Pyrido-piperazinone derivatives, preparation and therapeutic uses thereof

Abstract

Compounds are provided which can inhibit ERK5. Also provided are pharmaceutical compositions and medical uses of the same, including the use in treating or preventing conditions such as cancers.

Description

[0001] PYRIDO-PIPERAZINONE DERIVATIVES, PREPARATION AND THERAPEUTIC USES THEREOF

[0002] Compounds are provided which can inhibit ERK5. Also provided are pharmaceutical compositions and medical uses of the same, including the use in treating or preventing conditions such as cancers.

[0003] The mitogen-activated protein kinase (MAPK) cascade is a highly-conserved cellular pathway which transmits signals from the cell surface to the nucleus. The pathway plays an important role in cell proliferation, differentiation, and migration and it is well known to be involved in the development of cancer. Proteins in the pathway include the extracellular signal-regulated kinase (ERK) proteins; among those, ERK5 (expressed from the MAPK7 gene) plays an important role in cell proliferation, as well as epithelial development and neural differentiation. ERK5 is unique among the ERK proteins, having a large C-terminal domain which contains a transcriptional activation domain (TAD) as well as a nuclear localization signal and two pro line -rich regions. Autophosphorylation of the TAD is required for transcriptional activation.

[0004] ERK5 plays an important role in controlling cell proliferation and cell cycle progression, for example via direct or indirect phosphorylation of MEF2C, cMYC, SGK1, RSK, FOS, and FRA1 among others. The involvement of ERK5 in numerous biological pathways means that its activity is associated with many aspects of cancer progression, including tumour angiogenesis, metastasis, inflammation, sustained proliferation, and evasion of growth suppression. It therefore presents an attractive target for modulating disease pathology and treatment in a wide range of conditions. In previous studies, ERK5 inhibition or downregulation has been shown to block tumorigenesis in murine leukaemia cells, reduce growth of chronic myeloid leukaemia cells, inhibit growth of breast cancer and multiple myeloma cells, suppress colon cancer cell proliferation, and have to have an impact on renal cell carcinoma, mesothelioma, adenocarcinoma, neuroblastoma and hepatocellular carcinoma cell growth or survival, among others.

[0005] ERK5 inhibition thus represents a promising approach to tackle a broad range of cancers. Despite recent progress in cancer treatment with the development of targeted therapies and immunotherapies, not all cancer patients can be offered an efficient therapeutic solution. There is therefore a need to identify and develop new drugs. The present disclosure seeks to address this need by providing novel compounds, which comprise a 5-5 bicyclic piperidine core, for use as ERK5 inhibitors and for the treatment of ERK5 related diseases and conditions.

[0006] Accordingly, herein is provided a compound of Formula (I), comprising a piperazinone ring fused to a pyridine ring:

[0007]

[0008] or a pharmaceutically acceptable salt thereof, wherein:

[0009] R1 represents a hydrogen atom or a -NH2 group,

[0010] R2 and R3 are independently selected from a hydrogen atom or a (Cl-C4)alkyl group, or form together with the carbon atom to which they are attached a (C4- C6)cyloalkyl or 4 to 6-membered heterocycloakyl group,

[0011] the dotted line in the piperazinone ring represents a saturated or unsaturated bond; when the dotted line represents a saturated bond, X is selected from -CH2-, -O-, - S-, -SO2- or -NH-, and

[0012] when the dotted line represents an unsaturated bond, X represents -N= and R3 is absent.

[0013] In an embodiment of formula (I), R2 and R3 are independently selected from a hydrogen atom or methyl or ethyl group, or form together with the carbon atom to which they are attached a cyclobutyl, cyclopentyl, cyclohexyl or tetrahydropyranyl group.

[0014] In an embodiment of formula (I), X represents -N=, R3 is absent and R2 is selected from a hydrogen atom or methyl or ethyl group. A further aspect provides a compound of formula (II), or a pharmaceutically acceptable salt thereof, wherein Rl, R2 and R3 are defined as in formula (I) and wherein X is selected from -CH2-, -O-, -S-, -SO2- or -NH-:

[0015]

[0016] (ID

[0017] In an embodiment of formula (II), Rl represents -NH2.

[0018] In another embodiment of formula (II), X represents -S- or -SO2- and R2 and R3 represent hydrogen atoms or (Cl-C4)alkyl groups, such as methyl groups.

[0019] In another embodiment of formula (II), X represents -NH- and the compounds are those of formula (Ila), or a pharmaceutically acceptable salt thereof:

[0020]

[0021] wherein R1 represents a hydrogen atom or a -NH2 group; and wherein R2 and R3 are independently selected from a hydrogen atom or a (Cl -C4)alkyl group, or form together with the carbon atom to which they are attached a (C4-C6)cyloalkyl or 4 to 6-membered heterocycloalkyl group.

[0022] In an embodiment of formula (Ila), R2 and R3 are independently selected from a hydrogen atom or methyl group, or form together with the carbon atom to which they are attached a cyclobutyl, cyclopentyl, cyclohexyl or tetrahydropyranyl group.

[0023] A further aspect provides a compound of formula (III), or a pharmaceutically acceptable salt thereof, wherein R1 and R2 are defined as in formula (I):

[0024] O

[0025] NW I

[0026] ii TRl

[0027]

[0028] FX

[0029] F F°

[0030] (III)

[0031] In an embodiment of formula (III), R1 represents -NH2.

[0032] In another embodiment of formula (III), R2 represents a (Cl-C4)alkyl group, such as ethyl.

[0033] A further aspect provides a compound selected from the group consisting of:

[0034] 8-[ 1 -[2-amino-4-(trifluoromethoxy)benzoyl] -4-piperidyl]-7 -fluoro- 1 , 1 -dioxo-4H- pyrido[3,2-b][l,4]thiazin-3-one

[0035] 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-2-ethyl-7-fluoro-4H- pyrido[2,3-b]pyrazin-3-one

[0036] 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-2,2-dimethyl-4H- pyrido[3,2-b][l,4]thiazin-3-one 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-spiro[l,4- dihydropyrido[2,3-b]pyrazine-2,l'-cyclobutane]-3-one

[0037] 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-spiro[l,4- dihydropyrido[2,3-b]pyrazine-2,l'-cyclopentane]-3-one

[0038] 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-spiro[l,4- dihydropyrido[2,3-b]pyrazine-2,l'-cyclohexane]-3-one

[0039] 7 -fluoro-8-[ l-[4-(trifluoromethoxy)benzoyl]-4-piperidyl] spiro[ 1 ,4-dihydropyrido [2,3 - b]pyrazine-2,4'-tetrahydropyran] -3-one

[0040] 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-spiro[l,4- dihydropyrido[2,3-b]pyrazine-2,4'-tetrahydropyran]-3-one

[0041] 7-fluoro-2,2-dimethyl-8-[l-[4-(trifluoromethoxy)benzoyl]-4-piperidyl]-l,4- dihydropyrido[2,3-b]pyrazin-3-one

[0042] 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-2,2-dimethyl-l,4- dihydropyrido[2,3-b]pyrazin-3-one

[0043] 6-fluoro-5-[l-[4-(trifluoromethoxy)benzoyl]-4-piperidyl]-3,4-dihydro-lH-l,8- naphthyridin -2-one

[0044] 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-4H-pyrido[3,2- b][l,4]oxazin-3-one

[0045] 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-2,4-dihydro-lH- pyrido[2,3-b]pyrazin-3-one

[0046] 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-4H-pyrido[3,2- b][l,4]thiazin-3-one,

[0047] and the pharmaceutically acceptable salts thereof.

[0048] Definitions

[0049] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understoodby one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, exemplary methods, devices, and materials are now described. All technical and patent publications cited herein are incorporated herein by reference in their entirety. All numerical designations, e.g., pH, temperature, time, concentration, molecular weight, etc., including ranges, are approximations which are varied ( + ) or ( - ) by increments of, e.g., 0.1 or 1.0, where appropriate. It is to be understood, although not always explicitly stated , that all numerical designations are preceded by the term “about”, which is used to denote a conventional level of variability. For example, a numerical designation which is “about” a given value may vary by ± 10% of said value; alternatively, the variation may be ± 5%, ± 2%, or ± 1% of the value. It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0050] As used in the specification and claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof. Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. The term “including” is used herein to mean, and is used interchangeably with, the phrase “including but not limited to”.

[0051] As used herein, the term “comprising” or “comprises” is intended to mean that the compositions and methods include the recited elements, without excluding other elements. “Consisting essentially of’ when used to define compositions and methods, shall mean excluding other elements of any essential significance for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. “Consisting of’ shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this disclosure or process steps to produce a composition or achieve an intended result. Embodiments defined by each of these transition terms are within the scope of this disclosure. Use of the term “comprising” herein is intended to encompass, and to disclose, the corresponding statements in which the term “comprising” is replaced by “consisting essentially of’ or “consisting of’.

[0052] A “subject,” “individual”, or “patient” is used interchangeably herein, and refers to a vertebrate, such as a mammal. Mammals include, but are not limited to, rodents, farm animals, sport animals, pets, and primates; for example murines, rats, rabbit, simians, bovines, ovines, porcines, canines, felines, equines, and humans. In a particular embodiment, the mammal is a human.

[0053] “Administering” is defined herein as a means of providing an agent or a composition containing the agent to a subject in a manner that results in the agent being contacted with (e.g., being inside) the subject’s body. Such an administration can be by any route including, without limitation, oral, transdermal (e.g., by the vagina, rectum, or oral mucosa), by injection (e.g., subcutaneous, intravenous, parenteral, intraperitoneal, or into the central nervous system), or by inhalation (e.g., oral or nasal). Administration may also involve providing a substance or composition to a part of the surface of the subject’s body, for example by topical administration to the skin. Pharmaceutical preparations are, of course, given by forms suitable for each administration route.

[0054] “Treating” or “treatment” of a disease includes: (1) preventing the disease, i.e. causing the clinical symptoms of the disease not to develop in a patient that may be predisposed to the disease but does not yet experience or display symptoms of the disease; (2) inhibiting the disease, i.e. arresting or reducing the development of the disease or its clinical symptoms; and / or (3) relieving the disease, i.e. causing regression of the disease or its clinical symptoms.

[0055] The term “suffering” as it relates to the term “treatment” refers to a patient or individual who has been diagnosed with or is predisposed to the disease. A patient may also be referred to being “at risk of suffering” from a disease because of a history of disease in their family lineage or because of the presence of genetic mutations associated with the disease. A patient at risk of a disease has not yet developed all or some of the characteristic pathologies of the disease.

[0056] An “effective amount” or “therapeutically effective amount” is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications, or dosages. Such delivery is dependent on a number of variables including the time period for which the individual dosage unit is to be used, the bio availability of the therapeutic agent, the route of administration, etc. It is understood, however, that specific dose levels of the therapeutic agents of the present disclosure for any particular subject depends upon a variety of factors including, for example, the activity of the specific compound employed, the age, body weight, general health, sex, and diet of the subject, the time of administration, the rate of excretion, the drug combination, the severity of the particular disorder being treated and the form of administration. Treatment dosages generally may be titrated to optimize safety and efficacy. Typically, dosage -effect relationships from in vitro and / or in vivo tests initially can provide useful guidance on the proper doses for patient administration. In general, one will desire to administer an amount of the compound that is effective to achieve a serum level commensurate with the concentrations found to be effective in vitro. Determination of these parameters is well within the skill of the art. These considerations, as well as effective formulations and administration procedures are well known in the art and are described in standard textbooks. Consistent with this definition, as used herein, the term “therapeutically effective amount” is an amount sufficient to treat (e.g., improve) one or more symptoms associated with the condition. The total daily dose may be administered in single or divided doses and may, at the physician's discretion, fall outside of the typical range given herein.

[0057] As used herein, the terms "increased" and "elevated" are used interchangeably and encompass any measurable increase in a biological function and / or a biological activity and / or a concentration. For example, an increase can be by at least about 10%, e.g. at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, such as at least about 95%, 96%, 97%, 98%, 99%, or 100%. Thus, an increase can be by atleast about2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold, such as at least about 20-fold, 25 -fold, 50-fold, 100-fold, or higher, relative to a control or baseline amount or function, or activity, or concentration.

