PDEAR VEEFKLKKMWKSPNG IR ILGGTVFREPI ICK I PRLVPGWTKPI IGKHAHGDQYKA D FVADRAGTFKMVFTPKDGSGVKEWEVYNFPAGGVGMGMYNTDES I SGFAHSCFQYAIQKKW PLYMSTKNTILKAYDGRFKDIFQEIFDKHYKTDFDKNKIWYEHRLIDDMVAQVLKSSGGFV WACKNYDGDVQSDILAQGFGSLGLMTSVLVCPDGKTIEAEAAHGTVTRHYREHQKGRPTST NPIAS IFAWTRGLEHRGKLDGNQDLIRFAQMLEKVCVETVESGAMTKDLAGCIHGLSNVKL NEHFLNTTDFLDTIKSNLDRALGRQHHHHHH

Purification

Approximately 70g of cells (wet weight) were obtained from a 10 1 insect cell expression culture. Cell pellet was

resuspended in 360 ml of lysis buffer (25 mM HEPES/NaOH pH 7.5, 200 mM NaCl, 5% glycerol) with 1 Roche Complete protease inhibitor cocktail tablet added per 50 ml. Cells were lysed by sonication (40% amplitude, 2 min 30 sec of sonication time, 30 sec on/off cycles) in batches of 120 ml. The lysate was cleared by centrifugation for 60 min at 16500g.

The protein was purified by affinity chromatography,

hydrophobic interaction chromatography and size exclusion chromatography .

Protein was loaded onto a 5 ml HisTrap Crude FF column equilibrated in lysis buffer (25 mM HEPES pH 7.5, 200 mM NaCl, 5% glycerol) . The column was washed with 10 column volumes of lysis buffer. The protein was eluted in a linear gradient from 0% to 100% elution buffer (25 mM HEPES pH 7.5, 200 mM NaCl, 5% glycerol, 250 mM imidazole pH 8.0) over 10 column volumes.

Positive elution fractions were pooled. Ammonium sulphate salt was slowly added to the stirred and cooled protein solution until a final concentration of 2 M. The sample was cleared from precipitated protein by short centrifugation at 12000 rpm.

The protein was then loaded onto a 5 ml HiTrap Butyl

Sepharose column equilibrated in HIC loading buffer (25 mM HEPES/NaOH pH 7.5, 2 M Ammonium Sulfate, 1 mM DTT, 5%

glycerol) . After washing of the column with 10 volumes of HIC loading buffer the protein was eluted in a linear gradient from 0 to 100% HIC elution buffer (25 mM HEPES/NaOH pH 7.5, 1 mM DTT, 5% Glycerol) over 20 column volumes.

The protein in the positive elution fractions was pooled and concentrated using 30 K Amicon ultra centrifugal filters and then subjected to size exclusion chromatography on Superdex S75 26/60pg columns equilibrated in 25 mM HEPES/NaOH pH 7.5, 100 mM NaCl, 1 mM DTT, 10% glycerol.

Positive fractions were combined and the protein

Mutant IDH1 biochemical assay

The enzymatic reaction was performed at room temperature in 384-well plates using a final reaction volume of 16 ]i containing 20 mM Tris pH 7.5, 150 mM NaCI, 10 mM MgCI₂ and 0.03% BSA. A final concentration 0.5yg/mL IDH1 (R132H) was pre-incubated with compound for 90 minutes, followed by addition of substrate solution (20 μΜ NADPH and ImM - ketoglutarate) . The reaction was incubated for a further 90 minutes at room temperature, and then 48 ]i 100% methanol was added to quench the reaction. After termination, plates were sealed and stored at 4°C until analysis.

IDH1 (R132H) catalytic activity was measured using the

RapidFire™ mass spectrometry (RF/MS) platform. Plates were analysed on the Agilent RF300 integrated autosampler/solid- phase extraction (SPE) system coupled to an Agilent QQQ 6460 mass spectrometer for the conversion of substrate (a- ketoglutarate) to product (2-hydroxyglutarate) . Solvent A was water containing 0.1% (v/v) acetic acid. Solvent B was methanol/0.1% ammonium acetate (9:1, v/v) . More specifically, plates were centrifuged at 4350 rpm for 10 min, samples were aspirated under vacuum for 600 ms, then loaded onto a

graphite solid-phase extraction cartridge and washed for 3 s with solvent A at a flow rate of 1 mL/min. Retained substrate and product were eluted with solvent B at a flow rate of 0.8 mL/min for 6 s and finally the cartridge was reequilibrated with solvent A for 500 ms . The mass transition for 2-HG and -ketoglutarate were 146.9/128.9 and 144.9/100.9,

respectively. The relative responses 2-HG/ (2-HG+a-KG) were measured at varied inhibitor concentrations and used to calculate inhibitory IC5Q values (normalized IC5Q regression curves) .

