WO2015019037A1 - Pharmaceutical compounds - Google Patents

Pharmaceutical compounds Download PDF

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Publication number
WO2015019037A1
WO2015019037A1 PCT/GB2013/052080 GB2013052080W WO2015019037A1 WO 2015019037 A1 WO2015019037 A1 WO 2015019037A1 GB 2013052080 W GB2013052080 W GB 2013052080W WO 2015019037 A1 WO2015019037 A1 WO 2015019037A1
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WO
WIPO (PCT)
Prior art keywords
group
amino
pyrimidin
phenyl
methyl
Prior art date
Application number
PCT/GB2013/052080
Other languages
French (fr)
Inventor
James Samuel Shane Rountree
Colin Roderick O'dowd
Frank Burkamp
Mark Peter Bell
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Almac Discovery Limited
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Priority to PCT/GB2013/052080 priority Critical patent/WO2015019037A1/en
Publication of WO2015019037A1 publication Critical patent/WO2015019037A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/04—Ortho-condensed systems

Definitions

  • the present invention relates to compounds that are useful as inhibitors of the activity of Wee-1 kinase.
  • the present invention also relates to pharmaceutical compositions comprising these compounds and to methods of using these compounds in the treatment of cancer and methods of treating cancer.
  • Detection of DNA damage initiates a series of events which are key in maintaining the genome.
  • Cell cycle checkpoints are designed to stop the cell cycle and allow repair of the lesion before allowing the cell to continue into mitosis .
  • Two key checkpoints have been identified, one at the end of Gl phase and the second at G2, which work in tandem to ensure all lesions, are identified and repaired.
  • the Gl checkpoint is non-functional due to mutations in the tumour suppressor gene p53.
  • the G2 check-point is seldomly mutated, and often found to be activated in cancer cells. Cancer cells exploit this to confer resistance to treatment modalities
  • Inhibitors for the kinases are currently in clinical trials .
  • Wee-1 is a nuclear tyrosine kinase, which negatively regulates entry into mitosis, at the G2/M check-point by catalysing a phosphorylation of the cdc2 / cyclin B kinase complex. The phosphorylation occurs on the
  • Wee-1 is downstream of the Chk family and is a crucial component of the checkpoint signalling cascade as it prevents cells from entering mitosis if lesions are detected .
  • anti-cancer compounds induce DNA damage; including anti-metabolites, platiniums,
  • topoisomerase inhibitors and alkylating agents are used in combination with these agents to prevent DNA repair selectively in tumour cells.
  • Compounds which work in combination with these agents to prevent DNA repair selectively in tumour cells would be extremely beneficial.
  • the tumour suppressor gene p53 is commonly mutated in tumour cell lines, therefore the
  • Irradiation is known to increase phosphorylation of the Tyrl5 and Thrl4 residues of cdc2, leading to a
  • kinase inhibitory effect for example a Wee-1 kinase inhibitory effect, are described in WO2007/126122, US2010/0063024, EP2,213,673, W02008 / 133866 and US2007/0254892.
  • the compounds of WO2005/090344 are said to show activity as protein kinase inhibitors, in particular Src family tyrosine kinase inhibitors.
  • the compounds described in Bioorg & Med Chem Lett Vol 15, pp 1931-1935 are said to be 10-100-fold more potent inhibitors of c- Src than Weel, and variation of substituents on the 6- phenyl ring does not markedly alter this preference. It is said that solubilizing substituents off the 2-anilino ring in many cases increases Weel activity, lowering this preference to about 10-fold.
  • analogues are said to be generally Weel selective, but at the expense of absolute potency.
  • compositions are provided.
  • the present invention provides a compound of Formula (I) :
  • X is an oxygen atom or a nitrogen atom
  • Y is a carbon atom or a nitrogen atom;
  • R 1 is an optionally substituted aryl group;
  • R 2 is a hydrogen atom, an optionally substituted alkyl group or an optionally substituted amino group when Y is a carbon atom and R 2 is absent when Y is a nitrogen atom;
  • R 3 is a hydrogen atom, an optionally substituted alkyl group or an optionally substituted aryl group when X is a nitrogen atom and R 3 is absent when X is an oxygen atom;
  • R 4 is an optionally substituted alkyl group or an optionally substituted aryl group.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the compound of formula (I), or a pharmaceutically acceptable salt or N- oxide derivative thereof, and at least one
  • the present invention provides the compound of formula (I), or a pharmaceutically acceptable salt or N-oxide derivative thereof, or a pharmaceutical composition comprising the compound of formula (I) for use in therapy.
  • the present invention provides the compound of formula (I) for use as a medicament. In a fifth aspect the present invention provides the compound of formula (I) for use in treating or preventing cancer . In a sixth aspect the present invention provides the compound of formula (I) for the manufacture of a
  • the present invention provides the use of the compound of formula (I) for the manufacture of a medicament for treating or preventing cancer.
  • the present invention provides a method of treating or preventing cancer in a human or animal patient comprising administering to a patient in need thereof an effective amount of the compound of formula (I) or a pharmaceutical composition comprising the compound of formula (I) .
  • Other preferred embodiments of the compounds according to the invention appear throughout the specification and in particular in the examples. Particularly preferred are those named compounds having greater activity as tested. Compounds having higher activity are more preferred over those having lower activity.
  • the present inventors have surprisingly found that the compounds of the present invention show an improved selectivity towards Wee-1 kinase.
  • the compounds of the present invention show an improved selectivity towards Wee-1 kinase.
  • the compounds of the invention are selective over members of the Src family of kinases, for example LCK (Lymphocyte specific protein tyrosine kinase) and c- Src.
  • LCK Lymphocyte specific protein tyrosine kinase
  • c- Src kinases
  • the present inventors have surprisingly found that the compounds of the present invention show an improved or similar kinase-inhibitory effect compared to known compounds or compositions.
  • the compounds of the present invention preferably show an improved or similar Wee-1 kinase-inhibitory effect compared to known compounds or compositions.
  • the compounds of the present invention have an improved stability in human microsomes and/or an improved tolerability compared to known compounds or compositions .
  • alkyl group refers to an aliphatic group containing at least carbon and hydrogen and containing 1 to 15 carbon atoms, such as 1 to 10 carbon atoms.
  • a " C n alkyl” group refers to an aliphatic group containing n carbon atoms.
  • a Ci-Cio alkyl group
  • An alkyl group may be straight chained or it may be branched.
  • An alkyl group may contain no ring structures or it may contain one or more rings (i.e. "a cycloalkyl group") .
  • a "cycloalkyl group” contains at least one ring. It is understood that attachment to a cycloalkyl group is via a ring atom of the cycloalkyl group.
  • Each ring may contain 3 to 10 atoms, such as 4 to 8 or 5 to 7 atoms.
  • Each ring may be independently selected to contain just carbon atoms or to contain both carbon atoms and from 1 to 4 heteroatoms selected from 0, N and S.
  • attachment to the cycloalkyl group may occur either through a carbon atom or, if one or more heteroatoms are contained in a ring, attachment may also occur through a heteroatom contained in a ring.
  • a cycloalkyl group may be mono-cyclic or bi- cyclic.
  • the bicyclic cycloalkyl group may have a spiro structure in which the two rings share one and the same ring atom, or may have a bicyclo structure in which the two rings share two or more ring atoms.
  • a " C n cycloalkyl" group contains n carbon atoms. All n carbon atoms may be contained in the ring(s) of the cycloalkyl group or one or more of the carbons may not be contained in the ring(s) and may instead form one or more chains branching from the ring.
  • the two alkyl groups contain a total number of m + n carbon atoms.
  • an alkyl group may be saturated or unsaturated.
  • the alkyl group may be an alkenyl group (i.e. contain a carbon-carbon double bond) and / or an alkynyl group (i.e. contain a carbon-carbon triple bond) .