[0058] As used herein, the terms "increased expression" and / or "increased activity" of a substance, such as ERK5, in a sample or cancer or patient, typically refers to an increase in the amount of the substance (e.g., of the MAPK7 gene product or ERK5 protein), although it may also denote an increase in the biological activity of the substance (e.g., constitutive activation of phosphorylation and / or reduced discrimination of phosphorylation sites of ERK5). For example, an increase can be by an amount of about 5%, e.g., about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, such as about 96%, 97%, 98%, 99%, or 100%. Thus, the increase can be about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold, such as about 20-fold, 25-fold, 50-fold, 100-fold, or higher, relative to the amount (or activity) of the substance, such as ERK5, in a control sample or control samples, such as an individual or group of individuals who are not suffering from the disease or disorder (e.g. cancer) or an internal control, as determined by techniques known in the art. A subject can also be determined to have an "increased expression” or "increased activity" of ERK5 if the expression and / or activity of ERK5 is increased by one standard deviation, two standard deviations, three standard deviations, four standard deviations, five standard deviations, or more, relative to the mean (average) or median amount of ERK5 in a control group of samples or a baseline group of samples or a retrospective analysis of patient samples. As practiced in the art, such control or baseline expression levels can be previously determined, or measured prior to the measurement in the sample or cancer or subject, or can be obtained from a database of such control samples.

[0059] As used herein, the term “pharmaceutically acceptable excipient” encompasses any of the standard pharmaceutical excipients, for example as described in Remington’s Pharmaceutical Sciences (20th ed., Mack Publishing Co. 2000). Such excipients include carriers such as a phosphate buffered saline solution, water, and emulsions, such as an oil / water or water / oil emulsion, and various types of wetting agents. Pharmaceutical compositions also can include stabilizers, preservatives, adjuvants, fillers, binders, lubricants, and the like.

[0060] As used herein, the term “(Cl-C4)alkyl group” means a linear or branched saturated hydrocarbon group comprising from 1 to 4 carbon atoms, such as methyl or ethyl.

[0061] As used herein, the term “(C4-C6)cycloalkyl group” means a cyclic alkyl group, such as cyclobutyl, cyclopentyl and cyclohexyl.

[0062] As used herein, the term “4 to 6-membered heterocyclo akyl group” means a cycloalkyl group comprising one or more heteroatoms, such as an oxygen atom. An example may be a tetrahydropyranyl group.

[0063] The compounds of the present disclosure are described, inter alia, by way of structural formulae. It will be appreciated that these formulae typically show only one form (e.g., resonance form, tautomeric form, etc.) of the compound, whereas certain compounds may exist in more than one such form. This will be readily apparent to the skilled reader. The present disclosure includes all possible tautomers of the compounds characterised by the structural formulae hereinbefore and below, including as single tautomers, or as any mixture of tautomers in any ratio.

[0064] The compounds of the disclosure may exist in the form of free acids or bases, or may exist as addition salts with suitable acids or bases.

[0065] As used herein, the term “pharmaceutically acceptable” when used in connection with salts means a salt of a currently disclosed compound that may be administered without any resultant substantial undesirable biological effect(s) or any resultant deleterious interaction(s) with any other component of a pharmaceutical composition in which it may be contained.

[0066] The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0067] Compositions and methods provided herein maybe combined with one or more of any of the other compositions and methods provided herein.

[0068] The following abbreviations and empirical formulae are used herein:

[0069] °C degree Celsius

[0070] pm micrometer

[0071] Ac Acetyl

[0072] BOC tert-butoxycarbonyl

[0073] cat. catalytic amount

[0074] DCM dichloromethane

[0075] DiPEA Diisopropylethylamine

[0076] 4-DMAP 4-Dimethylaminopyridine

[0077] DMF dimethylformamide

[0078] DMSO dimethylsulfoxide

[0079] Dppf (diphenylphosphino)ferrocene

[0080] EA ethyl acetate

[0081] EDC1 l-ethyl-3-(3-dimethylaminopropyl)carbodiimide

[0082] Et Ethyl

[0083] G gram

[0084] HOBt 1 -Hydroxybenzo triazole

[0085] hrs hours

[0086] i-Pr iso-propyl

[0087] LC / MS Liquid chromatography / mass spectrometry

[0088] LDA lithium diisopropylamide

[0089] LiHMDS Lithium bis(trimethylsilyl)amide

[0090] Me Methyl

[0091] mg milligram Min minute

[0092] mL milliliter

[0093] mm millimeter

[0094] mmol millimoles

[0095] MW micro wave

[0096] NBS N-bro mo succinimide

[0097] n-Bu n-Butyl

[0098] NMM N-methylMorpholine

[0099] NMR Nuclear Magnetic Resonance

[0100] Pd Palladium

[0101] Pd / C Palladium on charcoal

[0102] PE petroleum ether

[0103] PG protective group

[0104] Ph phenyl

[0105] r.t. room temperature

[0106] SCX strong cation exchange

[0107] TATU 1 - [Bis(dimethylamino)methylene] - 1 H- 1 ,2,3 -triazolo [4,5 -b]pyridinium 3 -Oxide Tetrafluoroborate

[0108] TBTU O-(Benzotriazol- 1 -yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate

[0109] t-Bu tert-Butyl

[0110] TEA triethylamine

[0111] TFA trifluoro acetic acid

[0112] THF tetrahydrofuran

[0113] UV ultra-violet

[0114] General synthetic schemes

[0115] The following scheme, Scheme 1, illustrates an exemplary way of preparing compounds in accordance with the present disclosure and examples: STEP 1 STEP 2 STEP 3 O

[0116] R2 --R3 F. / j^ ^F n-BuLi, C2CI6H2SO4, K2S2O8F Compound 1D I NI 40 °C, 4 hrs TEA, DCM, NH2- THF, -78 °C to r.t.

[0117] r.t. 16 hrs 2 hrs Compound 1A Compound 1B Compound 1C Compound 1E or Fe / NH4CI STEP 4 1 ,4-dioxane EtOH or MeOH / water / H2O, 70-90 "C 90 °C, 4 hrs 3-48 hrs

[0118] Compound 1G Pd Catalyst Base, solvent Compound 11 100-110 'C. 2-5 hrs Compound 1F

[0119]

[0120] Compound I

[0121] SCHEME 1

[0122] According to Scheme 1 (in which X may be N, NH, O, S; R1is -H or -NH2, R2 and R3 are independently selected from a hydrogen atom or a (Cl -C4)alkyl group, or form together with the carbon atom to which they are attached a (C4-C6)cyloalkyl or 4 to 6-membered heterocycloakyl group, or R3 is absent, the dotted line in the piperazinone ring represents a saturated or unsaturated bond), Compound IB can be obtained in STEP 1 by substitution of Compound 1 A with C2CI6 using a base such as n-Buthyllithium for example. Compound 1 B can be converted into compound 1C in STEP 2 using K2S2O8 and sulfuric acid. Compound IE can be obtained by nucleophilic aromatic substitution of compound 1C with compound ID under basic conditions in STEP 3. Compound IF can be then obtained from compound IE by using Fc.NH4CI to perform the reduction-cyclization sequence in STEP 4. Compound IF can be then transformed into compound 1H in STEP 5 via a Suzuki coupling with compound 1G using, for example, a catalyst such as Pd(P(t-Bu)a)2, in a mixture of dioxane and water and in the presence of a base, such as potassium triphosphate, by heating to reflux.

[0123] Compound 1H canthen be converted to Compound II in STEP 6 by deprotection using TFA or HC1 in DCM for example. Compound IK can then be prepared from Compound II in STEP 7 with carboxylic acid Compound 1 J, using conditions known by the person skilled in the art such as TBTU in a solvent like DMF in presence of a base such as DIPEA. Compound I can be obtained by hydrogenation of compound 1 K in STEP 8 using Pd / C and Pd(OH)2 with H2 for example. STEP 8 may optionally further comprise subsequent oxidation of either the pyridine ring and the sulfur atom (when X = S), followed by a pyridinium-N-oxide deoxygenation step.

[0124] The preparation of compound I (in which X may be N, NH, O, S; R1is -H or -NH2, R2 and R3 are independently selected from a hydrogen atom or a (Cl-C4)alkyl group, or form together with the carbon atom to which they are attached a (C4-C6)cyloalkyl or 4 to 6-membered heterocycloakyl group, or R3 is absent, the dotted line in the piperazinone ring represents a saturated or unsaturated bond), maybe also effected according to the alternative following scheme, Scheme 2:

[0125]

[0126] SCHEME 2

[0127] According to Scheme 2 (in which X may be N, NH, O, S; R1is -H or -NH2, R2 and R3 are independently selected from a hydrogen atom or a (Cl -C4)alkyl group, or form together with the carbon atom to which they are attached a (C4-C6)cyloalkyl or 4 to 6-membered heterocycloakyl group, or R3 is absent, the dotted line in the piperazinone ring represents a saturated or unsaturated bond), Compound IL can be obtained by hydrogenation of compound 1H in STEP 1 using Pd / C and Pd(OH)2 with H2, for example. Compound IL can then be converted to Compound IM in STEP 2 by deprotection using TFA or HC1 in DCM, for example. Compound I can then be prepared from Compound IM in STEP 3 with carboxylic acid Compound 1 J, using conditions known by the person skilled in the art such as TBTU in a solvent like DMF in presence of a base such as DIPEA. STEP 3 may optionally further comprise subsequent oxidation of either the pyridine ring and the sulfur atom (when X = S), followed by a pyridinium-N-oxide deoxygenation step.

[0128] The preparation of compound I (in which X may be CH2; R1is -H or -NH2,), may be also effected according to the alternative following scheme, Scheme 3:

[0129] STEP 1 STEP 3

[0130] O

[0131] (BOC)2O, DCM LDA, C2C|6Compound 1Q 4-DMAP THF, -78 °C Pd catalyst r.t., 12 hrs 3 hrs Base, solvent 100 °C, 6 hrs Compound 1N Compound 10 Compound 1R STEP 4

[0132] Compound 1G Pd Catalyst Base, solvent 100-110 °C, 2-12 hrs STEP 7 STEP 5 NMM Pd / C,H2MeOH solvent, 70 °C 70 °C, 12 hrs 5 days

[0133] Compound 1V Compound 1U Compound 1T

[0134] Compound 1J EDCI, HOBt DIPEA DMF, r.t., 12 hr

[0135]

[0136] Compound I SCHEME 3 According to Scheme 3 (in which X may be CH2; R1is -H or -NH2,), Compound 10 can be obtained in STEP 1 by N-BOC protection of compound IN using BOC2O under basic conditions. Compound IP can be obtained in STEP 2 by substitution of Compound 10 with C2CI6 using a base such as LDA, for example. Compound IP can be then transformed into compound 1R in STEP 3 via a Suzuki coupling with compound IQ using, for example, a catalyst such as PdChCdppf), in a mixture of dioxane and water and in the presence of a base, such as sodium carbonate, by heating to reflux. Compound 1R can be then transformed into compound IS in STEP 4 via a Suzuki coupling with compound 1G using, for example, a catalyst such as Pd(P(t-Bu)a)2, in a mixture of dioxane and water and in the presence of a base, such as potassium triphosphate, by heating to reflux. Compound IT can be obtained by hydrogenation of compound IS in STEP 5 using Pd / C with H2 for example. Compound IT can then be converted to Compound 1U in STEP 6 by deprotection using TFA or HC1 in DCM for example. Intramolecular cyclization of compound 1U into compound IV in STEP 7 can be then performed with NMM in MeOH. Compound I can then be prepared from Compound IV in STEP 8 with carboxylic acid Compound 1 J, using conditions known by the person skilled in the art such as EDC1 and HOBt in a solvent like DMF in presence of a base such as DIPEA.

[0137] The preparation of compound I (in which X may be N, NH, O, S; R1is -H or -NH2, R2 and R3 are independently selected from a hydrogen atom or a (Cl-C4)alkyl group, or form together with the carbon atom to which they are attached a (C4-C6)cyloalkyl or 4 to 6-membered heterocycloakyl group, or R3 is absent, the dotted line in the piperazinone ring represents a saturated or unsaturated bond), maybe also effected according to the alternative following scheme, Scheme 4: STEP 1 STEP 2

[0138]

[0139] Compound I Compound 1H

[0140] SCHEME 4

[0141] According to Scheme 4 (in which X may be N, NH, O, S; R1is -H or -NH2, R2 and R3 are independently selected from a hydrogen atom or a (Cl -C4)alkyl group, or form together with the carbon atom to which they are attached a (C4-C6)cyloalkyl or 4 to 6-membered heterocycloakyl group, or R3 is absent, the dotted line in the piperazinone ring represents a saturated or unsaturated bond), Compound 1C can be transformed into compound 1 W in STEP 1 via a Suzuki coupling with compound 1G using, for example, a catalyst such as Pd(P(t-Bu)a)2, in a mixture of dioxane and water and in the presence of a base, such as potassium triphosphate, by heating to reflux. Compound IX can be obtained by nucleophilic aromatic substitution of compound 1W with compound ID under basic conditions in STEP 2. Compound 1H can be obtained from compound IX in STEP 3 using iron with ammonium chloride. Compound IL can be obtained by hydrogenation of compound 1H in STEP 4 using Pd / C and Pd(OH)2 with H2, for example. Compound 1 H can then be converted to Compound IM in STEP 5 by deprotection using TFA or HC1 in DCM for example. Compound I can then be prepared from Compound IM in STEP 6 with carboxylic acid Compound 1J, using conditions known by the person skilled in the art such as EDC1 and HOBt in a solvent like DMF in presence of a base such as DIPEA. STEP 6 may optionally further comprise subsequent oxidation of either the pyridine ring and the sulfur atom (when X = S), followed by a pyridinium-N-oxide deoxygenation step. Pharmaceutical compositions, uses

[0142] A further aspect provides a pharmaceutical composition comprising a compound of formula (I) and at least one pharmaceutically acceptable excipient or carrier.