Mutant IDH2 biochemical assay

The enzymatic reaction was performed at room temperature in 384-well plates using a final reaction volume of 16 ]i containing 20 mM Tris pH 7.5, 150 mM NaCI, 10 mM MgCI₂ and 0.03% BSA. A final concentration 0.5yg/mL IDH2 (R172K) was pre-incubated with compound for 90 minutes, followed by addition of substrate solution (20 μΜ NADPH and ImM - ketoglutarate) . The reaction was incubated for a further 90 minutes at room temperature, and then 48 ]i 100% methanol was added to quench the reaction. After termination, plates were sealed and stored at 4°C until analysis.

IDH2 (R172K) catalytic activity was measured using the

RapidFire™ mass spectrometry (RF/MS) platform. Plates were analysed on the Agilent RF300 integrated autosampler/solid- phase extraction (SPE) system coupled to an Agilent QQQ 6460 mass spectrometer for the conversion of substrate ( - ketoglutarate) to product (2-hydroxyglutarate) . Solvent A was water containing 0.1% (v/v) acetic acid. Solvent B was methanol/0.1% ammonium acetate (9:1, v/v) . More specifically, plates were centrifuged at 4350 rpm for 10 min, samples were aspirated under vacuum for 600 ms, then loaded onto a graphite solid-phase extraction cartridge and washed for 3 s with solvent A at a flow rate of 1 mL/min. Retained substrate and product were eluted with solvent B at a flow rate of 0.8 mL/min for 6 s and finally the cartridge was reequilibrated with solvent A for 500 ms . The mass transition for 2-HG and -ketoglutarate were 146.9/128.9 and 144.9/100.9,

Results

The results of the mIDHI R132H and mIDH2 R172K biochemical inhibition assays with exemplary compounds of the present invention are summarized in Table 1.

The compounds according to the present invention were active inhibitors in the employed assays. For example, Compounds 1 and 4 of the present invention are particularly efficient dual mIDHl/mIDH2 inhibitors, whereas compounds 2, 3, 5-10 of the present invention are specific mIDHI inhibitors with considerable mIDH2 inhibitory activity.

Table 1 . IC50 values (μΜ) for the inhibition of neomorphic activity of mlDHl and mIDH2 calculated from the biochemical assay data for different compounds with mlDHl and mIDH2.

(500 MHz, DMSO-d6)_ 11.19 (s, 1H), 7.19 (d, J = 7.9 Hz, 2H), 7.16 (t, J = 6.5 Hz, 1H), 7.08 (d, J = 7.8 Hz, 2H), 6.91 (dd, J= 3.6, 2.0 Hz, 1H), 6.41 (dd, J= 3.6, 1.7 Hz, 1H), 4.75 (s, 1H), 4.52 (dd, J= 15.5, 3.37

108 19.222 3.72 6.5 Hz, 1H), 4.47 (t, J = 8.9 366

Hz, 1H), 4.38 (dd, J= 15.5,

6.1 Hz, 1H), 4.30 (dd, J= 8.9,

5.2 Hz, 1H), 2.25 (s, 3H),

2.12 (d, J = 31.5 Hz, 1H),

0.75 (s, 3H), 0.69 (d, J = 6.7

Hz, 3H).

Claims

1. A compound of general Formula (I)

(I)

or a salt or solvate thereof, wherein n is 0 or 1 ;

R¹ is N (R¹¹ ) or CH;

R² is N or C (R¹²) ;

R³ is S or C (R¹³) ;

provided that

R¹ is CH and R² is CR¹² when R³ is S; and

R¹ is N(R^1X) when R³ is C(R¹³);

R¹¹ is a hydrogen atom or C¾;

R¹² is a hydrogen atom, a cyano group or C¾;

R¹³ is a hydrogen atom or a halogen atom;

R⁴, R⁵ are independently selected from a hydrogen atom; CH₂OH; CH₂OCH₃; a Ci-6 alkyl group, wherein the Ci-6 alkyl group is unsubstituted or substituted with at least one halogen atom; or R⁴, R⁵ may be joined together to form a C3-6 cycloalkyl group;

R⁶ is an aromatic group, a heteroaromatic group or a C3-6 cycloalkyl group, wherein R⁶ is unsubstituted or

substituted with one or two R^6a, which are the same or different ;

R^6a is a halogen atom, a Ci-6 alkyl group, a Ci-6 alkoxy group, a S(0)₂-Ci-₆ alkyl group, or R^6aa;

R^6aa is an aromatic group, a heteroaromatic group, or a heteroalicyclylalkyl group, wherein R^6aa is unsubstituted or substituted with one or two R^6b, which are the same or different;

R^6b is a halogen atom; a Ci-6 alkyl group, which is unsubstituted or substituted with at least one halogen atom; cyclopropyl or benzyloxycarbonyl ;

R^7a is a Ci-6 alkyl group, an aromatic group, a

heteroaromatic group, or a heteroalicyclic group, wherein R^7a is unsubstituted or substituted with a substituent selected from a halogen atom, C¾, or OH;

R⁹, R¹⁰ are independently selected from a hydrogen atom, or R^9a, or R⁹ and R¹⁰ may be joined together to form a C3- 6 cycloalkyl group or a heteroalicyclic group;