  • the alkyl group may contain at least 2 carbon atoms. It is understood that any unsaturated portions of an alkyl group are non-aromatic (aromatic groups fall within the scope of the definition of "aryl") .
  • Any part of the alkyl group may be unsaturated, for example the straight, branched or cyclic portion of an alkyl group may contain a carbon-carbon double bond or a carbon- carbon triple bond. Attachment to an unsaturated alkyl group may occur through the unsaturated part of the alkyl group or may occur through the unsaturated part of the group .
  • an unsaturated alkyl group may contain 1 to 4 carbon-carbon double bonds or 1 to 3 carbon-carbon triple bonds or 1 to 4 of a combination of carbon-carbon double bonds and carbon-carbon triple bonds.
  • An alkyl or cycloalkyl group can be optionally
  • substituted (a "substituted alkyl” or "substituted cycloalkyl") with one or more alkyl group substituents , which can be the same or different.
  • An alkyl group substituent can be attached to the alkyl or cycloalkyl group through a carbon atom in the alkyl or cycloalkyl group, or, if one or more heteroatoms are contained in a cycloalkyl group, attachment may also occur through a heteroatom contained in the cycloalkyl group.
  • alkyl group substituent includes but is not limited to alkyl, substituted alkyl, aralkyl, substituted aralkyl, halo, cyano, amino, alkylamino, arylamino, aryl,
  • arylsulfonyl and cycloalkyl If a group, for example an alkyl group, is "optionally substituted” , it is understood that the group has one or more substituents attached (substituted) or does not have any substituents attached (unsubstituted) . Examples of unsubstituted saturated alkyl groups
  • containing no cyclic structures include methyl, ethyl, n- propyl, sec-propyl, n-butyl, sec-butyl, tert-butyl, pentyl (branched or unbranched) , hexyl (branched or unbranched) , heptyl (branched or unbranched) , octyl
  • unsubstitued saturated cycloalkyl groups include cyclopropyl, cylcobutyl, cyclopentyl and
  • unsaturated alkyl groups include ethenyl, trimethenyl, tetramethenyl , propenyl, butenyl, 2- methybutenyl , pentamethenyl , hexamethenyl and
  • cyclohexenyl examples include azetidinyl, pyrrolodinyl , piperidinyl, piperizinyl, tetrahydrofuranyl , tetrahydropyranyl , morpholine, thiomorpholine, homopiperaz ine , homo- piperidine, homomorpholine, homothiomorpholine, S,S- dioxythiomorpholine, S, S-dioxyhomothiomorpholine .
  • aryl group refers to a group containing at least one ring that is aromatic and containing 1 to 15 carbon atoms, such as 1 to 10 carbon atoms.
  • the aryl group can be optionally substituted (a
  • substituted aryl with one or more aryl group
  • aryl group substituent includes but is not limited to alkyl, substituted alkyl aryl, substituted aryl, aralkyl, hydroxyl, alkoxyl, haloalkoxyl perhaloalkoxyl , aryloxyl, aralkyloxyl, carboxyl, alkanoyl, halo, nitro,
  • alkoxycarbonyl aryloxycarbonyl , aralkoxycarbonyl , acyloxyl, cyano, acylamino, amido, carbamoyl,
  • alkylcarbamoyl dialkylcarbamoyl , arylthio and alkylthio.
  • Each ring may be independently selected to contain only carbon atoms or to contain both carbon atoms and from 1 to 4 heteroatoms selected from 0, N and S.
  • heteroaryl groups i.e. aryl groups that contain one or more heteroatoms
  • attachment to the aryl group may occur either through a carbon atom or, if one or more
  • heteroatoms are contained in a ring, attachment may also occur through a heteroatom contained in a ring.
  • heteroatoms contained in a ring of a heteroaryl group may be substituted, for example forming an iV-oxide.
  • the aromatic group may be mono-cyclic or bi- cyclic, wherein one or both of the rings of a bi-cyclic system is aromatic. If one ring is non-aromatic, the non- aromatic ring may be further substituted with one or more substituents , which can be the same or different.
  • the substituents include, but are not limited to alkyl, spirocyclic alkyl, aralkyl, substituted aralkyl, halo, cyano, amino, alkylamino, arylamino, aryl, substituted aryl, nitro, thio, acyl, hydroxyl, aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio, carboxyl, alkoxycarbonyl , oxo, alkylsulfonyl , arylsulfonyl and cycloalkyl .
  • aryl groups include acridinyl, phenyl, carbazolyl, cinnolinyl, quinoxalinyl , pyrrazolyl, benzotriazolyl , furanyl, naphthyl, thienyl, thiazolyl, benzothienyl , benzofuranyl , quinolinyl, isoquinolinyl , oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, tetrahydroquinoline, tetrahydroisoquinoline, benzimidazolyl and melaminyl.
  • heterocycle includes within its scope both cycloalkyl groups containing one or more heteroatoms within the ring system and aryl groups containing one or more heteroatoms within the ring system.
  • Heterocyclic groups may be, for example, pyrazoles, imidazoles and any of the various triazoles and may include the oxygen and/or sulphur containing analogues that is oxazoles, isoxazoles, thiazoles and isothiazoles and derivatives.
  • nitrogen-containing heterocyclic group refers to a monocyclic or bicyclic heterocyclic group containing at least one nitrogen atom, in which each ring comprises from 3 to 7 ring atoms and optionally contains, in addition to the nitrogen atom, zero or one or two or more, the same or different hetero atoms, but preferably one hetero atom selected from the group consisting of an oxygen atom, a nitrogen atom and a sulfur atom; and the heterocyclic group may be aromatic or aliphatic.
  • the bicyclic heterocyclic group may have a spiro structure of which the two rings share one and the same ring atom, or may have a bicyclo structure of which the rings share two or more ring atoms.
  • the nitrogen-containing heterocyclic group include, for example, a pyrrolyl group, an imidazolyl group, a pyrazolyl group, a
  • thiazolyl group an isothiazolyl group, an oxazolyl group, an isoxazolyl group, a triazolyl group, a
  • tetrazolyl group an oxadiazolyl group, a 1,2,3- thiadiazolyl group, a 1 , 2 , 4-thiadiazolyl group, a 1,3,4- thiadiazolyl group, a pyridyl group, a pyrazinyl group, a pyrimidinyl group, a pyridazinyl group, a 1 , 2 , 4-triazinyl group, a 1 , 3 , 5-triaz inyl group, an indolyl group, a benzimidazolyl group, a benzoxazolyl group, a
  • benzisothiazolyl group an indazolyl group, a purinyl group, a quinolyl group, an isoquinolyl group, a
  • phthalazinyl group a naphthyridinyl group, a quinoxalinyl group, a quinazolinyl group, a cinnolinyl group, a pteridinyl group, a pyrido [ 3 , 2-b] pyridyl group, an azetidinyl group, a pyrrolidinyl group, a dihydro- 1 , 2 , 4-triazolyl group, a dihydro-1 , 2 , 4-oxadiazolyl group, a dihydro-1 , 3 , 4-oxadiazolyl group, a dihydro-1 , 2 , 4- thiadiazolyl group, a dihydro-1 , 2 , 3 , 5-oxathiadiazolyl group, a piperidinyl group, a piperazinyl group, a dihydropyridyl group, a morpholinyl group, a
  • thiomorpholinyl group a 2 , 6 -diazaspiro [ 3.5 ] nonyl group, a 2 , 7-diazaspiro [ 3.5 ] nonyl group, a 2,7- diazaspiro [ 4.5 ] decyl group, or a 2,7- diazabicyclo [ 3.3.0 ] octyl group, a 3,6- diazabicyclo [ 3.3.0 ] octyl group.
  • the nitrogen-containing heterocyclic group can be
  • amino group refers to the -NH 2 group.
  • the amino group can be optionally substituted (a "substituted amino") with one or more amino group substituents, which can be the same or different, wherein “amino group substituent” includes but is not limited to alkyl and aryl .