[0143] The pharmaceutical compositions of the disclosure may be formulated for administration in solid or liquid form, e.g., using conventional carriers or excipients. Compositions may be adapted for, e.g., oral administration (e.g., as a solution, suspension, tablet, or capsule), parenteral administration (e.g., as a solution, dispersion, suspension, or emulsion, or as a dry powder for reconstitution), or topical application (e.g., as a cream, ointment, patch, or spray to be applied to the skin) using techniques known in the art.

[0144] Compounds of the present disclosure act as inhibitors of ERK5, which gives them utility in the treatment of ERK5 -associated disorders and conditions. In particular, compounds of the disclosure are useful in the treatment of cancers.

[0145] Viewed from this aspect, the disclosure provides a method of treatment comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the disclosure (e.g., a compound of Formula (I), (II), (Ila) or (III), or a pharmaceutically acceptable salt thereof). In a related aspect, the disclosure provides the use of a compound of the disclosure (e.g., a compound of Formula (I), (II), (Ila) or (III), or a pharmaceutically acceptable salt thereof) in the manufacture of a medicament. In a further related aspect, the disclosure provides a compound of the disclosure (e.g., a compound of Formula (I), (II), (Ila) or (III), or a pharmaceutically acceptable salt thereof) for use in therapy.

[0146] Compounds of the present disclosure are useful for treating or preventing: diseases or deleterious conditions in which ERK5, or a variant or mutant thereof, is known to play a role ; diseases or disorders associated with increased MAPK7 (i.e., ERK5 gene) expression and / or increased ERK5 activity; and diseases or disorders in which inhibition or antagonism of ERK5 activity is beneficial.

[0147] In one aspect, the present disclosure provides a method of treating or preventing a disease or disorder mediated by ERK5, or a disease or disorder in which ERK5 is implicated, in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound of the disclosure (e.g., a compound of Formula (I), (II), (Ila) or (III), or a pharmaceutically acceptable salt thereof). In a related aspect, the disclosure provides the use of a compound of the disclosure (e.g., a compoundof Formula (I), (II), (Ila) or (III), or a pharmaceutically acceptable salt thereof) in the manufacture of a medicament for the treatment or prevention of a disease or disorder mediated by ERK5, or a disease or disorder in which ERK5 is implicated. In a further related aspect, the disclosure provides a compound of the disclosure (e.g., a compound of Formula (I), (II), (Ila) or (III), or a pharmaceutically acceptable salt thereof) for use in the treatment or prevention of a disease or disorder mediated by ERK5, or a disease or disorder in which ERK5 is implicated.

[0148] In another aspect, the present disclosure provides a method of treating or preventing a disease or disorder associated with ERK5 (e.g., cancer) in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound of the disclosure (e.g., a compound of Formula (I), (II), (Ila) or (III), or a pharmaceutically acceptable salt thereof). In a related aspect, the disclosure provides the use of a compound of the disclosure (e.g., a compound of Formula (I), (II), (Ila) or (III), or a pharmaceutically acceptable salt thereof) in the manufacture of a medicament for the treatment or prevention of a disease or disorder associated with ERK5 (e.g., cancer). In a further related aspect, the disclosure provides a compound of the disclosure (e.g., a compound of Formula (I), (II), (Ila) or (III), or a pharmaceutically acceptable salt thereof) for use in the treatment or prevention of a disease or disorder associated with ERK5 (e.g., cancer).

[0149] In another aspect, the present disclosure provides a method of treating or preventing cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a compound of the disclosure (e.g., a compound of Formula (I), (II), (Ila) or (III), or a pharmaceutically acceptable salt thereof). In a related aspect, the disclosure provides the use of a compound of the disclosure (e.g., a compoundof Formula (I), (II), (Ila) or (III), or a pharmaceutically acceptable salt thereof) in the manufacture of a medicament for the treatment or prevention of cancer. In a further related aspect, the disclosure provides a compound of the disclosure (e.g., a compound of Formula (I), (II), (Ila) or (III), or a pharmaceutically acceptable salt thereof) for use in the treatment or prevention of cancer.

[0150] In embodiments, the compound reduces angiogenesis, reduces or prevents metastasis, reduces inflammation, blocks tumorigenesis (e.g., in part or completely), reduces evasion of growth suppression, reduces or inhibits growth of cancerous or pre-cancerous cells, supresses proliferation of cancerous or pre-cancerous cells, and / or reduces the survival of cancerous or pre-cancerous cells. In embodiments, the cancer is characterized by increased MAPK7 (i.e., ERK5 gene) expression and / or increased ERK5 activity. In embodiments, the cancer has elevated ERK5 activity. In embodiments, the cancer overexpresses ERK5. In embodiments, the cancer is characterised by MAPK7 genomic amplification and / or constitutively active ERK5 signalling. In embodiments, the cancer has genomically amplified ERK5. In embodiments, the cancer has constitutively active ERK5 signalling.

[0151] In embodiments, the cancer is a solid tumour (e.g., a melanoma, carcinoma, or blastoma). In other embodiments, the cancer is leukaemia (e.g., chronic lymphocytic leukaemia, CLL; acute myelogenous leukaemia, AML; or chronic myelogenous leukaemia, CML).

[0152] In embodiments, the cancer is a primary tumour. In other embodiments, the cancer is a secondary tumour (e.g., a metastatic tumour).

[0153] In embodiments, the cancer is selected from breast cancer (e.g., ductal breast carcinoma, or breast adenocarcinoma), liver cancer, kidney cancer (e.g., hepatocellular carcinoma), prostate cancer, colorectal cancer (CRC), lung cancer (e.g., non-small cell lung cancer, NSCLC; lung adenocarcinoma; or lung squamous cell carcinoma), pancreatic cancer (e.g., adenocarcinoma), ovarian cancer, brain cancer (e.g., glioblastoma), cervical cancer (e.g., adenocarcinoma), gastric cancer, skin cancer (e.g., melanoma), bile duct cancer (e.g., cholangiocarcinoma), nervous system cancer (e.g., neuroblastoma), and melanoma.

[0154] In embodiments, the cancer is selected from leukaemia (e.g., acute leukaemia, acute lymphocytic leukaemia, acute myelocytic leukaemia, acute myeloblastic leukaemia, acute promyelocytic leukaemia, acute myelomonocytic leukaemia, acute monocytic leukaemia, acute erythroleukemia, chronic leukaemia, chronic myelocytic leukaemia, or chronic lymphocytic leukaemia), polycythaemia vera, lymphoma (e.g., Hodgkin's disease or nonHodgkin's disease), Waldenstrom macroglobulinemia, and multiple myeloma.

[0155] In embodiments, the cancer is selected from leukaemia (e.g., chronic myeloid leukaemia), breast cancer, multiple myeloma, colon cancer, colorectal cancer, lung cancer, pancreatic cancer, renal cell carcinoma, mesothelioma, adenocarcinoma, neuroblastoma, melanoma, and hepatocellular carcinoma.

[0156] In embodiments, the cancer is selected from leukaemia (e.g., chronic myeloid leukaemia), breast cancer, multiple myeloma, colon cancer, renal cell carcinoma, mesothelioma, adenocarcinoma, neuroblastoma, and hepatocellular carcinoma. In another aspect, the disclosure provides a method of inhibiting ERK5 activity, the method comprising contacting ERK5 (e.g., a cell comprising ERK5) with a compound of the present disclosure (e.g., a compound of Formula (I) or a pharmaceutically acceptable salt thereof) . In embodiments, the method is an in vitro or ex vivo method. In other embodiments the method is an in vivo method. In a related aspect, the disclosure provides an in vitro method of inhibiting ERK5 activity in a cell, the method comprising contacting the cell with a compound of the present disclosure (e.g., a compound of Formula (I) or a pharmaceutically acceptable salt thereof).

[0157] The compounds of the disclosure may be used alone (e.g., as a monotherapy) or in combination with one or more cancer therapies.

[0158] Having been generally described herein, the follow non-limiting examples are provided to further illustrate this disclosure.

[0159] Although specific embodiments of the present disclosure will now be described with reference to the description and examples, it should be understood that such embodiments are by way of example only and merely illustrative of but a small number of the many possible specific embodiments which can represent applications of the principles of the present disclosure. Various changes and modifications will be obvious to those of skill in the art given the benefit of the present disclosure and are deemed to be within the spirit and scope of the present disclosure as further defined in the appended claims.

[0160] EXAMPLES

[0161] Examples 1 to 14 - Compounds

[0162] All synthetic reactions were performed under an inert atmosphere, unless otherwise stated. In the following examples, when the source of the starting products is not specified, it should be understood that said products are known compounds (e.g., commercially available compounds from suppliers such as Sigma- Aldrich).

[0163] Analytical data have been obtained as follows:

[0164] Proton NMR: 'H NMR Spectra at 400 and 500 MHz were performed on a Bruker Avance DRX-400and Bruker AvanceDPX-500 spectrometer, respectively, with the chemical shifts (5 in ppm) in the solvent dimethyl sulfoxide-de (DMSO-de) referenced at 2.5 ppm at the quoted temperatures. Coupling constants (J) are given in Hertz.

[0165] LC-MS: The liquid chromatography / mass spectra (LC / MS) were obtained using three different methods:

[0166] o Method A (examples 1 to 6): LC / MS were performed on a SHIMADZU 2020 LCMS-008 instrument, PDA detector and Mass spectra: MSD1 -Positive mode / MSD2-Negative mode using UV detection DAD 210<l<400 nm and column Acquity BEH C18 1.7 pm, dimension 2.1x50 mm, mobile phase H2O + 0.1% HCO2H / CH3CN + 0.1% HCO2H;

[0167] o Method B (examples 7 to 10): LC / MS were performed on a UPLC Acquity Waters instrument, light scattering detector Sedere and SQD Waters mass spectrometer using UV detection DAD 210<l<400 nm and column Acquity UPLC CSH Cl 8 1.7 pm, dimension 2.1x50 mm, mobile phase H2O + 0.1% HCO2H / CH3CN + 0.1% HCO2H;

[0168] o Method C (examples 11 to 14): LC / MS were performed on an Agilent instrument, light scattering detector MSD G6110A and DAD G1315D mass spectrometer with Mass spectra in ESI Positive mode 110 to 1000 amu, using UV detection DAD 210<l<400 nm and column XBridge C18, 3.5 pm, mobile phase H2O + 10 mM NH4HCO3 / CH3CN, UV detection 214, 4 nm.

[0169] Table 1 below lists the compounds of formula (I) synthesized as in the previous general schemes or in the following synthetic examples. Table 1:

[0170] Example No. Structure Name

[0171] 8-[l-[2-amino-4- (trifluoromethoxy)benzoyl]-4-piperidyl]-7- I. , - fluoro- 1 , 1 -dioxo-4H-pyrido [3 ,2- 1 b][l,4]thiazin-3-one

[0172] ’ Qvy ,, =.

[0173] F A 0,

[0174] 8-[l-[2-amino-4- (trifluoromethoxy)benzoyl]-4-piperidyl]-2- ethyl-7-fluoro-4H-pyrido[2,3-b]pyrazin-3- 2 1' Ov= one

[0175] [I .J

[0176] F A

[0177] *

[0178] 8-[l-[2-amino-4- (trifluoromethoxy)benzoyl]-4-piperidyl]-7- fluoro-2,2-dimethyl-4H-pyrido[3,2- 3 T ' ..-O b][l,4]thiazin-3-one OX

[0179] F* Fs

[0180] 8-[l-[2-amino-4- (trifluoromethoxy)benzoyl]-4-piperidyl]-7- f luoro - spiro [ 1 ,4 -dihydropyrido [2,3- b]pyrazine-2,l'-cyclobutane]-3-one 4

[0181]

[0182] Example No. Structure Name

[0183] 8-[l-[2-amino-4- (trifluoromethoxy)benzoyl]-4-piperidyl]-7- XY .... f luoro - spiro [ 1 ,4 -dihydropyrido [2,3- b]pyrazine-2, 1 '-cyclopentane] -3-one 5

[0184] X(“ 1 n -U -n .