R^9a is a Ci-6 alkyl group, an aromatic group, a

heteroaromatic group, or a heteroalicyclic group, wherein R^9a is unsubstituted or substituted with a substituent selected from a halogen atom, C¾, or OH. The compound according to claim 1 or a salt or solvate thereof, wherein

(i) R¹ is NH, R² is CH, R³ is CH; or

(ii) R¹ is NH, R² is N, R³ is CH; or

(iii) R¹ is CH, R² is CH, R³ is S ; or

(iv) R¹ is NH, R² is CH, R³ is CF; or

(v) R¹ is NH, R² is CCN, R³ is CH; or

(vi) R¹ is NH, R² is CCH₃, R³ is CH; or

(vii) R¹ is NCH₃, R² is CH, R³ is CH.

The compound according to claim 1 or 2 or a salt or solvate thereof, wherein one of R⁴, R⁵ is a hydrogen atom or C¾ and the other is a hydrogen atom, C¾,

CH₂CH₃, CH₂OH, CH₂OCH₃, CH₂F or CF₃; or R⁴, R⁵ are joined together to form a cyclopropyl group.

The compound according to any of claims 1 to 3 or a salt or solvate thereof, wherein R⁶ is an aromatic group or a heteroaromatic group and wherein R⁶ is unsubstituted or substituted with one or two R^6a, which are the same or different .

The compound according to any of claims 1 to 4 or a salt or solvate thereof, wherein R⁶ is imidazolyl, pyridyl, thiazolyl, pyrazolyl, phenyl, oxadiazolyl, oxazolyl, cyclohexyl, thiophenyl or furanyl and wherein R⁶ is unsubstituted or substituted with one or two R^6a, which are the same or different.

6. The compound according to any of claims 1 to 5 or a salt or solvate thereof, wherein R^6a is CH₃, OCH₃, F, CI, S (0) ₂CH₃ or R^6aa.

7. The compound of any of claims 1 to 6 or a salt or solvate thereof, wherein R^6aa is phenyl, pyridyl, thiophenyl or piperazinylmethyl and wherein R^6aa is unsubstituted or substituted with one or two R^6b, which are the same or different.

8. The compound according to any of claims 1 to 7 or a salt or solvate thereof, wherein R^6b is CI, F, CF₃,

cyclopropyl or benzyloxycarbonyl .

9. The compound according to any of claims 1 to 8 or a salt or solvate thereof, wherein one of R⁷, R⁸ is a hydrogen atom or C¾ and the other is C¾F, a fluoroethyl, methyl, ethyl, isopropyl, tert. -butyl, 1-methyl-propyl, a hydroxyethyl , phenyl, pyridyl or methylimidazolyl ; or R⁷ and R⁸ are joined together to form a cyclopropyl, tetrahydrofuranyl or tetrahydropyranyl group.

10. The compound according to any of claims 1 to 9 or a salt or solvate thereof, wherein R⁹ and R¹⁰ are hydrogen atoms, and at least one of R⁷ and R⁸ is other than a hydrogen atom.

11. The compound according to any of claims 1 to 10 or a salt or solvate thereof, wherein n is 1.

12. The compound according to any of claims 1 to 11 or a salt or solvate thereof of Formula (I), wherein n is 1, R⁴ is a hydrogen atom and R⁵ is other than a hydrogen atom to give Formula (la)

The compound according to any of claims 1 to 12 salt or solvate thereof, wherein the compound is selected from one of the following:

14. A compound according to any of claims 1 to 13 or a salt or solvate thereof for use in the treatment or

prophylaxis of a disorder of a human or animal body.

15. A compound according to any of claims 1 to 13 or a salt or solvate thereof for use in the treatment or

prophylaxis of a disorder associated with a mutant isocitrate dehydrogenase having a neomorphic activity.

16. The compound according to any of claims 1 to 13 or a salt or solvate thereof for use according to Claim 15, wherein the disorder associated with a mutant isocitrate dehydrogenase having a neomorphic activity is a cell proliferation disorder.

17. The compound according to any of claims 1 to 13 or a

salt or solvate thereof for use according to Claim 15 or 16, wherein the disorder associated with a mutant isocitrate dehydrogenase having a neomorphic activity is one of the following: brain cancer, such as glioma, glioblastoma multiforme, paraganglioma, and

supratentorial primordial neuroectodermal tumors (pNET) , leukemia, such as acute myeloid leukemia (AML) ,

myelodysplasia syndrome, and chronic myelogenous

leukemia (CML) , skin cancer such as melanoma, prostate cancer, thyroid cancer, colon cancer, lung cancer, sarcoma, including central chondrosarcoma, central and periosteal chondroma, and fibrosarcoma.

18. A pharmaceutical composition comprising the compound of general Formula (I) according to any of Claims 1 to 13 or of a salt or solvate thereof as active ingredient.

19. The pharmaceutical composition according to Claim 18, wherein said pharmaceutical composition is an oral dosage form.