  • alkoxy group refers to an alkyl-0 group. The alkoxy group can refer to linear, branched, or cyclic, saturated or unsaturated oxo-hydrocarbon chains,
  • alkoxy group can be optionally substituted (a "substituted alkoxy") with one or more alkoxy group substituents , which can be the same or different, wherein "alkoxy group substituent" includes but is not limited to alkyl, amino and aryl .
  • alkanoyl group refers to an organic acid group wherein the -OH of the carboxyl group has been replaced with another substituent.
  • alkanoyl groups include an acetyl group, a propionyl group, a butyryl group, an isobutyryl group, a valeryl group, an isovaleryl group, a pivaloyl group.
  • sulfonyl group refers to a sulfonic acid group wherein the wherein the -OH of the sulfonyl group has been replaced with another substituent.
  • the substitutent may be an alkyl group ("an alkylsufonyl group”) .
  • An alkylsulfonyl group can be represented by the formula S ⁇ 0) 2 ⁇ , wherein R is an alkyl group,
  • alkylsulfonyl groups include a methylsulfonyl group, an ethylsulfonyl group, a
  • propylsulfonyl group an isopropylsulfonyl group, a butylsulfonyl group, a sec-butylsulfonyl group, an isobutylsulfonyl group, a tert-butylsulfonyl group, a pentylsulfonyl group, an isopentylsulfonyl group, a hexylsulfonyl group and an isohexylsulfonyl group.
  • halo refers to a group selected from chlorine, fluorine, bromine and iodine.
  • aromaticity may indicate the presence of, for
  • a mono-, di- or tri-cyclic system that contains (4n+2) electrons where n is an integer.
  • these systems may display Hiickel aromaticity.
  • invention may possess some aspect of stereochemistry.
  • the compounds may possess chiral centres and / or planes and / or axes of symmetry.
  • the compounds may be provided as single stereoisomers, single diastereomers , mixtures of stereoisomers or as racemic mixtures.
  • Stereoisomers are known in the art to be molecules that have the same molecular formula and sequence of bonded atoms, but which differ in their spatial orientations of their atoms and / or groups.
  • the compounds of the present invention may possess tautomerism. Each tautomeric form is intended to fall within the scope of the invention.
  • the compounds of the present invention may be provided as a pro-drug.
  • Pro-drugs are transformed, generally in vivo, from one form to the active forms of the drugs described herein.
  • a prodrug may be formed by protecting the -N-H group to which R 4 is attached with a hydrolysable group that gives -NH on hydrolysis.
  • X, Y or Z and/or any moieties appended thereto taken separately or together are -N-H, one or more of these may be
  • the elements of the compounds of the present invention may be provided as isotopes.
  • hydrogen may also be deuterium.
  • the compounds of the present invention may be provided in the form of their pharmaceutically
  • the compounds may be provided having protonated amine groups.
  • pharmaceutically acceptable salt refers to ionic compounds formed by the addition of an acid to a base.
  • the term refers to such salts that are considered in the art as being suitable for use in contact with a patient, for example in vivo and pharmaceutically
  • acceptable salts are generally chosen for their non- toxic, non-irritant characteristics.
  • co-crystal refers to a multi- component molecular crystal, which may comprise non-ionic
  • compositions may be prepared by ion exchange chromatography or by reacting the free base or acidic form of a compound with
  • Salts known in the art to be generally suitable for use in contact with a patient include salts derived from inorganic and / or organic acids, including the
  • hydrobromide hydrochloride, sulphate, bisulphate, nitrate, acetate, oxalate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate and tartrate.
  • alkali and alkaline earth metals such as sodium, potassium, calcium and magnesium, as well as ammonium, tetramethylammonium
  • the compounds of the present invention may sometimes exist as zwitterions, which are considered as part of the invention.
  • the present inventors have discovered that the compounds of the present invention are useful in the treatment of medical conditions associated with disordered cell growth, including, but not restricted to, cancer, in particular cancers associated with mutations in the tumour suppressor gene p53.
  • cancers include cardiac cancers, lung cancers, gastrointestinal cancers, genitourinary tract cancers, liver cancers, bone cancers, nervous system cancers, gynecological cancers, hematologic cancers, skin cancers and adrenal gland cancers.
  • cancers include adrenal tumors, bile duct, bladder, blood, bone and connective tissue, brain and central nervous system, breast, cervical, colon and rectal (colorectal), endometrial, esophageal, gallbladder, head and neck, Hodgkin's Lymphoma,
  • hypopharangeal kidney, laryngeal, leukemias, liver, lung, lymphoma, mediastinal tumors, melanoma (malignant melanoma) , mesothelioma, multiple myeloma, nasal cavity, nasopharyngeal, neuroendocrine tumors, non-Hodgkin ' s lymphoma, oral, oesophagus, oropharyngeal, ovarian, pancreas, paranasal sinus, parathyroid, penis, pituitary tumors, prostate, salivary gland, sarcoma, skin, spine, stomach, testicular, thyroid, urethra, uterine, vaginal and vulvar.
  • the compounds of the present invention are also useful in preparing a medicament that is useful in treating the diseases described above, in particular cancer.
  • the present invention is further directed to a method of inhibiting Wee-1 activity which comprises administering to a mammal in need thereof a pharmaceutically effective amount of the compound of the present invention.
  • the compounds of this invention may be administered to mammals, including humans, either alone or, in
  • the compounds can be administered orally or parenterally, including the intravenous, intramuscular,
  • the present invention also includes within its scope the use of the compounds of the present invention in
  • the present invention also includes the use of the compounds of the invention in a regime including the step of radiotherapy.
  • the radiotherapy maybe an ordinary method of treatment by x-ray, ⁇ -ray, neutron, proton or electron beam irradiation.
  • the co-administration of compounds contained in this invention may lead to the potentiation of the radiation therapy, thus classifying them as radio-sensitizers .
  • cancers often become resistant to therapy.
  • the development of resistance may be delayed or overcome by the administration of a combination of drugs that includes the compounds of the present invention for example in cancers which are known to be resistant to DNA damaging agents or radiotherapy.
  • drugs that may be used in combination with the compounds of the present invention may target the same or a similar biological pathway to that targeted by the compounds of the present invention or may act on a different or unrelated pathway.
  • the second active ingredient may include, but is not restricted to:
  • alkylating agents including cyclophosphamide
  • antimitotic agents including vinca alkaloids (vincristine, vinorelbine and vinblastine), taxanes
  • paclitaxel docetaxel
  • epothilones and inhibitors of mitotic kinases including aurora and polo kinases
  • topoisomerase inhibitors including anthracyclines , epipodophyllotoxins , camptothecin and analogues of camptothecin ; antimetabolites, including 5-fluorouracil, capecitabine , cytarabine, gemcitabine, 6-mercaptopurine, 6 -thioguanine , fludarabine, methotrexate and premetrexed; protein kinase inhibitors, including imatinib, gefitinib, sorafenib, sunitinib, erlotinib, dasatinib, and
  • proteosome inhibitors including bortezomib
  • histone deacetylase inhibitors including valproate and SAHA
  • antiangiogenic drugs including bevacizumab
  • monoclonal antibodies including trastuzumab, rituximab, alemtuzumab, tositumomab, cetuximab, panitumumab;
  • myoclonal antibodies including Gemtuzumab ozogamicin, Ibritumomab tiuxetan; hormonal therapies, including antiestrogens (tamoxifen, raloxifen,
  • the compounds of the present invention may be administered separately,
  • the present invention provides a compound of Formula ( I ) :
  • X is an oxygen atom or a nitrogen atom
  • Y is a carbon atom or a nitrogen atom
  • R 1 is an optionally substituted aryl group
  • R 2 is a hydrogen atom, an optionally substituted alkyl group or an optionally substituted amino group when Y is a carbon atom and R 2 is absent when Y is a nitrogen atom;
  • R 3 is a hydrogen atom, an optionally substituted alkyl group or an optionally substituted aryl group when X is a nitrogen atom and R 3 is absent when X is an oxygen atom;
  • R 4 is an optionally substituted alkyl group or an optionally substituted aryl group.