[0185] X m — - / — ■. o r \

[0186] / ^1'ft; I--,

[0187] HHF ^ / \

[0188] 8-[l-[2-amino-4- X. j (trifluoromethoxy)benzoyl]-4-piperidyl]-7- f luoro - spiro [ 1 ,4 -dihydropyrido [2,3- 6 b]pyrazine-2, 1 '-cyclohexane] -3-one ; j

[0189] J! ,-A 7-fluoro-8-[l-[4- ■':A 1 J.«»» ■■■"> . - (trifluoromethoxy)benzoyl] -4- piperidyl] spiro [ 1 ,4-dihydropyrido [2,3- b]pyrazine-2,4'-tetrahydropyran]-3-one 7

[0190] ^■■■■' " .r

[0191] o

[0192] 8-[l-[2-amino-4- (trifluoromethoxy)benzoyl]-4-piperidyl]-7- f luoro - spiro [ 1 ,4-dihydropyrido [2,3- b]pyrazine-2,4'-tetrahydropyran]-3-one 8

[0193]

[0194] Example No. Structure Name

[0195] 7-fluoro-2,2-dimethyl-8-[l-[4- , -X 'is~ (trifluoromethoxy)benzoyl] -4-piperidyl]- l,4-dihydropyrido[2,3-b]pyrazin-3-one 9

[0196] Ci

[0197] T

[0198] F-. . :

[0199] / 3 )0=== F-J

[0200] / \ T) T|

[0201] o v / v Z j—-—

[0202] 8-[l-[2-amino-4- (trifluoromethoxy)benzoyl]-4-piperidyl]-7- ex'" 1-. fluoro-2,2-dimethyl-l,4-dihydropyrido[2,3- b]pyrazin-3-one

[0203] 10

[0204] 1 J

[0205] Fi. O

[0206] F

[0207] 6-fluoro-5-[l-[4- (trifluoromethoxy)benzoyl] -4-piperidyl]- 3,4-dihydro-lH-l,8-naphthyridin-2-one 11

[0208] Ht*xls8-[l-[2-amino-4- (trifluoromethoxy)benzoyl]-4-piperidyl]-7- oX fluoro-4H-pyrido[3,2-b][l,4]oxazin-3-one f: k. Al, .<□

[0209] 12

[0210] « . "*i 3

[0211]

[0212] Example No. Structure Name

[0213] 8-[l-[2-amino-4- (trifluoromethoxy)benzoyl]-4-piperidyl]-7- f luoro-2,4-dihydro - 1 H-pyrido [2,3 - b]pyrazin-3-one

[0214] F k.

[0215] 13

[0216] VI " "-a ( I

[0217] I z,

[0218] \ / O

[0219] 8-[l-[2-amino-4- (trifluoromethoxy)benzoyl]-4-piperidyl]-7- fluoro-4H-pyrido[3,2-b][l,4]thiazin-3-one 14

[0220]

[0221] Examples 1 & 14: 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-l,l-dioxo-4H-pyrido[3,2-b][l,4]thiazin-3-one & 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-4H-pyrido[3,2-b][l,4]thiazin-3-one

[0222]

[0223] METHOD A (STEPS 1 to 5 of Scheme 1, then STEPS 1 to 3 of Scheme 2)

[0224] STEP 1: 4-chloro-3,5-difluoro-pyridin-2-amine

[0225] ci

[0226] Fx X zF

[0227]

[0228] X NI^NH2

[0229] To a mixture of 3,5-difluoropyridin-2-amine (10 g, 76.9 mmol) in THF (200 mL) was added n-butyllithium (2.5 Msolution in hexane, 61.5 mL, 653.3 mmol) at -78 °C and the resulting mixture stirred for 1 hour under nitrogen atmosphere. Then, 1,1,1,2,2,2-hexachloroethane (36.4 g, 153.7 mmol) was added and the reaction mixture was stirred for 6 hours at -78 °C. After 6 hours, a saturated aqueous ammonium chloride solution was added carefully to quench the reaction and the mixture was diluted with water (200 mL) and extracted with ethyl acetate (3x100 mL). The combined organic layers were washed with brine (2x150 mL), dried over sodium sulfate, filtered and concentrated to dryness. The resulting residue was purified by flash chromatography on silica gel eluting with 0 to 12% of ethyl acetate in petroleum ether, to give 11 g (87% yield) of 4-chloro-3,5-difluoro-pyridin-2-amine as a yellow solid. LC / MS (m / z, M+H): calc. 165.0, found 165.2.

[0230] STEP 2: 4-chloro-3,5-difluoro-2-nitro-pyridine

[0231] ci

[0232]

[0233] A mixture of potassium persulfate (90.6 g, 334 mmol) in sulfuric acid (94 mL) was stirred for 30 minutes, then, 4-chloro-3,5-difhioro-pyridin-2-amine (11g, 0.06685 mol) was added at 40 °C and the resulting mixture was stirred for 2 hours. Another mixture of potassium persulfate (45.4 g, 167 mmol) in sulfuric acid (46 mL) was prepared, stirred for 30 minutes and merged with the first one. The whole mixture was stirred for 2 additional hours. The mixture was then diluted with iced water (200 mL) and extracted with ethyl acetate (3x100 mL). The combined organic layers were washed with brine (2x50 mL), dried over sodium sulfate, filtered, concentrated to dryness. The resulting residue was purified by flash chromatography on silica gel eluting with 0 to 5% of ethyl acetate in petroleum ether to give 2.8 g (21% yield) of 4-chloro-3,5-difluoro-2-nitro-pyridineas a yellow oil. LC / MS (m / z, M+H): calc. 195.0, found 195.7.

[0234] STEP 3: ethyl 2-[(4-chloro-5-fluoro-2-nitro-3-pyridyl)sulfanyl]acetate

[0235]

[0236] To a solution of 4-chloro-3,5-difluoro-2-nitro-pyridine (3 g, 15.4 mmol) in DCM (50 mL) were added ethyl 2-sulfanylacetate (1.85 g, 15.4 mmol) and triethylamine (4.67 g, 46.3 mmol) and the resulting reaction mixture was stirred at 0 °C for 2 hours. Then, the reaction mixture was concentrated and the residue was purified by flash chromatography on silica gel to give 1.5 g (33% yield) of ethyl 2-[(4-chloro-5-fluoro-2-nitro-3-pyridyl)sulfanyl]acetate. LC / MS (m / z, M+H): calc. 295.0, found 295.0. STEP 4: 8-chloro-7-fluoro-4H-pyrido[3,2-b][l,4]thiazin-3-one

[0237]

[0238] To a mixture of ethyl 2-[(4-chloro-5-fluoro-2-nitro-3-pyridyl)sulfanyl]acetate (1.5 g, 5.09 mmol) in water (8 mL) and methanol (40 mL) were added iron (1.42 g, 25.5 mmol) and ammonium chloride (1.36 g, 25.5 mmol). The resulting mixture was stirred at 60 °C for 2 hours under nitrogen atmosphere. The mixture was then diluted with water (30 mL) and extracted with dichloromethane (3x40 mL). The combined organic layers were washed with brine (2x20 mL), dried over sodium sulfate, filtered and concentrated. The resulting residue was purified by flash chromatography on silica gel eluting with 0 to 50% of ethyl acetate in petroleum ether to give 800 mg (72% yield) of 8-chloro-7-fluoro-4H-pyrido[3,2-b][l,4]thiazin-3-one. LC / MS (m / z, M+H): calc. 219.0, found 219.1.

[0239] STEP 5: tert-butyl 4-(7-fluoro-3-oxo-4H-pyrido[3,2-b][l,4]thiazin-8-yl)-3,6-dihydro-2H-pyridine- 1 -carboxylate

[0240]

[0241] To a mixture of 8-chloro-7-fluoro-4H-pyrido[3,2-b][l,4]thiazin-3-one (800 mg, 3.66 mmol) in 1,4-dioxane (10 mL) and water (2 mL), were added tert-butyl 4-(4, 4, 5, 5-tetramethyl- 1,3,2-dioxaborolan-2-yl)-3,6-dihydro-2H-pyridine-l -carboxylate (1.13 g, 3.66 mmol), palladium bis(tri-tert-butylphosphine) (93.5 mg, 0.18m mol) and potassium triphosphate (2.33 g, 11 mmol) and the resulting reaction mixture was stirred at 100 °C for 16 hours under argon atmosphere. Then, the reaction mixture was diluted with water (50 mL) and extracted with ethyl acetate (3x30 mL). The combined organic layers were washed with brine (2x30 mL), dried over sodium sulfate, filtered and concentrated to dryness. The resulting residue was purified by flash chromatography on silica gel eluting with 0 to 40% of ethyl acetate in petroleum ether, to give 700 mg (52% yield) of tert-butyl 4-(7-fluoro-3-oxo-4H-pyrido[3,2-b][l,4]thiazin-8-yl)-3,6-dihydro-2H-pyridine-l-carboxylate. LC / MS (m / z, M+H): calc. 366.1, found 366.3. STEP 6: tert-butyl 4-(7-fluoro-3-oxo-4H-pyrido[3,2-b][l,4]thiazin-8-yl)piperidine-l-carboxylate

[0242]

[0243] To a mixture of tert-butyl 4-(7-fluoro-3-oxo-4H-pyrido[3, 2-b] [1 , 4]thiazin-8-yl)-3, 6-dihydro-2H-pyridine-l -carboxylate (700 mg, 1.92 mmol) in methanol (50 mL) was added 5% Pd / C (1 g,) and the resulting reaction mixture was stirred at room temperature for 24 hours under one atmosphere of hydrogen. Then, the reaction mixture was filtered, concentrated and the resulting residue was purified by flash chromatography on silica gel eluting with 0 to 50% of ethyl acetate in petroleum ether to give 700 mg (99% yield) of crude tert-butyl 4-(7-fluoro-3-oxo-4H-pyrido[3,2-b][l,4]thiazin-8-yl)piperidine-l-carboxylate as a white solid. LC / MS (m / z, M+H): calc. 368.1, found 368.0.

[0244] STEP 7: 7-fluoro-8-(4-piperidyl)-4H-pyrido[3,2-b][l,4]thiazin-3-one;2,2,2-trifluoroacetic acid

[0245]

[0246] A mixture of tert-butyl 4-(7-fluoro-3-oxo-4H-pyrido[3,2-b][l,4]thiazin-8-yl)piperidine-l-carboxylate (700 g, 1.91 mmol) in trifluoroacetic acid (4 mL) and DCM (2 mL) was stirred at room temperature for 2 hours. Then, the mixture was concentrated to dryness to give 500 mg (68% yield) of the crude product as a yellow oil which was engaged in the next step without further purification. LC / MS (m / z, M+H - TFA): calc. 268.1, found 268.2.

[0247] STEP 8 (example 14): 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-4H-pyrido[3,2-b][l,4]thiazin-3-one

[0248]

[0249] To a solution of 2-amino-4-(trifluoromethoxy)benzoic acid (110 mg, 0.5 mmol) and 7-fluoro-8-(4-piperidyl)-4H-pyrido[3,2-b][l,4]thiazin-3-one;2,2,2-trifluoroacetic acid (133 mg, 0.35 mol) in DMF (5 mL) were added 1 -hydro xybenzotriazole;hydrate (114 mg, 0.75 mmol), N'-(ethylcarbonimidoyl)-N,N-dimethylpropane-l,3-diamine hydrochloride (143 mg, 0.75 mmol) and DIPEA (642 mg, 4.97 mmol). The resulting mixture was stirred for 16 hours at room temperature. Then, the reaction mixture was diluted with water (50 mL) and extracted with EA (3x20 mL). The combined organic layers were concentrated and the resulting residue was dissolved in a mixed solution of methanol (10 mL) and aqueous ammonia purified (15 mL) and stirred for 1 hour. The resulting solution was diluted with water and extracted with EA. The combined organic layers were washed with water, brine, dried over sodium sulfate, filtered and concentrated. The resulting residue was purified by flash chromatography on silica gel eluting with 3% methanol in dichloromethane to give 18 mg (11% yield) of 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-4H-pyrido[3,2-b][l,4]thiazin-3-one. iH NMR (400 MHz, DMSO-76) 6 ppm 8.12 (d, 7 = 2.4 Hz, 1H), 7.11 (d, 7= 8.4 Hz, 1H), 6.66 (d, 7= 1.2 Hz, 1H), 6.51-6.49 (m, 1H), 5.60 (s, 2H), 4.89-3.68 (m, 2H), 3.57 (s, 2H), 3.27-3.21 (m, 1H), 3.03-2.99 (m, 2H), 1.94-1.89 (m, 2H), 1.76-1.73 (m, 2H). LC / MS (m / z, M+H): calc. 471.1, found 471.0.