  • Y is a carbon atom.
  • R 2 is a hydrogen atom, an amino group or an alkyl group when Y is a carbon atom. More preferably, R 2 is a hydrogen atom, an NH 2 group or a methyl group when Y is a carbon atom. Most preferably, R 2 is a hydrogen atom when Y is a carbon atom.
  • Y is a nitrogen atom.
  • R 2 is absent when Y is a nitrogen atom.
  • R 1 is a mono-halogenated aryl group, for example a 2-chlorophenyl group .
  • X is a nitrogen atom.
  • R 3 is a hydrogen atom or an optionally
  • R 3 is a hydrogen atom or a substituted alkyl group wherein the alkyl group substituent is selected from the group consisting of an alkoxy-subst ituted aryl group, a carboxyl group, an alkoxycarbonyl group and a hydroxyl group. More
  • R 3 is a hydrogen atom, a methyl group, an ethyl group, a propenyl group, a hydroxyl-subst ituted propyl group or a cyclopropyl group. More preferably still, R 3 is a hydrogen atom or a methyl group. Most preferably, R 3 is a methyl group.
  • R 4 is a group represented by the formula a ) :
  • Z is a nitrogen atom or an optionally substituted methine group
  • R 4a is a hydrogen atom, a halo group, an optionally substituted C 1 -C6 alkyl group, an optionally substituted C 1 -C6 alkoxy group or is a nitrogen-containing
  • heterocyclic group optionally substituted with a
  • R 4 is a hydrogen atom, a halo group, an optionally substituted C 1 -C6 alkyl group or an optionally substituted C 1 -C6 alkoxy group;
  • R a and R and the ring atoms to which they are attached may form, as taken together, a C3-C7 alkyl group, in which one or two methylene groups constituting the C3- C 7 alkyl group may be each independently replaced by an oxygen atom or a group of -N(R le )-, and the C3-C7 alkyl group may be substituted with one or more substituents selected from the group consisting of a halo group and a C 1 -C6 alkyl group;
  • R 4a and R 4 and the ring atoms to which they are attached may form, as taken together, a spiro ring or a bicyclo ring to be formed of a 5-membered to 7-membered aliphatic ring and any other 3-membered to 7-membered aliphatic ring, in which one or two or more methylene groups constituting the spiro ring or the bicyclo ring may be each independently replaced by an oxygen atom, a sulfur atom, a sulfinyl group, a sulfonyl group, an oxo group or a group of -N(R lf )-, and the spiro ring or the bicyclo ring may be each independently substituted with a substituent selected from the group consisting of a halo group, a hydroxyl group or a C 1 -C6 alkyl group; and
  • R le and R lf are each independently a hydrogen atom or a Ci- C6 alkyl group optionally substituted with a substituent selected from the group consisting of a halo group, a hydroxyl group, a cyano group, an oxo group, a C 1 -C6 alkyl group, a C 1-C6 alkoxy group, an amino group, a substituted amino group and a nitrogen-containing heterocyclic group.
  • R 4 is a group represented by the formula ( c ) :
  • R 4a is a hydrogen atom, a halo group, a C 1-C6 alkyl group or an optionally substituted C 1-C6 alkoxy group, or is a nitrogen-containing heterocyclic group optionally substituted with a substituent selected from the group consisting of a halo group, a C 1-C6 alkyl group, an oxo group and a group of -Q 1 -N (R la ) R l ;
  • R 4 is a hydrogen atom, a halo group, an optionally substituted C 1-C6 alkyl group or an optionally substituted C1-C6 alkoxy group;
  • R la and R l each independently is a hydrogen atom, a C1-C6 alkyl group, or as taken together, they may form an optionally substituted C2-C6 alkyl group;
  • Q 1 is a single bond or a C 1-C3 alkyl group.
  • R 4a is a substituted C 1-C6 alkoxy group or a nitrogen-containing heterocyclic group
  • R 4 is a hydrogen atom.
  • R 4a is a substituted C 1-C6 alkoxy group, preferably a substituted C i_C2 alkoxy group, the
  • R a is a nitrogen-containing heterocyclic group optionally substituted with a C1-C6 alkyl group
  • R 4 is a hydrogen atom, a methyl group, a methoxy group or a methyl group substituted by a hydroxy group.
  • R 4a is a nitrogen-containing heterocyclic group optionally substituted with a methyl group
  • R 4 is a hydrogen atom, a methyl group, a methoxy group or a methyl group substituted by a hydroxy group.
  • R 4 is a group represented by the formula d) :
  • R m is selected from the group consisting of a hydrogen atom and an optionally substituted C1-C6 alkyl group. More preferably R lm is a hydrogen atom or a methyl group .
  • R 4 is a substituted alkyl group wherein the alkyl group contains no ring structures or is a cycloalkyl group, said cycloalkyl group containing carbon atoms and 0 or 1 or 2 heteroatoms elected from 0, N and S, more preferably containing carbon atoms and 1 nitrogen atom.
  • R 4 is a substituted alkyl group
  • at least one of the alkyl group substituents is an optionally substituted Ci-6 alkyl group or a substituted aryl group containing carbon atoms and 0 or 1 or 2 heteroatoms elected from 0, N and S, more preferably containing carbon atoms and 1 nitrogen atom.
  • R 1 is a group represented by the formula (e) :
  • R 2a is a hydrogen atom, a halo group, a hydroxyl group, a cyano group, a C 1-C6 alkyl group, a C 1-C6 alkoxy group or a C 1-C6 alkoxy-Ci-C6 alkyl group;
  • R 3a and R 3 are each independently selected from the group consisting of a hydrogen atom, a hydroxyl group, an amino group, an alkoxy group and a C 1-C6 alkyl group;
  • T is a nitrogen atom, or a methine group which may be substituted with a halo group, a hydroxyl group, a cyano group, a C 1-C6 alkyl group, a C 1-C6 alkoxy group or a C1-C6 alkoxy-Ci-C6 alkyl group.
  • R a and R are each
  • T is a methine group and R 3a is a hydroxyl group.
  • R 1 is a group represented by the formula (f ) :
  • R a and R are each independently selected from the group consisting of a hydrogen atom and a
  • R 3a and R 3 are a hydrogen atom and the other is a hydroxyl group. More preferably R 3a is a hydroxyl group and R 3 is a hydrogen atom.
  • R 1 is a group represented by the formula (g) :
  • R 2a is a hydrogen atom, a halo group, a hydroxyl group, a cyano group, a C 1-C6 alkyl group, a C 1-C6 alkoxy group or a C 1-C6 alkoxy-Ci-C6 alkyl group;
  • T is a nitrogen atom, or a methine group which may be substituted with a halo group, a hydroxyl group, a cyano group, a C 1-C6 alkyl group, a C 1-C6 alkoxy group or a C1-C6 alkoxy-Ci-C6 alkyl group.
  • R a is a halo group, a C 1-C6 alkyl group or a C 1-C6 alkoxy group
  • T is a methine group or is a methine group
  • R 2a is a halo group
  • T is a methine group substituted with a halo group.
  • R 1 is a 2 , 6-dichlorophenyl group, that is wherein R 1 wherein is a group represented by the formula (g) and R 2a is a chlorine atom and T is a methine group substituted with a chlorine atom.
  • Y is a nitrogen atom and R 1 is a group represented by the formula h) :
  • R 2a is a hydrogen atom, a hydroxyl group, a cyano group, a C1-C6 alkyl group, a C1-C6 alkoxy group or a C1-C6 alkoxy-Ci-C6 alkyl group;
  • R 3a and R 3 are each independently selected from the group consisting of a hydrogen atom, a hydroxyl group, an amino group, an alkoxy group and a C1-C6 alkyl group; and T' is a nitrogen atom, or a methine group which may be substituted with a halo group, a hydroxyl group, a cyano group, a Ci ⁇ C6 alkyl group, a Ci ⁇ C6 alkoxy group or a Ci ⁇ C6 alkoxy-Ci-C6 alkyl group. More preferably, R 2a , R 3a and R 3 are independently a hydrogen atom, and T' is a methine group substituted with a halo group. Most preferably, Y is a nitrogen atom and R 1 is a 2- chlorophenyl group.
  • the present invention provides a compound of Formula (II) :
  • R 1 is an optionally substituted aryl group
  • R 2 is a hydrogen atom
  • R 3 is a hydrogen atom or an optionally substituted alkyl group
  • R 4 is an optionally substituted alkyl group or an optionally substituted aryl group.
  • R 1 is a group represented by the formula (e) as defined above and/or R 4 is a group represented by the formula (a) as defined above. More preferably, R 1 is a group represented by the formula (f) or the formula (g) as defined above and/or R 4 is a group represented by the formula (c) or formula (d) as defined above. More preferably still, R 1 is a group represented by the formula (g) as defined above and/or R 4 is a group represented by the formula (d) as defined above.
  • R 1 is a 2 , 6-dichlorophenyl group or a 4-hydroxyphenyl group and/or R 2 is a hydrogen atom and/or R 3 is a hydrogen atom, a methyl group, an ethyl group, a propenyl group, a hydroxyl-substituted propyl group or a cyclopropyl group and/or R 4 is a group
  • the present invention provides a compound of Formula (III) :
  • R 1 is an optionally substituted aryl group
  • R 3 is a hydrogen atom or an optionally substituted alkyl group
  • R 4 is an optionally substituted aryl group.
  • R 1 is a group represented by the formula (h) as defined above and/or R 4 is a group represented by the formula (a) as defined above. More preferably, R 1 is a group represented by the formula (h) as defined above and/or R 4 is a group represented by the formula (c) as defined above. More preferably still, R 1 is a group represented by the formula (h) as defined above and/or R 3 is a hydrogen atom or a methyl group and/or R 4 is a group represented by the formula (c) wherein R 4a is a nitrogen-containing
  • R 4 is a hydrogen atom, a methyl group, a methoxy group or a methyl group substituted by a hydroxy group. More preferably still, R 1 is a 2-chlorophenyl group and/or R 3 is a methyl group and/or R 4 is a group re resented by the formula (i) :
  • R 5a is a hydrogen atom, a methyl group, a methoxy group or a methyl group substituted by a hydroxy group
  • R 6a is a hydrogen atom or a methyl group.
  • R 1 is a 2-chlorophenyl group and/or R 3 is a methyl group and/or R 4 is a group represented by the formula (i) as defined above, wherein R 5a is a hydrogen atom, a methyl group, a methoxy group or a methyl group substituted by a hydroxy group; and R 6a is a hydrogen atom .
  • the compound of formula (I) or formula (II) is selected from the following:
  • the compound of formula (I) or formula (III) is selected from the following:
  • Suitable pharmaceutically acceptable excipients would be known by the person skilled in the art, for example, fats, water, physiological saline, alcohol (e.g.,
  • glycerol glycerol
  • polyols aqueous glucose solution
  • extending agent disintegrating agent, binder, lubricant, wetting agent, stabilizer, emulsifier, dispersant, preservative, sweetener, colorant, seasoning agent or aromatizer, concentrating agent, diluent, buffer
  • substance, solvent or solubilizing agent, chemical for achieving storage effect salt for modifying osmotic pressure, coating agent or antioxidant, saccharides such as lactose or glucose; starch of corn,
  • fatty acids such as stearic acid
  • inorganic salts such as magnesium metasilicate aluminate or anhydrous calcium phosphate
  • synthetic polymers such as polyvinylpyrrolidone or polyalkylene glycol
  • alcohols such as stearyl alcohol or benzyl alcohol
  • synthetic cellulose derivatives such as methylcellulose
  • hydroxypropylmethylcellulose hydroxypropylmethylcellulose
  • other conventionally used additives such as gelatin, talc, plant oil and gum arabic .
  • composition comprising the compound of formula (I), or a pharmaceutically acceptable salt or N- oxide derivative thereof, and at least one
  • composition comprising the compound of formula (I) comprising one or more further
  • composition comprising the compound of formula (I) for use in therapy.
  • the compound of formula (I) for use as a medicament. In a preferred embodiment there is provided the compound of formula (I) for use in treating or preventing cancer.
  • composition comprising the compound of formula (I) for use as a medicament and/or for use in treating or preventing cancer.
  • a method of treating or preventing cancer in a human or animal patient comprising administering to a patient in need thereof an effective amount of a compound of formula ⁇ 1 ⁇ or a pharmaceutical composition comprising formula (I) .
  • the compounds of the present invention have an IC 5 o value for Wee-1 kinase of about 1 nM to about 7000 nM, more preferably from about 1 nM to about 1500 nM, or from about 1 nM to about 500 nM, or from about 1 nM to about 250 nM, or from about 1 nM to about 100 nM, or from about 1 nM to about 50 nM, or from about 1 nM to about 10 nM, most preferably less than ⁇ .
  • IC 5 o corresponds to an IC 5 o in the range of 100 nM to 10 ⁇ and '++' corresponds to an IC 5 o of ⁇ 100 nM.
  • HT29 pCDC2 activity is classified as the following: '+' corresponds to an IC 50 >500 nM and '++' corresponds to an IC 50 ⁇ 500 nM.
  • '+' corresponds to an IC 50 >4 ⁇
  • '++' corresponds to an IC 50 in the range of 1 ⁇ to 4 ⁇
  • '+++' corresponds to an IC 50 ⁇ 1 ⁇ .
  • Representative compounds (e.g. Example 2) caused dose dependent inhibition of phosphorylation of cdc2 in HT29 and HCT-116 cells, and inhibition of proliferation of
  • HT29 cells at concentrations ⁇ 10 ⁇ .
  • inhibitor MK1775 has a CLint value of 28uL/min/mg .
  • microwave experiments were carried out using a CEM DiscoverTM/Explorer24TM system controlled by Synergy 1.5 software. In other cases a Biotage InitiatorTM Eight was used. Both machines give good reproducibility and control at temperature ranges from 60-250°C and pressures of up to maximum of 20 bar.
  • Biotage KP-Sil SNAP cartridge columns (10-340 g) were used along with the stated solvent system and an appropriate solvent gradient depending on compound polarity. In the case of more polar and basic compounds, Biotage KP-NH SNAP cartridge columns (11 g) were used.
  • Method A The system consists of an Agilent Technologies 6140 single quadrupole mass spectrometer linked to an Agilent Technologies 1290 Infinity LC system with UV diode array detector and autosampler.
  • the spectrometer consists of a multimode ionization source ( electrospray and atmospheric pressure chemical ionizations) operating in positive and negative ion mode.
  • LCMS experiments were performed on each sample submitted using the following conditions: LC Column: Zorbax Eclipse Plus C18 RRHD 1.8 micron 50 x 2.1 mm maintained at 40°C. Mobile phases: A) 0.1% (v/v) formic acid in water; B) 0.1% (v/v) formic acid in acetonitrile .
  • Method B The system consisted of a ThermoFinnigan LCQ Advantage Mass Spectrometer with Surveyor LC system and 200 position autosampler. The LC system was coupled to an inline Surveyor DAD detector and ESI source operating in positive and negative ion mode. LCMS experiments were performed on each sample submitted using the following conditions: LC Column: Luna 3 micron C18 50 x 2 mm.
  • the system consisted of an Agilent Technologies 6120 single quadrupole mass spectrometer linked to an Agilent Technologies 1200 Preparative LC system with Multiple Wavelength detector and autosampler.