[0250] STEP 9: 8-[ 1 -[2-amino-4-(trifluoromethoxy)benzoyl] -4-piperidyl]-7 -fluoro-5 -oxido- 1 , 1 -dioxo-4H-pyrido[3,2-b][l,4]thiazin-5-ium-3-one

[0251]

[0252] To a stirred solution of 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-4H-pyrido[3,2-b][l,4]thiazin-3-one (140 mg, 0.3 mmol) in DCM (3 mL), was added 3-chlorobenzenecarboperoxoic acid (205 mg, 1.19 mmol), and the resulting reaction mixture was stirred for 2 hours at room temperature. After completion, the reaction mixture was quenched with an aqueous saturated solution of NaHCOa and extracted with DCM (2x10 mL). The combined organic layers were dried over NaaSO^ filtered and concentrated under vacuum to give 160 mg (purity 43%, 45% yield) of 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl] -4-piperidyl]-7-fluoro-5-oxido-l,l-dioxo-4H-pyrido[3,2-b][l,4]thiazin-5-ium-3-one as an off white solid which was engaged in the next step without further purification.

[0253] STEP 10 (example 1): 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro- 4H-pyrido[3,2-b][l,4]thiazin-3-one

[0254]

[0255] To a stirred solution of 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-5-oxido-l,l-dioxo-4H-pyrido[3,2-b][l,4]thiazin-5-ium-3-one (purity 43%, 150mg, 0.12 mmol) in DMF (3 mL), was added tetrahydrodiboron (31 mg, 0.35 mmol) and the resulting reaction mixture was stirred for 30 minutes at room temperature. After completion of the reaction, the reaction mixture was filtered through celite bed, washed with ethanol and concentrated to dryness. The resulting residue was purifiedby preparative HPLC, to give 3 mg (5% yield) of 8-[l - [2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7 -fluoro- 1 ,1 -dioxo-4H-pyrido[3,2-b][l,4]thiazin-3-oneas an off white solid. ’H NMR (400 MHz, DMSO-t / 6) 5 ppm 11.74 (s, 1 H), 8.62 (d, 7= 30.0 Hz, 1 H), 7.12 (d, 7 = 8.4 Hz, 1 H), 6.65 (s, 1 H), 6.49 (d, 7= 8.4 Hz, 1 H), 5.61 (d, 7= 10.8 Hz, 2 H), 4.95 (s, 1 H), 4.07 (s, 1 H), 2.97 (s, 2 H), 2.05 (s, 2 H), 1.95 (d, 7= 13.2 Hz, 2 H), 1.80 (d, 7= 11.6 Hz, 2 H). LC / MS (m / z, M+H): calc. 503.1, found 503.1.

[0256] Example 2: 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-2-ethyl-7-fluoro-4H-pyrido[2,3-b]pyrazin-3-one

[0257]

[0258] METHOD B (Scheme 1)

[0259] STEP 1: ethyl l-[(4-chloro-5-fluoro-2-nitro-3-pyridyl)amino]cyclopropanecarboxylateClu O

[0260] F. X A

[0261]

[0262] STEP 1 of example 2 was performed following the protocol described in STEP 3 of examples 1 & 14 using ethyl 1 -aminocyclo propan ecarboxylate (1330 mg, 10.3 mmol), to give 1100 mg (70% yield) of ethyl l-[(4-chloro-5-fhioro-2-nitro-3-pyridyl)amino]cyclopropanecarboxylate as a white solid. LC / MS (m / z, M+H): calc. 304.0, found 303.9.

[0263] STEP 2: 8-chloro-7-fluoro-spiro[l,4-dihydropyrido[2,3-b]pyrazine-2,T-cyclopropane]-3-one

[0264]

[0265] STEP 2 of example 2 was performed following the protocol described in STEP 4 of examples 1 & 14, to give 500 mg (89% yield) of 8-chloro-7-fluoro-spiro[l,4-dihydropyrido[2,3-b]pyrazine-2,l'-cyclopropane]-3-one as a yellow solid. LC / MS (m / z, M+H): calc. 228.0, found 227.9.

[0266] STEP 3 : tert-butyl 4-(7 -fluoro-3-oxo-spiro[ 1 ,4-dihydropyrido [2,3-b]pyrazine-2, 1 cyclopropane] -8-yl)-3,6-dihydro-2H-pyridine-l -carboxylate

[0267]

[0268] STEP 3 of example 2 was performed following the protocol described in STEP 5 of examples 1 & 14, to give 170 mg (50% purity, 10% yield) of tert-butyl4-(7-fluoro-3-oxo-spiro[l,4-dihydropyrido[2,3-b]pyrazine-2,T-cyclopropane]-8-yl)-3,6-dihydro-2H-pyridine-l-carboxylate as a brown solid. LC / MS (m / z, M+H): calc. 375.2, found 375.1.

[0269] STEP 4: 7-fluoro-8-( 1,2,3, 6-tetrahydropyridin-4-yl)spiro[l,4-dihydropyrido [2,3 -b]pyrazine-2,l'-cyclopropane]-3-one hydrochloride

[0270]

[0271] STEP 4 of example 2 was performed following the protocol described in STEP 7 of examples 1 & 14, to give 150 mg (70% purity, 100% yield) of 7-fluoro-8-(l,2,3,6-tetrahydropyridin-4-yl)spiro[l,4-dihydropyrido[2,3-b]pyrazine-2,l'-cyclopropane]-3-one hydrochloride as a brown solid which was engaged in the next step without further purification .

[0272] STEP 5: 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-3,6-dihydro-2H-pyridin-4-yl]-7-fluoro-spiro[l,4-dihydropyrido[2,3-b]pyrazine-2,r-cyclopropane]-3-one

[0273]

[0274] To a stirred solution of 7-fluoro-8-(l,2,3,6-tetrahydropyridin-4-yl)spiro[l,4-dihydropyrido [2,3 -b]pyrazine-2,l'-cyclopro pane] -3-one hydrochloride (0.150g (70% purity), 0.33 mmol) in DMF (2 mL), were added DIPEA (165 mg, 1.63 mmol) and 2-amino-4-(trifluoromethoxy)benzoic acid (120 mg, 0.54 mmol) at 0 °C followed by TBTU (209 mg, 0.65 mmol). Then, the reaction mixture was stirred at room temperature for 1 hour, diluted with water (10 mL), extracted with EtOAc (2 x 20 mL), and the combined organic layers were dried overNa2SO4 and concentrated under vacuum. The resulting residue was purified by flash chromatography on silica gel eluting with 5% of MeOH in DCM, to give 28 mg (18% yield) of 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-3,6-dihydro-2H-pyridin-4-yl]-7-fluoro-spiro[l,4-dihydropyrido[2,3-b]pyrazine-2,l'-cyclopropane]-3-one as a light brown solid. LC / MS (m / z, M+H): calc. 478.1, found 478.1.

[0275] STEP 6: 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-2-ethyl-7-fluoro-4H-pyrido[2,3-b]pyrazin-3-one

[0276]

[0277] To a stirred solution of 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-3,6-dihydro-2H-pyridin-4-yl]-7-fluoro-spiro[l,4-dihydropyrido[2,3-b]pyrazine-2,r-cyclopropane]-3-one (28 mg, 0.06 mmol) in EtOH / MeOH (1 mL / 1 mL) was added 10% Pd / C (12.5 mg, 0.12 mmol) under an inert atmosphere and then the reaction mixture was stirred overnight under one atmosphere of hydrogen atroom temperature for 16 hours. Then, the reaction mixture was filtered through celite bed, washed with ethanol (20 mL) and concentrated under reduced pressure to dryness. The resulting residue was purified by preparativ HPLC (Mobile Phase: A= 0.1% HCOOH in water, B= MeCN, Column: Gemini NX (250 mm x 21.2 mm), Flow rate : 18 mL / min), to give 7 mg (25% yield) of 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-2-ethyl-7-fluoro-4H-pyrido[2,3-b]pyrazin-3-one as an off white solid.JH NMR (300 MHz, DMSO-d6) 5 ppm 8.48 (s, 1 H), 7.12-7.10 (d, 1 H), 6.66 (s, 1 H), 6.51-6.49 (s, 1 H), 5.60 (s, 2 H), 3.92-3.86 (t, 3 H), 3.07 (bs, 2 H), 2.87-2.82 (q, 2 H), 2.25-2.22 (m, 2 H), 1.75-1.73 (d, 2 H), 1.26-1.23 (t, 3 H). LC / MS (m / z, M+H): calc. 480.2, found 480.1.

[0278] Example 3 : 8 - [ 1 - [2-amino-4-(trif luoromethoxy)benzoyl] -4-piperidyl] -7-fluoro-2,2-dimethyl-4H-pyrido[3,2-b][l,4]thiazin-3-one

[0279]

[0280] Example 3 was prepared following Method B using methyl 2-methyl-2-sulfanyl-propanoate (1 g, 7.45 mmol) in STEP 1, to give 90 mg (30% yield) of 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-2,2-dimethyl-4H-pyrido[3,2-b][l,4]thiazin-3-one as a white solid. ’H NMR (400 MHz, DMSO-A) 5 ppm 11.05 (s, 1 H), 8.14 (d, J = 2.4 Hz, 1 H), 7.10 (d, 7=8.4 Hz, 1 H), 6.66 (q, J= 1.0 Hz, 1 H), 6.52 - 6.49 (m, 1 H), 5.60 (s, 2 H), 4.18 (s, 2 H), 3.22 (s, 1 H), 3.00 (s, 2 H), 1.95 (q, 7= 11.0 Hz, 2 H), 1.73 (d, 7 = 12.4 Hz, 2 H), 1.38 (d, 7= 10.8 Hz, 6 H). LC / MS (m / z, M+H): calc. 499.1, found 499.2. Example 4: 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-spiro[l,4-dihydropyrido[2,3-b]pyrazine-2,l'-cyclobutane]-3-one

[0281]

[0282] Example 4 was prepared following Method B using ethyl 1 -aminocyclobutanecarboxylate (1.47 g, 10.3 mmol) in STEP 1, and sodium dithionite (1.64 g, 9.44 mmol) in 1,4-dioxane / water at 90 °C for 4 hours in STEP 2 instead of iron and ammonium chloride, to give in the last step, 60 mg (15% yield) of 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-spiro[l,4-dihydropyrido[2,3-b]pyrazine-2,r-cyclobutane]-3-one as a white solid. ’H NMR (400 MHz, DMSO-t / 6) 6 ppm 10.66 (s, 1 H), 7.50 (d, 7=2.64 Hz, 1 H), 7.07-7.16 (m, 1 H), 6.62-6.72 (m, 1 H), 6.49-6.60 (m, 1 H), 6.31-6.40 (m, 1 H), 5.49-5.72 (m, 2 H), 3.35-3.44 (m, 2 H), 3.14-3.20 (m, 1 H), 2.95-3.14 (m, 2 H), 2.40-2.47 (m, 2 H), 2.16-2.26 (m, 2 H), 1.75-1.91 (m, 2 H), 1.64-1.71 (m, 2 H), 1.06-1.12 (m, 2 H). LC / MS (m / z, M+H): calc. 494.2, found 494.3.

[0283] Example 5: 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-spiro[l,4-dihydropyrido[2,3-b]pyrazine-2,l'-cyclopentane]-3-one

[0284]

[0285] Example 5 was prepared following Method A using ethyl 1 -aminocyclopentanecarboxylate (1.21 g,7.71 mmol)) in STEP 3, sodium dithionite (1.57 g, 9.04 mmol) in 1,4-dioxane / water at 90 °C for 4 hours in STEP 4 instead of iron and ammonium chloride, and XphosPdG2 as Pd catalyst in STEP 5, to give in the last step, 150 mg (30% yield) of 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-spiro[l,4-dihydropyrido[2,3-b]pyrazine-2,l'-cyclopentane]-3-one as a white solid. ’H NMR (400 MHz, DMSO-t / 6) 6 ppm 10.84-10.58 (m, 1 H), 7.60-7.46 (m, 1 H), 7.18-7. 04 (m, 1 H), 6.75-6.61 (m, 1 H), 6.57-6.42 (m, 1 H), 5.95-5.85 (m, 1 H), 5.69-5.54(m, 2 H), 4.39-3.65 (m, 2 H), 3.16-2.89 (m, 2 H), 2.10-1.94 (m, 2 H), 1.95-1.72 (m, 5 H), 1.68-1.49 (m, 6 H). LC / MS (m / z, M+H): calc. 508.2, found 508.1.

[0286] Example 6: 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-spiro[l,4-dihydropyrido[2,3-b]pyrazine-2,l'-cyclohexane]-3-one

[0287]

[0288] Example 6 was prepared following Method A using ethyl 1 -aminocyclohexanecarboxylate (2.14 g, 10.3 mmol) in STEP 3, sodium dithionite (1.51 g, 8.68 mmol) in 1,4-dioxane / water at 90 °C for 4 hours in STEP 4 instead of iron and ammonium chloride, and XphosPdG2 as Pd catalyst in STEP 5, to give in the las step, 51 mg (16% yield) of 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-spiro[l,4-dihydropyrido[2,3-b]pyrazine-2,l'-cyclohexane]-3-one as a white solid. ’H NMR (400 MHz, DMSO-t e) 6 ppm 10.61 (s, 1 H), 7.47-7.67 (m, 1 H), 6.99-7.16 (m, 1 H), 6.60-6.70 (m, 1 H), 6.37-6.55 (m, 1 H), 5.45-5.64 (m, 3 H), 3.69-4.49 (m, 2 H), 3.66-3.54 (m, 1 H), 3.12-2.98 (m, 2 H), 1.80-1.95 (m, 2 H), 1.62-1.78 (m, 4 H), 1.54-1.62(m, 4 H), 1.43-1.53 (m, 3 H), 1.27-1.40(m, 1 H). LC / MS (m / z, M+H): calc. 522.2, found 522.1.

[0289] Example 7 : 7 -f luoro-8 -[ 1 -[4-(trif luoromethoxy)benzoyl] -4-piperidyl] spiro [1,4-dihydropyrido[2,3-b]pyrazine-2,4'-tetrahydropyran]-3-one

[0290]

[0291] Example 7 was prepared following Method A using ethyl 4-aminotetrahydropyran-4-carboxylate hydrochloride (929 mg, 4.43 mmol) with DIPEA as a base and DMSO as solvent at room temperature in STEP 3, and XphosPdG4 as Pd catalyst in STEP 5, HC14M in 1,4-dioxane instead of TFA in STEP 7, and 4-(trifluoromethoxy)benzoic acid (62 mg, 0.29 mmol) in STEP 8, to give in the last step, 65 mg (69% yield) of 7-fluoro-8-[l-[4-(trifluoromethoxy)benzoyl] -4-piperidyl]spiro[l,4-dihydropyrido[2,3-b]pyrazine-2,4'-tetrahydropyran] -3-one as a white solid. 'H NMR (400 MHz, DMSO-t e, 100°C) 5 ppm 1.55 (dt, 7=13.5, 5.0 Hz, 2 H), 1.71 (br d, 7=13.1 Hz, 2 H), 1.86-2.01 (m, 4 H), 3.08 (br t, 7=12.5 Hz, 2 H), 3.33-3.48 (m, 1 H), 3.74 (t, 7=5.4 Hz, 4 H), 4.08-4.34 (m, 2 H), 5.44 (s, 1 H), 7.39 (brd, 7=8.5 Hz, 2 H), 7.53 (brd, 7=8.5 Hz, 2 H), 7.59 (d, 7=2.5 Hz, 1 H), 10.21-10.31 (m, 1 H). LC / MS (m / z, M+H): calc. 509.2, found 509.4.

[0292] Example 8: 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-spiro[l,4-dihydropyrido[2,3-b]pyrazine-2,4'-tetrahydropyran]-3-one

[0293]

[0294] Example 8 was prepared following Method A using ethyl 4-aminotetrahydropyran-4-carboxylate (929 mg, 4.43 mmol) with DIPEA as a base and DMSO as solvent at room temperature in STEP 3, and XphosPdG4 as Pd catalyst in STEP 5, HC14M in 1,4-dioxane instead of TFA in STEP 7, to give 75 mg (61% yield) of 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-spiro[l,4-dihydropyrido[2,3-b]pyrazine-2,4'-tetrahydropyran]-3-one as a white solid. 'H NMR (400 MHz, DMSO-6?6, 100°C) 5 ppm 1.50- 1.62 (m, 2 H), 1.73 (br d, 7=11.4 Hz, 2 H), 1.88-2.02 (m, 4 H), 3.10 (br t, 7=12.8 Hz, 2 H), 3.31-3.44 (m, 1 H), 3.77 (t, 7=5.4 Hz, 4 H), 4.18 (brd, 7=12.8 Hz, 2 H), 5.35 (br s, 2 H), 5.45 (s, 1 H), 6.52 (brd, 7=8.4 Hz, 1 H), 6.71 (br s, 1 H), 7.13 (d, 7=8.4 Hz, 1 H), 7.61 (d, 7=2.5 Hz, 1 H), 10.17-10.33 (m, 1 H). LC / MS (m / z, M+H): calc. 524.2, found 524.4.

[0295] Example 9: 7-fluoro-2,2-dimethyl-8-[l-[4-(trifluoromethoxy)benzoyl]-4-piperidyl]-l,4-dihydropyrido [2,3 -b]pyrazin-3 -one

[0296]

[0297] Example 9 was prepared following Method A using ethyl 2-amino-2-methyl-propanoate (646 mg, 3.85 mmol) with DIPEA as a base and DMSO as solvent at room temperature in STEP 3, HC14M in 1,4-dioxane instead of TFA in STEP 7, and 4-(trifluoromethoxy)benzoic acid (50 mg, 0.24 mmol) with TATU (92 mg, 0.28 mmol) instead of TBTU in STEP 8, to give 78 mg (70% yield) of 7-fluoro-2,2-dimethyl-8-[l-[4-(trifluoromethoxy)benzoyl]-4-piperidyl]-l,4-dihydropyrido[2,3-b]pyrazin-3-one as a white solid.JH NMR (400 MHz, DMSO-6?6, 100°C) 5 ppm 1.31 (s, 6 H), 1.69 (br d, 7=12.3 Hz, 2 H), 1.83-1.98 (m, 2 H), 3.07 (br t, 7=12.6 Hz, 2 H), 3.21-3,38 (m, 1 H), 4.02-4.27 (m, 2 H), 5.57 (s, 1 H), 7.39 (br d, 7=8.4 Hz, 2 H), 7.49 (d, 7=2.6 Hz, 1 H),7.53 (br d, 7=8.4 Hz, 2 H), 10.04-10.18 (m, 1 H). LC / MS (m / z, M+H): calc. 467.2, found 467.4.

[0298] Example 10: 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-2,2-dimethyl-l,4-dihydropyrido[2,3-b]pyrazin-3-one

[0299]

[0300] Example 10 was prepared following Method A using ethyl 2-amino-2-methyl-propanoate (646 mg, 3.85 mmol) with DIPEA as a base and DMSO as solvent at room temperature in STEP 3, HC1 4M in 1,4-dioxane instead of TFA in STEP 7, and 2-amino-4-(trifluoromethoxy)benzoic acid (50 mg, 0.23 mmol) with TATU (86 mg, 0.27 mmol) instead of TBTU in STEP 8, to give 75 mg (70% yield) of 7-fluoro-2,2-dimethyl-8-[l-[4-(trifluoromethoxy)benzoyl]-4-piperidyl]-l,4-dihydropyrido[2,3-b]pyrazin-3-one as a white solid. >HNMR (400 MHz, DMSO-76, 100°C) 5 ppm 1.31 (s, 6 H), 1.68 (br d, 7=11.8 Hz, 2 H), 1.82-2.00 (m, 2 H), 3.05 (brt, 7=12.7 Hz, 2 H), 3.20-3.31 (m, 1 H), 4.13 (brd, 7=13.1 Hz, 2 H), 5.33 (br s, 2 H), 5.53 (s, 1 H), 6.49 (brd, 7=8.4 Hz, 1 H), 6.68 (br s, 1 H), 7.10 (d, 7=8.4 Hz, 1 H), 7.48 (d, 7=2.6 Hz, 1 H), 10.03-10.19 (m, 1 H). LC / MS (m / z, M+H): calc. 482.2, found 482.3.

[0301] Example 11: 6-fluoro-5-[l-[4-(trifluoromethoxy)benzoyl]-4-piperidyl]-3,4-dihydro-lH-l,8-naphthyridin-2-one

[0302]

[0303] METHOD C (Scheme 3) STEP 1: tert-butyl N-(3-bromo-4-chloro-5-fluoro-2-pyridyl)-N-tert-butoxycarbonyl-carbamate

[0304]

[0305] To a solution of 3-bromo-5-fluoro-pyridin-2-amine (15 g, 78.5 mmol) in DCM (300 mL) were added N,N-dimethylpyridin-4-amine (21.1 g, 173 mmol) and tert-butoxycarbonyl tertbutylcarbonate (37.7 g, 173 mmol). The resulting mixture was stirred at 40 °C for 16 hours. The reaction mixture was then concentrated, treated with water (200 mL) and extracted with ethyl acetate (3x100 mL). The combined organic layers were washed with water, brine, dried over sodium sulfate and concentrated to dryness. The resulting residue was purified by falsh chromatography on silica gel eluting with 0-15% of EA in PE to give 26.2 g (84% yield) of tert-butyl N-(3-bromo-5-fluoro-2-pyridyl)-N-tert-butoxycarbonyl-carbamate. LC / MS (m / z, M+H - BOC-t-Bu): calc. 236.0, found 236.9.

[0306] STEP 2: tert-butyl N-(3-bromo-4-chloro-5-fluoro-2-pyridyl)-N-tert-butoxycarbonyl-carbamate

[0307]

[0308] To a stirred solution of tert-butyl N-(3-bromo-5-fluoro-2-pyridyl)-N-tert-butoxycarbonyl-carbamate (10 g, 25.4 mmol) in anhydrous tetrahydrofuran (120 mL) was added LDA (2 mol / L in THF, 19 mL, 38 mmol) dropwise under nitrogen atmosphere at -78 °C and the resulting reaction mixture was stirred for 1.5 hours at -78 °C. Then, a solution of 1, 1,1, 2,2,2-hexachloroethane (15 g, 63.4 mmol) in anhydrous tetrahydrofuran (30 mL) was added and the whole mixture was stirred for 0.5 hour at -78 °C, then slowly warmed to room temperature and stirred for 3 additional hours. The reaction was quenched with a saturated solution of ammonium chloride (200 mL) and extracted with ethyl acetate (3x100 mL). The combined organic layers were washed with brine (100 mL), dried over sodium sulfate and concentrated to dryness. The resulting residue was purified by flash chromatography on silica gel eluting with 0-10% of EA in PE to give 5.7 g (48% yield) of tert-butyl N-(3-bromo-4- chloro-5-fluoro-2-pyridyl)-N-tert-butoxycarbonyl-carbamate as a white solid. LC / MS (m / z, M+H - BOC-t-Bu): calc. 270.4, found 270.9.

[0309] STEP 3: ethyl (E)-3-[2-[bis(tert-butoxycarbonyl)amino]-4-chloro-5-fluoro-3-pyridyl]prop-2-enoate

[0310]

[0311] To a solution of tert-butyl N-(3-bromo-4-chloro-5-fluoro-2-pyridyl)-N-tert-butoxycarbonyl-carbamate (6.64 g, 14.3 mmol) and ethyl (E)-3-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)prop-2-enoate (3.55 g, 15.7 mmol) in a mixture of 1,4-dioxane (10 mL) and whate (100 mL), were added sequentially [l,l'-bis(diphenylphosphino)ferrocene]dicholoropalladium(II) (1.05 g, 1.43 mmol), sodium carbonate (4.54 g, 42.9 mmol). The resulting reaction mixture was refluxed for 6 hours under nitrogen atmosphere. Then, the reaction mixture was diluted with water (100 mL) and extracted with ethyl acetate (3x100 mL). The combined organic layers were washed with brine (100 mL), dried over sodium sulfate and concentrated to dryness. The resulting residue was purified by flash chromatography on silica gel eluting with 0-15% of EA in PE, to give 4.23 g (57% purity, 38% yield) of ethyl (E)-3-[2-[bis(tert-butoxycarbonyl)amino]-4-chloro-5-fluoro-3-pyridyl]prop-2-enoate as a yellow solid. LC / MS (m / z, M+- BOC-t-Bu): calc. 288.6, found 289.0.