  • Acidic conditions LC Column: Waters XBridgeTM Prep C18 5 ⁇ OBDTM 19 x 50 mm column at RT .
  • Mobile phase A) Water 0.1% (v/v) formic acid in water; B) 0.1% (v/v) formic acid in 95:5, acetonitrile/water .
  • Total experiment time was ca. 10 min and an example method is given:
  • Example 1 8-Methyl-2- ( ( 4- ( 4-methylpiperazin-l- yl ) phenyl ) amino ) -6 -phenylpyrido [ 2 , 3-d] pyrimidin-5 ( 8H) -one
  • Step 1 Ethyl 4- ( ( 3-ethoxy-3-oxopropyl ) (methyl ) amino) -2- (methylthio ) pyrimidine-5-carboxylate : Ethyl 4-chloro-2- (methylthio ) pyrimidine-5-carboxylate (426 mg, 1.833 mmol) [commercially available] was added to a stirred solution of ethyl 3- (methylamino ) propanoate (240 mg, 1.83 mmol) [prepared according to the literature procedure: J. Org. Chem., 1985, 50, p3979-3982; J. Med. Chem., 2005, 48, p4100-4110] in THF (5.0 mL) at RT under nitrogen.
  • Step 2 Ethyl 5-hydroxy-8-methyl-2- (methylthio) -7, 8- dihydropyrido [ 2 , 3-d] pyrimidine-6 -carboxylate : Potassium tert-butoxide solution (1.0 M in THF) (0.767 mL, 0.767 mmol) was added to a stirred solution of ethyl 4-((3- ethoxy-3-oxopropyl ) (methyl) amino) -2-
  • Step 3 Ethyl 8-methyl-2- (methylthio ) -5-oxo-5 , 8- dihydropyrido [ 2 , 3-d] pyrimidine-6-carboxylate : Bromine
  • Step 4 8-Methyl-2- (methylthio ) -5-oxo-5 , 8- dihydropyrido [ 2 , 3-d] pyrimidine-6-carboxylic acid: 1M HC1 (aq) solution (17.4 mL, 17.4 mmol) was added to a stirred solution of ethyl 8-methyl-2- (methylthio) -5-oxo-5, 8- dihydropyrido [ 2 , 3-d] pyrimidine-6-carboxylate (1.62 g, 5.79 mmol) in 1,4-dioxane (16.0 mL) at RT under nitrogen. The temperature was increased to 95 °C.
  • Step 6 8-Methyl-2- (methylthio) -6 -phenylpyrido [2, 3- d] pyrimidin-5 ( 8H) -one : PdCl 2 (dppf ) -DCM adduct (10.4 mg, 0.013 mmol) was added to a pre-degassed solution of 6- iodo-8-methyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) - one (42.3 mg, 0.127 mmol), phenylboronic acid (20.1 mg, 0.165 mmol) and sodium carbonate (40.4 mg, 0.381 mmol) in DMF (2.0 mL) in a 10 mL vial.
  • Step 7 8-Methyl-2- ( ( 4- ( 4-methylpiperazin-l- yl ) phenyl ) amino ) -6 -phenylpyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) - one: mCPBA ( ⁇ 77% pure) (17.3 mg, 0.077 mmol) in DCM (1.0 mL) was added to a stirred solution of 8-methyl-2- (methylthio ) -6 -phenylpyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one
  • Step 1 6- ( 2 , 6-Dichlorophenyl ) -8-methyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one : Pd2 (dba ) 3 (4.2 mg, 4.6 pmol) was added to a pre-degassed solution of 6 -iodo-8-methyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one (153 mg, 0.460 mmol), (2,6- dichlorophenyl ) boronic acid (114 mg, 0.599 mmol), SPhos (7.6 mg, 0.018 mmol) and potassium phosphate, tribasic (293 mg, 1.38 mmol) in a, , -trifluorotoluene (2.0 mL) in a 10 mL vial. The vessel was sealed and the reaction mixture was stirred at 80 °C. After 16 h
  • Step 1 6- ( 2-Chlorophenyl ) -8-methyl-2-
  • Step 2 6- ( 2-Chlorophenyl ) -8-methyl-2- ( ( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one: mCPBA ( ⁇ 77% pure) (16.4 mg, 0.073 mmol) in DCM (0.5 mL) was added to a stirred solution of 6-(2- chlorophenyl ) -8-methyl-2- (methylthio ) pyrido [2,3- d] pyrimidin-5 ( 8H) -one (20.1 mg, 0.063 mmol) in DCM (2.0 mL) at 0 °C under nitrogen.
  • Step 1 6- ( 2-Methoxyphenyl ) -8-methyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one : Pd 2 (dba)3 (3.3 mg, 3.6 pmol) was added to a pre-degassed solution of 6 -iodo-8-methyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one (30.0 mg, 0.090 mmol), ( 2-methoxyphenyl ) boronic acid (41.1 mg, 0.270 mmol), SPhos (3.0 mg, 7.2 pmol) and potassium phosphate, tribasic (57.3 mg, 0.270 mmol) in toluene (1.0 mL) in a 4 mL vial.
  • Step 2 6- ( 2-Methoxyphenyl ) -8-methyl-2- (( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one: mCPBA ( ⁇ 77% pure) (16.8 mg, 0.075 mmol) in DCM (0.5 mL) was added to a stirred solution of 6-(2- methoxyphenyl ) -8-methyl-2- (methylthio ) pyrido [2,3- d] pyrimidin-5 ( 8H) -one (20.3 mg, 0.065 mmol) in DCM (2.0 mL) at 0 °C under nitrogen.
  • Step 1 8-Methyl-2- (methylthio) -6- (o-tolyl) pyrido [2, 3- d] pyrimidin-5 ( 8H) -one : Pd 2 (dba)3 (3.3 mg, 3.6 pmol) was added to a pre-degassed solution of 6-iodo-8-methyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one (30.0 mg, 0.090 mmol), o-tolylboronic acid (36.7 mg, 0.270 mmol), SPhos (3.0 mg, 7.2 pmol) and potassium phosphate, tribasic (57.3 mg, 0.270 mmol) in toluene (1.0 mL) in a 4 mL vial.
  • Step 2 8-Methyl-2- (methylsulfonyl ) -6- ( o- tolyl ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H) -one : mCPBA ( ⁇ 77% pure) (49.0 mg, 0.219 mmol) was added to a stirred solution of 8-methyl-2- (methylthio) -6- (o-tolyl) pyrido [2,3- d] pyrimidin-5 ( 8H) -one (16.9 mg, 0.057 mmol) in DCM (1.5 mL) at RT under nitrogen. After 16 h, the reaction mixture was partitioned between DCM and sodium
  • Step 3 8-Methyl-2- ( ( 4- ( 4-methylpiperazin-l- yl ) phenyl ) amino ) -6- (o-tolyl) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) - one: TFA (0.021 mL, 0.272 mmol) was added to a stirred solution of 8-methyl-2- (methylsulfonyl ) -6- ( o- tolyl ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H) -one (17.9 mg, 0.054 mmol) and 4- ( 4-methylpiperazin-l-yl ) aniline (11.4 mg, 0.060 mmol) in 2-propanol (1.0 mL) in a 10 mL vial.
  • the vessel was sealed and irradiated at 120 °C for 20 min (CEM Discover/Explorer24 ) .
  • LCMS analysis showed residual starting material.
  • the reaction was rerun under the same conditions.
  • LCMS analysis showed further conversion, but still some starting material.
  • the reaction was rerun at 150 °C for 20 min.
  • the solvents were removed in vacuo and the remaining residue was partitioned between
  • Example 6 6- ( 2 , 4-Dichlorophenyl ) -8-methyl-2- (( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one
  • Step 1 6- ( 2 , 4-Dichlorophenyl ) -8-methyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one : Pd 2 (dba ) 3 (3.3 mg, 3.6 pmol) was added to a pre-degassed solution of 6 -iodo-8-methyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one (30.0 mg, 0.090 mmol), (2,4- dichlorophenyl ) boronic acid (172 mg, 0.900 mmol), SPhos (3.0 mg, 7.2 pmol) and potassium phosphate, tribasic (57.3 mg, 0.270 mmol) in toluene (1.0 mL) in a 4 mL vial.