[0312] STEP 4: tert-butyl 4-[2-[bis(tert-butoxycarbonyl)amino]-3-[(E)-3-ethoxy-3-oxo-prop-l-eny l]-5-fluoro -4-pyridyl] -3, 6-dihydro-2H-pyridine-l -carboxylate

[0313]

[0314] To a solution of ethyl (E)-3-[2-[bis(tert-butoxycarbonyl)amino]-4-chloro-5-fluoro-3-pyridyl]prop-2-enoate (4.23 g, 5.47 mmol) and tert-butyl 4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-3,6-dihydro-2H-pyridine-l-carboxylate(2.03 g, 6.56 mmol) in a mixture of 1,4-dioxane (50 mL) and water (10 mL), were added sequentially palladium bis(tri-tert-butylphosphine) (280 mg, 0.55 mol), potassium triphosphate (3.48 g, 16.4 mmol). The resulting reaction mixture was refluxed for 5 hours under argon atmosphere. Then, the reaction mixture was treated with water (50 mL) and extracted with EA (3x50 mL). The combined organic layers were washed with brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated. The resulting residue was purified by flash chromatography on silica gel eluting with EA 0 to 20% in PE to give 2.06 g (56 % yield) of tert-butyl 4-[2-[bis(tert-butoxycarbonyl)amino]-3-[(E)-3-ethoxy-3-oxo-prop-l-enyl]-5-fluoro-4-pyridyl]-3,6-dihydro-2H-pyridine-l -carboxylate as a yellow oil. LC / MS (m / z, M+H - BOC-t-Bu): calc.

[0315] 436.2, found 436.1.

[0316] STEP 5: tert-butyl 4-[2-[bis(tert-butoxycarbonyl)amino]-3-(3-ethoxy-3-oxo-propyl)-5-fluoro-4-pyridyl]piperidine-l -carboxylate

[0317]

[0318] To a solution of tert-butyl 4-[2-[bis(tert-butoxycarbonyl)amino]-3-[(E)-3-ethoxy-3-oxo-prop-l-enyl]-5-fluoro-4-pyridyl]-3,6-dihydro-2H-pyridine-l-carboxylate (170 mg, 0.29 mmol) in methanol (20 mL) was added Pd on carbon (30 wt %, 50 mg). The mixture was stirred for a week at 40 °C under one atmosphere of hydrogen. The reaction mixture was filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give the 170 mg (92% yield) of tert-butyl 4-[2-[bis(tert-butoxycarbonyl)amino]-3-(3-ethoxy-3-oxo-propyl)-5-fluoro-4-pyridyl]piperidine-l-carboxylate which was used in the next step without further purification. LC / MS (m / z, M+H - BOC): calc. 496.3, found 496.4.

[0319] STEP 6: ethyl 3-[2-amino-5-fluoro-4-(4-piperidyl)-3-pyridyl]propanoate;2,2,2-trifluoroacetic acid

[0320]

[0321] To a solution of tert-butyl 4-[2-[bis(tert-butoxycarbonyl)amino]-3-(3-ethoxy-3-oxo-propyl)-5-fluoro-4-pyridyl]piperidine-l -carboxylate (170 mg, 0.26 mmol) in DCM (5 mL) was added trifluoro acetic acid (2 mL) at 0 °C. The resulting mixture was stirred for 2 hours at room temperature then concentrated to give 78 mg (73% yield) of ethyl 3-[2-amino-5-fluoro-4-(4-piperidyl)-3-pyridyl]propanoate;2,2,2-trifluoroacetic acid as a yellow oil which was used in the next step without further purification. LC / MS (m / z, M+H - TFA): calc. 296.2, found 296.1.

[0322] STEP 7: 6-fluoro-5-(4-piperidyl)-3,4-dihydro-lH-l,8-naphthyridin-2-one

[0323]

[0324] A solution of ethyl 3-[2-amino-5-fluoro-4-(4-piperidyl)-3-pyridyl]propanoate;2,2,2-trifluoro acetic acid (78.0 mg, 0.19 mmol) and 4-methylmorpholine (2 mL) in methanol (10 mL) was stirred fori 6 hours at 70 °C. The resulting reaction mixture was concentrated to dryness to give 50 mg (100% yield) of 6-fluoro-5-(4-piperidyl)-3,4-dihydro-lH-l,8-naphthyridin-2-one which was used in the next step without purification. LC / MS (m / z, M+H): calc. 250.1, found 250.0.

[0325] STEP 8: 6-fluoro-5-[l-[4-(trifluoromethoxy)benzoyl]-4-piperidyl]-3,4-dihydro-lH-l,8-naphthyridin-2-one

[0326] 0.

[0327] N^ N^O

[0328]

[0329] To a solution of 6-fluoro-5-(4-piperidyl)-3,4-dihydro-lH-l,8-naphthyridin-2-one (50 mg, 0.19 mmol) and 4-(trifluoromethoxy)benzoic acid (43 mg, 0.21 mmol) in DMF (5 mL) was added 1 -hydroxybenzotriazole (38 mg, 0.28 mmol), 3-(ethyliminomethyleneamino)-N,N-dimethyl-propan-l-amine;hydrochloride (55 mg, 0.28 mmol) and N-ethyl-N-isopropyl-propan-2-amine (123 mg, 0.95 mmol). The resulting mixture was stirred for 16 hours at room temperature. The residue was treated with water (20 mL) and extracted with EA (3x20 mL). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered and concentrated to dryness. The resulting residue was purified by preparative HPLC (water in CAN, 10 mM NH4HCO3) to give 14 mg (17% yield) of 6-fluoro-5-[l-[4-(trifluoromethoxy)benzoyl]-4-piperidyl]-3,4-dihydro-lH-l,8-naphthyridin-2-one as a white solid. 'H NMR (500 MHz, DMSO-76) 6 ppm 10.46 (s, 1H), 8.04 (d, 7=3.0 Hz, 1H), 7.57-7.45 (m,4H), 4.62-4.60 (m, 1H), 3.65-3.63 (m, 1H), 3.27-3.22 (m, 2H), 3.02 (t, 7=7.5 Hz, 2H), 2.93-2.87 (m, 1H), 2.51-2.47 (m, 2H), 1.95-1.63 (m, 4H). LC / MS (m / z, M+H): calc. 438.2, found 438.1.

[0330] Example 12: 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-4H-pyrido[3,2-b][l,4]oxazin-3-one

[0331]

[0332] METHOD D (SCHEME 4)

[0333] STEP 1: tert-butyl 4-(3,5-difluoro-2-nitro-4-pyridyl)-3,6-dihydro-2H-pyridine-l -carboxy late

[0334]

[0335] A mixture of 4-chloro-3,5-difluoro-2-nitro-pyridine (2.8 g, 14.4 mmol), tert-butyl 4-(4,4,5,5-tetramethyl- 1 , 3, 2-dioxaborolan-2-yl)-3,6-dihydro-2H-pyridine-l -carboxylate (4.9 g, 15.8 mmol), palladium bis(tri-tert-butylphosphine) (368 mg, 0.72 mol), potassium triphosphate (9.17 g, 43.2 mmol) in 1,4-dioxane (40 mL) and water (8 mL) was stirred at 100 °C for 12 hours under argon atmosphere. Then, the mixture was diluted with water (200 mL), and extracted with ethyl acetate (3x100 mL). The combined organic layers were washed with brine (2x100 mL), dried over Na2SO4, filtered and concentrated to dryness. The resulting residue was purified by flash chromatography on silica gel eluting with 0-40% of ethyl acetate in petroleum ether, to give 2 g (41% yield) of tert-butyl 4-(3,5-difluoro-2-nitro-4-pyridyl)-3,6-dihydro-2H-pyridine-l -carboxy late (as a yellow solid. LC / MS (m / z, M+H): calc.

[0336] 342.2, found 342.0. STEP 2: tert-butyl 4-[3-(2-ethoxy-2-oxo-ethoxy)-5-fluoro-2-nitro-4-pyridyl]-3,6-dihydro- 2H-pyridine- 1 -carboxylate

[0337]

[0338] A mixture of ethyl 2-hydroxyacetate (1.22 g, 11.72 mmol) in tetrahydrofuran (40 mL) was added LDA (IM solution in tetrahydrofuran) (8.8 mL, 8.79 mmol) at 0 °C and stirred for 1 hour under nitrogen atmosphere. Then, tert-butyl 4-(3,5-difluoro-2-nitro-4-pyridyl)-3,6-dihydro-2H-pyridine-l-carboxylate(2 g, 5.86 mmol) was added and the reaction mixture was stirred for 4 hours at room temperature, A saturated aqueous ammonium chloride solution was added carefully to quench the reaction, the whole mixture was then diluted with water (200 mL) and extracted with ethyl acetate (3x100 mL). The combined organic layers were washed with brine (2xl50mL), dried over Na2SO4, filtered and concentrated to dryness. The resulting residue was purified by flash chromatography on silica gel eluting with 0 to 30% of ethyl acetate in petroleum ether to give 970 mg (39% yield) of tert-butyl 4-[3-(2-ethoxy-2-oxo-ethoxy)-5-fluoro-2-nitro-4-pyridyl]-3,6-dihydro-2H-pyridine-l-carboxylate as a yellow oil. LC / MS (m / z, M+H): calc. 426.2, found 426.0.

[0339] STEP 3: tert-butyl 4-(7-fluoro-3-oxo-4H-pyrido[3,2-b][l,4]oxazin-8-yl)-3,6-dihydro-2H-pyridine- 1 -carboxylate

[0340]

[0341] A mixture of tert-butyl 4-[3-(2-ethoxy-2-oxo-ethoxy)-5-fluoro-2-nitro-4-pyridyl]-3,6-dihydro-2H-pyridine-l -carboxylate (970 mg, 2.28 mmol), iron (637 mg, 11.4 mmol) and ammonium chloride (610 mg, 11.4 mmol) in water (4 mL) and methanol (20 mL), was stirred at 60 °C for 2 hours under nitrogen atmosphere. Then, the reaction mixture was diluted with water (30 mL) and extracted with dichloromethane (3x40 mL). The combined organic layers were washed with brine (2x20 mL), dried over Na2SO4, filtered and concentrated to dryness. The resulting residue was purified by flash chromatography on silica gel eluting with 0 to 50% of ethyl acetate in petroleum ether, to give 650 mg, (82% yield) of tert-butyl 4-(7- fluoro-3-oxo-4H-pyrido[3,2-b][l,4]oxazin-8-yl)-3,6-dihydro-2H-pyridine-l -carboxylate as a yellow oil. LC / MS (m / z, M+H): calc. 350.1, found 350.1.

[0342] STEP 4: tert-butyl 4-(7-fluoro-3-oxo-4H-pyrido[3,2-b][l,4]oxazin-8-yl)piperidine-l-carboxylate

[0343]

[0344] A mixture of tert-butyl 4-(7-fluoro-3-oxo-4H-pyrido[3,2-b][l,4]oxazin-8-yl)-3,6-dihydro-2H-pyridine-1 -carboxylate (650 mg, 1.86 mmol) and Pd / C (1 g,), Pd(OH)2 (1 g,) in methanol (50 mL) and tetrahydrofuran (50 mL) was stirred at room temperature for 24 hours under 1 atmosphere of H2. Then, the mixture was filtered, concentrated and the resulting residue was purified by flash chromatography on silica gel eluting with 0 to 50% of ethyl acetate in petroleum ether, to give 560 mg (86% yield) of tert-butyl 4-(7-fluoro-3-oxo-4H-pyrido[3,2-b][l,4]oxazin-8-yl)piperidine-l -carboxylate as a white solid. LC / MS (m / z, M+H): calc.

[0345] 352.2, found 352.1.

[0346] STEP 5: 7-fluoro-8-(4-piperidyl)-4H-pyrido[3,2-b][l,4]oxazin-3-one;2,2,2-trifluoroacetic acid

[0347]

[0348] A mixture of tert-butyl 4-(7-fluoro-3-oxo-4H-pyrido[3,2-b][l,4]oxazin-8-yl)piperidine-l-carboxylate (300 mg, 0.85 mmol) in TFA (5 mL) and DCM (5 mL) was stirred at room temperature for 2 hours. Then, the mixture was concentrated to give 210 mg (68% yield) of 7-fluoro-8-(4-piperidyl)-4H-pyrido[3,2-b][l,4]oxazin-3-one;2,2,2-trifluoroacetic acid as a yellow oil. LC / MS (m / z, M+H -TFA): calc. 252.2, found 252.1.

[0349] STEP 6: 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-4H-pyrido[3,2-b][l,4]oxazin-3-one

[0350]

[0351] To a solution of 7-fluoro-8-(4-piperidyl)-4H-pyrido[3,2-b][l,4]oxazin-3-one;2,2,2-trifluoro acetic acid (214 mg, 0.59 mmol) and 2-amino-4-(trifluoromethoxy)benzoic acid (188 mg, 0.85 mmol) in DMF (10 mL), was added l-hydroxybenzotriazole;hydrate (196 mg, 1.27 mmol), N'-(ethylcarbonimidoyl)-N,N-dimethylpropane-l,3-diamine hydrochloride (224 mg, I.27 mmol) and N-ethyl-N-isopropyl-propan-2-amine (1.1 g, 8.52 mmol). The resulting mixture was stirred for 16 hours at room temperature. Then, the resulting reaction mixture was treated with water (50 mL) and extracted with EA (3x20 mL). The combined organic layers were concentrated and the resulting residue was dissolved in a mixed solution of methanol (10 mL) and aqueous purified ammonia (15 mL) and the whole was stirred for one hour. Then, the resulting solution was treated with water and extracted with EA. The combined organic layers were washed with water, brine, dried over anhydrous Na2SO4, filtered and concentrated. The resulting residue was purified by preparative-HPLC to give 70 mg (26% yield) of 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-4H-pyrido[3,2-b][l,4]oxazin-3-one as a colorless solid.JH NMR (400 MHz, DMSO-t / e) 8 ppm II.28 (s, lH),7.90(s, 1H), 7.10 (d, J= 8.4 Hz, 1H), 6.66 (s, 1H), 6.50 (d, 7 = 8.2 Hz, 1H), 5.59 (s, 2H), 4.68 (s, 2H), 3.34 (s, 2H), 3.00 (s, 2H), 2.08 (d, J = 3.8 Hz, 1H), 1.93 (d, J = 10.7 Hz, 2H), 1.69 (d, J= 11.7 Hz, 2H). LC / MS (m / z, M+H): calc. 455.1, found 455.2.

[0352] Example 13: 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-2,4-dihydro-lH-pyrido[2,3-b]pyrazin-3-one

[0353]

[0354] Example 13 was prepared following METHOD A, using ethyl 2-aminoacetate (1.99 g, 19.2 mmol) in STEP 3, to give in the last step, 41 mg (13% yield) of 8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-2,4-dihydro-lH-pyrido[2,3-b]pyrazin -3-one as a white solid. iH NMR (400 MHz, DMSO-76) 6 ppm 10.71 (s, 1H), 7.46 (d, J = 2.4 Hz, 1H), 7.11 (d, 7 = 8.4 Hz, 1H),6.65 (d, 7= 1.1 Hz, 1H),6.5O (dd, 7=8.4, 1.2 Hz, 1H), 6.2O (s, 1H), 5.61 (s, 2H), 3.83 (s, 2H), 3.81 (d, 7= 1.6 Hz, 2H), 3.08 (dd, 7 = 38.5, 26.6 Hz, 3H), 1.86 (d, 7= 11.1 Hz, 2H), 1.66 (d, 7= 11.3 Hz, 2H). LC / MS (m / z, M+H): calc. 454.1, found 454.1.

[0355] ERK5 inhibitory activity of compounds

[0356] Two assays were performed to assess the ERK5 inhibitory activity of compounds of the Examples, a cell-based assay and a cell-free biochemical assay. Results of the assays are shown below in Table 2. Where repeat measurements were taken, the value reported is a mean average.

[0357] Inhibition of cellular ERK5 activity

[0358] The renal cancer cell line SN12C was transduced by a lentivirus pGreenFirel MEF2 EFl Neo (ref TR030VA-N) from SBI using standard infection protocol. pGreenFirel MEF2 EFl Neo allows the expression of luciferase gene under the control of minimal promoter with MEF2 transcriptional response elements. Cells harboring the reporter construct were selected by geneticin treatment. The selected cells were then transposed by a piggyback based plasmid pCM4007 allowing the expression of constitutively activated MEK5DD under control of TREG3 promoter, a doxycycline regulated promoter. Transposed cells were selected by puromycin treatment. Upon doxycycline treatment (Ipg / ml) MEK5DD was expressed. MEK5DD activates ERK5 that phosphorylates MEF2C protein. Activated MEF2C protein can bind to its transcriptional response elements. Then the luciferase is expressed. In a 96-well plate (96F nuncleon refl37101 thermofisher), 50,000 cells were inoculated in 142.5plof RPMI medium containing 10% fetal calf serum, 1% glutamine, and Ipg / ml doxycycline. After 24 hours, compounds were added in 7.5pl culture medium (with 2% DMSO to give a final concentration of 0.1%) in order to obtain the desired concentration (0.3-10000nM). The luciferase activity was measured using the Kit Bright Gio Luminescent Cell Assay Cat E2610 (Promega) accordingto the manufacturer’s protocol. Luminescence was determined using 0.2 second reading / well using a Tecan SPARK. IC50 values were calculated using XLFIT5 for Microsoft Excel using method 205. The IC50 values represent the concentration of compound which inhibits the measurable luminescence signal by 50% as compared to DMSO-treated control cells.

[0359] Cell-free assay ofERK5 inhibition

[0360] An assay was performed to measure the capacity of each compound to inhibit ERK5 enzymatic activity. Compound potency was evaluated by Time-Resolved Forster Resonance Energy Transfer (FRET system). The activated catalytic domain of protein ERK5 (CamaBiosciences#04-146) was mixed at 4 nM with varying concentrations of compound and incubated for 30 minutes at room temperature. A mixture of ImM ATP and IpM biotinylated synthetic peptide was added (Biosyntan GmbH). This synthetic peptide represents amino acids 30-52 of Eukaryotic translation initiation factor 4E-binding protein 1 (see, e.g., the sequence under accession No. NP_004086.1) biotinylated at the N-terminus. After 30 minutes at 37°C, the peptide phosphorylation by ERK5 was measuredby addition of FRET reagents consisting of 12.5 pg / ml Streptavidin -XL665, 1 nM Anti-P-4EBP1 antibody and 300ng / ml Anti-rabbit-K antibody. Following 90 minutes at room temperature fluorescent signals were read on the Pherastar FSX multimod detector from BMG Labtech (Exc° 340 nm, Eml 620 nm, Em2665 nm). The IC50 values represent the concentration of compound which inhibits the measurable fluorescence signal by 50% as compared to the DMSO only control.

[0361] Table 2: Results of cell-based and cell-free assays

[0362] Compound Cell-free assay Cell-based assay (Example No.) (IC50 in nM) (ICso in nM)

[0363] 1 1808 546

[0364] 2 275 246

[0365] 3 659 114

[0366] 4 84 16

[0367] 5 173 30

[0368] 6 391 48

[0369] 7 951 193

[0370] 8 185 67

[0371] 9 736 117

[0372] 10 205 29

[0373] 11 223 186

[0374] 12 184 199

[0375] 13 74 91

[0376]

[0377] 14 126 64 The data in Table 2 indicate that the compounds synthesised are active in the micromolar or nanomolar concentration range in cell-based and / or cell-free systems. All of the compounds synthesised have an IC50 value below 600 nM in at least one of the cell-based and cell-free assays.

Claims

CLAIMS1. A compound of Formula (I), comprising a piperazinone ring fused to a pyridine ring:(I)wherein:R1 represents a hydrogen atom or a -NH2 group,R2 and R3 are independently selected from a hydrogen atom or a (Cl-C4)alkyl group, or form together with the carbon atom to which they are attached a (C4- C6)cyloalkyl or 4 to 6-membered heterocycloakyl group,the dotted line in the piperazinone ring represents a saturated or unsaturated bond; when the dotted line represents a saturated bond, X is selected from -CH2-, -O-, - S-, -SO2- or -NH-, andwhen the dotted line represents an unsaturated bond, X represents -N= and R3 is absent;or a pharmaceutically acceptable salt thereof.

2. A compound of Formula (I) according to claim 1, wherein R2 and R3 are independently selected from a hydrogen atom or methyl or ethyl group, or form together with the carbon atom to which they are attached a cyclobutyl, cyclopentyl, cyclohexyl or tetrahydropyranyl group,or a pharmaceutically acceptable salt thereof.

3. A compound according to claim 1 or claim 2, which is represented by formula (II) :(II)wherein Rl, R2 and R3 are defined according to claim 1 and wherein X is selected from -CH2-, -O-, -S-, -SO2- or -NH- or a pharmaceutically acceptable salt thereof.

4. A compound according to claim 3, wherein X represents -S- or -SO2- and wherein R2 and R3 represent hydrogen atoms or (Cl -C4)alkyl groups,or a pharmaceutically acceptable salt thereof.

5. A compound according to any of claims 1 to 3, which is represented by formula (Ila):(Ila)wherein R1 represents a hydrogen atom or a -NH2 group; and wherein R2 and R3 are independently selected from a hydrogen atom or a (Cl -C4)alkyl group, or form together with the carbon atom to which they are attached a (C4-C6)cyloalkyl or 4 to 6-membered heterocycloalkyl group,or a pharmaceutically acceptable salt thereof.

6. A compound according to claim 5, wherein R2 and R3 are independently selected from a hydrogen atom or methyl group, or form together with the carbon atom to which they are attached a cyclobutyl, cyclopentyl, cyclohexyl or tetrahydropyranyl group,or a pharmaceutically acceptable salt thereof.

7. A compound according to claim 1 or claim 2, which is represented by formula (III), wherein R1 and R2 are defined according to claim 1 or claim 2, and R3 is absent:or a pharmaceutically acceptable salt thereof.

8. A compound according to claim 7, wherein R1 represents -NH2,or a pharmaceutically acceptable salt thereof.

9. A compound according to claim 7 or claim 8, wherein R2 represents a (Cl-C4)alkyl group,or a pharmaceutically acceptable salt thereof.

10. A compound according to any of claims 1 to 9, which is selected from the groupconsisting of:8-[ 1 -[2-amino-4-(trifluoromethoxy)benzoyl] -4-piperidyl]-7 -fluoro- 1 , 1 -dioxo-4H- pyrido[3,2-b][l,4]thiazin-3-one8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-2-ethyl-7-fluoro-4H- pyrido[2,3-b]pyrazin-3-one8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-2,2-dimethyl-4H- pyrido[3,2-b][l,4]thiazin-3-one8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-spiro[l,4- dihydropyrido[2,3-b]pyrazine-2,l'-cyclobutane]-3-one8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-spiro[l,4- dihydropyrido[2,3-b]pyrazine-2,l'-cyclopentane]-3-one8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-spiro[l,4- dihydropyrido[2,3-b]pyrazine-2,l'-cyclohexane]-3-one7 -fluoro-8-[ l-[4-(trifluoromethoxy)benzoyl]-4-piperidyl] spiro[ 1 ,4-dihydropyrido [2,3 - b]pyrazine-2,4'-tetrahydropyran] -3-one8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-spiro[l,4- dihydropyrido[2,3-b]pyrazine-2,4'-tetrahydropyran]-3-one7-fluoro-2,2-dimethyl-8-[l-[4-(trifluoromethoxy)benzoyl]-4-piperidyl]-l,4- dihydropyrido[2,3-b]pyrazin-3-one8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-2,2-dimethyl-l,4- dihydropyrido[2,3-b]pyrazin-3-one6-fluoro-5-[l-[4-(trifluoromethoxy)benzoyl]-4-piperidyl]-3,4-dihydro-lH-l,8- naphthyridin -2-one8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-4H-pyrido[3,2- b][l,4]oxazin-3-one8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-2,4-dihydro-lH- pyrido[2,3-b]pyrazin-3-one8-[l-[2-amino-4-(trifluoromethoxy)benzoyl]-4-piperidyl]-7-fluoro-4H-pyrido[3,2- b][l,4]thiazin-3-one,and the pharmaceutically acceptable salts thereof.

11. A pharmaceutical composition comprising the compound of any one of claims 1 -10, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient or carrier.

12. A compound according to any of claims 1 to 10, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 11, for use in therapy.

13. A compound according to any of claims 1 to 10, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 11, for use in the treatment or prevention of cancer.

14. The compound or pharmaceutical composition for use according to claim 13, wherein the cancer is characterized by increased MAPK7 expression and / or increased ERK5 activity.

15. The compound or pharmaceutical composition for use according to claim 13 or claim 14, wherein the cancer is selected from leukaemia, breast cancer, multiple myeloma, colon cancer, colorectal cancer, lung cancer, pancreatic cancer, renal cell carcinoma, mesothelioma, adenocarcinoma, neuroblastoma, melanoma, and hepatocellular carcinoma.

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