  • Step 2 6- ( 2 , 4-Dichlorophenyl ) -8-methyl-2- ( ( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one: mCPBA ( ⁇ 77% pure) (19.0 mg, 0.085 mmol) in DCM (0.5 mL) was added to a stirred solution of 6-(2,4- dichlorophenyl ) -8-methyl-2- (methylthio ) pyrido [2,3- d] pyrimidin-5 ( 8H) -one (22.1 mg, 0.063 mmol) in DCM (2.0 mL) at 0 °C under nitrogen. After 30 min, the
  • Example 7 6- ( 2 , 5-Dichlorophenyl ) -8-methyl-2- ( ( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one
  • Step 1 6- ( 2 , 5-Dichlorophenyl ) -8-methyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one : Pd 2 (dba ) 3 (3.3 mg, 3.6 pmol) was added to a pre-degassed solution of 6 -iodo-8-methyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one (30.0 mg, 0.090 mmol), (2,5- dichlorophenyl ) boronic acid (172 mg, 0.900 mmol), SPhos (3.0 mg, 7.2 pmol) and potassium phosphate, tribasic (57.3 mg, 0.270 mmol) in toluene (1.0 mL) in a 4 mL vial.
  • Step 2 6- ( 2 , 5-Dichlorophenyl ) -8-methyl-2- (( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one: mCPBA ( ⁇ 77% pure) (25.0 mg, 0.112 mmol) in DCM (0.5 mL) was added to a stirred solution of 6-(2,5- dichlorophenyl ) -8-methyl-2- (methylthio ) pyrido [2,3- d]pyrimidin-5 (8H) -one (29.1 mg, 0.083 mmol) in DCM (2.0 mL) at 0 °C under nitrogen.
  • Example 8 6- ( 2 , 3-Dichlorophenyl ) -8-methyl-2- ( ( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin-
  • Step 1 6- ( 2 , 3-Dichlorophenyl ) -8-methyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one : Pd 2 (dba)3 (3.3 mg, 3.6 pmol) was added to a pre-degassed solution of 6 -iodo-8-methyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one (30.0 mg, 0.090 mmol), (2,3- dichlorophenyl ) boronic acid (172 mg, 0.900 mmol), SPhos (3.0 mg, 7.2 pmol) and potassium phosphate, tribasic
  • Step 2 6- ( 2 , 3-Dichlorophenyl ) -8-methyl-2- (( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one: mCPBA ( ⁇ 77% pure) (20.4 mg, 0.091 mmol) in DCM (0.5 mL) was added to a stirred solution of 6-(2,3- dichlorophenyl ) -8-methyl-2- (methylthio ) pyrido [2,3- d] pyrimidin-5 ( 8H) -one (20.8 mg, 0.059 mmol) in DCM (2.0 mL) at 0 °C under nitrogen.
  • Step 1 8-Methyl-2- (methylthio) -6- (thiophen-3- yl) pyrido [2, 3-d] pyrimidin-5 ( 8H) -one : Pd 2 (dba)3 (3.3 mg,
  • Step 2 8-Methyl-2- ( ( 4- ( 4-methylpiperazin-l- yl ) phenyl ) amino ) -6 - ( thiophen-3-yl ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one: mCPBA ( ⁇ 77% pure) (21.0 mg, 0.094 mmol) was added to a stirred solution of 8-methyl-2- (methylthio) -6- (thiophen-3-yl ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H) -one (17.6 mg, 0.061 mmol) in toluene (2.0 mL) at RT under nitrogen.
  • Example 10 8-Methyl-6- ( 5-methyl-l , 3 , 4-oxadiazol-2-yl ) -2- ( ( 4- ( piperaz in- 1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 (8H) -one
  • Step 1 Ethyl 2 , 4-dichloropyrimidine-5-carboxylate : Ethyl 4-chloro-2- (methylthio ) pyrimidine-5-carboxylate (50.0 g, 215 mmol) in sulfuryl chloride (50.0 mL, 615 mmol) was heated at 50 °C under an atmosphere of nitrogen. After 2 hours, the reaction mixture was cooled and carefully poured onto saturated sodium bicarbonate (aq) solution and was extracted using DCM (x 3) . The combined organic phase was dried (Phase Separator) and the solvents were removed in vacuo to give title compound (crude, ca. 50 g) that carried through to the next step without further purification.
  • ⁇ NMR (500 MHz, CDCI 3 ) ⁇ 9.02 (s, 1H) , 4.46 (dd, 2H) , 1.43 (t, 3H) .
  • Step 2 Ethyl 2-chloro-4- ( ( 3-ethoxy-3- oxopropyl) (methyl ) amino ) pyrimidine-5-carboxylate : Ethyl 3- (methylamino ) propanoate (17.2 g, 131 mmol) in DCM (100 mL) was added to a stirred solution of ethyl 2,4- dichloropyrimidine-5-carboxylate (crude, 25.2 g, 114 mmol) and DIPEA (43.8 mL, 251 mmol) in DCM (100 mL) in a 500 mL RB flask at 0 °C under nitrogen.
  • Step 3 Ethyl 2-chloro-5-hydroxy-8-methyl-7 , 8- dihydropyrido [ 2 , 3-d] pyrimidine-6-carboxylate : Potassium tert-butoxide (1 M in THF) solution (114 mL, 114 mmol) was added to a stirred solution of ethyl 2-chloro-4- ( ( 3- ethoxy-3-oxopropyl ) (methyl ) amino ) pyrimidine-5-carboxylate (36.0 g, 114 mmol) in toluene (360 mL) at RT under nitrogen.
  • Step 4 Ethyl 2-chloro-8-methyl-5-oxo-5 , 8- dihydropyrido [ 2 , 3-d] pyrimidine-6 -carboxylate : Bromine (5.87 mL, 114 mmol) in chloroform (200 mL) was added to a stirred solution of ethyl 2-chloro-5-hydroxy-8-methyl- 7 , 8-dihydropyrido [ 2 , 3-d] pyrimidine-6 -carboxylate (30.7 g, 114 mmol) and triethylamine (47.7 mL, 342 mmol) in chloroform (400 mL) at 0 °C under nitrogen.
  • Step 5 Ethyl 2- ( ( 4- ( 4- ( tert-butoxycarbonyl ) piperaz in-1- yl ) phenyl ) amino ) -8-methyl-5-oxo-5 , 8-dihydropyrido [2,3- d] pyrimidine-6 -carboxylate : tert-Butyl 4- (4- aminophenyl ) piperazine-l-carboxylate (1.12 g, 4.03 mmol) was added to a stirred solution of ethyl 2-chloro-8- methyl-5-oxo-5 , 8-dihydropyrido [ 2 , 3-d] pyrimidine-6 - carboxylate (1.08 g, 4.03 mmol) in DMF (10.0 mL) in a 50 mL RB flask.
  • Step 6 Sodium 2- (( 4- ( 4- (tert-butoxycarbonyl) piperazin-1- yl ) phenyl ) amino ) -8-methyl-5-oxo-5 , 8-dihydropyrido [2,3- d] pyrimidine-6 -carboxylate : 2 M sodium hydroxide (aq) solution (4.92 mL, 9.83 mmol) was added to a stirred solution of ethyl 2- (( 4- ( 4- ( tert- butoxycarbonyl ) piperaz in-1-yl ) phenyl ) amino ) -8-methy1-5- oxo-5 , 8-dihydropyrido [ 2 , 3-d] pyrimidine-6 -carboxylate (1.0 g, 1.966 mmol) in 1,4-dioxane (5.0 mL) in a 50 mL RB flask. The reaction mixture was heated at 50 °C. After 16
  • Step 7 8-Methyl-6- ( 5-methyl-l , 3 , 4-oxadiazol-2-yl ) -2- ( ( 4- (piperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one: Acetohydrazide (8.1 mg, 0.109 mmol) was added to sodium 2- (( 4- ( 4- (tert-butoxycarbonyl) piperazin-1- yl ) phenyl ) amino ) -8-methyl-5-oxo-5 , 8-dihydropyrido [2,3- d] pyrimidine-6 -carboxylate (50.0 mg, 0.100 mmol) in POCI 3 (1.0 mL, 10.7 mmol) at RT in a 10 mL vial. The vessel was sealed and the temperature was increased to 100 °C with stirring. After 2 h, the reaction mixture was poured onto ice water, neutralised using 2 M Na
  • Step 1 6- ( 3-Chloropyridin-2-yl ) -8-methyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one :
  • Example 12 6- ( 2 , 6 -Dichlorophenyl ) -8-methyl-2- ( ( 4- (piperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one :
  • Step 1 tert-Butyl 4- ( 4- ( ( 6-iodo-8-methyl-5-oxo-5, 8- dihydropyrido [ 2 , 3-d] pyrimidin-2- yl ) amino ) phenyl ) piperaz ine-l-carboxylate : To a stirred solution of 6 -iodo-8-methyl-2- (methylthio ) pyrido [ 2 , 3- d] pyrimidin-5 ( 8H) -one (100 mg, 0.30 mmol) in toluene (2 mL) at 0 °C under nitrogen was added mCPBA ( ⁇ 77% pure) (78 mg, 0.35 mmol) in DCM (2 mL).
  • Step 2 tert-Butyl 4- ( 4- (( 6- ( 2 , 6-dichlorophenyl ) -8- methyl-5-oxo-5 , 8-dihydropyrido [ 2 , 3-d] pyrimidin-2- yl ) amino ) phenyl ) piperaz ine-l-carboxylate : Pd 2 (dba)3 (3.9 mg, 3.91 pmol) was added to a pre-degassed solution of 6- iodo-8-methyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) - one (44 mg, 0.078 mmol), ( 2 , 6-dichlorophenyl ) boronic acid (149 mg, 0.782 mmol), SPhos (2.6 mg, 6.26 pmol) and potassium phosphate, tribasic (50 mg, 0.235 mmol) in a, , -trifluorotoluene (
  • Step 3 6- ( 2 , 6-Dichlorophenyl ) -8-methyl-2- ( ( 4- (piperazin 1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one : To a solution of tert-butyl 4- ( 4- (( 6- ( 2 , 6-dichlorophenyl ) -8- methyl-5-oxo-5 , 8-dihydropyrido [ 2 , 3-d] pyrimidin-2- yl ) amino ) phenyl ) piperaz ine-l-carboxylate (20 mg, 0.034 mmol) in DCM (2 mL) at 0 °C was added TFA (1 mL, 12.98 mmol) .
  • Example 13 8-Allyl-6- ( 2 , 6 -dichlorophenyl ) -2- ( ( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one
  • Step 1 Ethyl 3- ( allylamino ) propanoate : Prop-2-en-l-amine (15.1 mL, 202 mmol) was added dropwise to a solution of ethyl acrylate (20 mL, 184 mmol) in EtOH (100 mL) and the resulting solution was stirred for 18 h at RT . The reaction mixture was concentrated in vacuo to afford the title compound (28 g, 97%) which was used without further purification.
  • Step 2 Ethyl 4- ( allyl ( 3-ethoxy-3-oxopropyl ) amino ) -2- (methylthio ) pyrimidine-5-carboxylate : Following the procedure for ethyl 4- ( ( 3-ethoxy-3- oxopropyl ) (methyl ) amino ) -2- (methylthio ) pyrimidine-5- carboxylate, ethyl 3- ( allylamino ) propanoate (28 g, 178 mmol) was reacted to afford the title compound as a yellow oil which was used without further purification (61 g, 97%).
  • Step 3 Ethyl 8-allyl-5-hydroxy-2- (methylthio ) -7 , 8- dihydropyrido [ 2 , 3-d] pyrimidine-6 -carboxylate : Following the procedure for ethyl 5-hydroxy-8-methyl-2-
  • Step 4 Ethyl 8-allyl-2- (methylthio ) -5-oxo-5 , 8- dihydropyrido [ 2 , 3-d] pyrimidine-6 -carboxylate : Following the procedure for ethyl 8-methyl-2- (methylthio ) -5-oxo- 5 , 8-dihydropyrido [ 2 , 3-d] pyrimidine-6 -carboxylate , ethyl 8-allyl-5-hydroxy-2- (methylthio ) -7 , 8-dihydropyrido [2,3- d] pyrimidine-6 -carboxylate (20.0 g, 65 mmol) was reacted to afford the title compound (19 g, 96%) as a yellow solid which was used without further purification.
  • Step 5 8-Allyl-2- (methylthio ) -5-oxo-5 , 8- dihydropyrido [ 2 , 3-d] pyrimidine-6 -carboxylie acid :
  • Step 6 8-Allyl-6- ( 2 , 6 -dichlorophenyl ) -2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one : To a solution of 8-allyl-2- (methylthio ) -5-OXO-5 , 8- dihydropyrido [ 2 , 3-d] pyrimidine-6-carboxylic acid (50 mg, 0.18 mmol) in toluene (2 mL) and DMA (0.22 mL) was added 1 , 3-dichloro-2-iodobenzene (98 mg, 0.36 mmol) and silver carbonate (49.7 mg, 0.18 mmol) .
  • the suspension was degassed for 5 min and (oxybis(2,l- phenylene) ) bis (diphenylphosphine) (9.7 mg, 0.018 mmol) and palladium (II) chloride (1.6 mg, 9.0 pmol) added.
  • the reaction mixture was then heated in a microwave
  • Step 7 8-Allyl-6- ( 2 , 6 -dichlorophenyl ) -2- ( ( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one: Following the procedure for 8-methyl-2- ( ( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) -6 -phenylpyrido [2,3- d] pyrimidin-5 ( 8H) -one, 8-allyl-6- (2, 6 -dichlorophenyl ) -2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one (50.0 mg, 0.13 mmol) was reacted to give crude material which was purified by flash chromatography using a KP-NH column (0- 100% EtOAc in cyclohexane) to afford the title compound (4.5
  • Step 1 Ethyl 3- (( 4-methoxybenzyl ) amino ) propanoate :
  • Step 2 Ethyl 4- ( ( 3-ethoxy-3-oxopropyl ) (4- methoxybenzyl ) amino ) -2- (methylthio ) pyrimidine-5- carboxylate :
  • Step 3 Ethyl 5-hydroxy-8- ( 4-methoxybenzyl ) -2- (methylthio ) -7 , 8-dihydropyrido [ 2 , 3-d] pyrimidine-6 - carboxylate: Following the procedure for ethyl 5-hydroxy- 8-methyl-2- (methylthio) -7, 8-dihydropyrido [2,3- d] pyrimidine-6 -carboxylate , ethyl 4- ( ( 3-ethoxy-3- oxopropyl ) ( 4-methoxybenzyl ) amino ) -2-
  • Step 4 Ethyl 8- ( 4-methoxybenzyl ) -2- (methylthio ) -5-oxo- 5, 8-dihydropyrido [ 2 , 3-d] pyrimidine-6 -carboxylate :
  • Step 5 8- ( 4-Methoxybenzyl ) -2- (methylthio ) -5-oxo-5 , 8- dihydropyrido [ 2 , 3-d] pyrimidine-6 -carboxylie acid :
  • Step 7 6- ( 2 , 6-Dichlorophenyl ) -8- ( 4-methoxybenzyl ) -2- (( 4- ( 4-methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [2,3- d] pyrimidin-5 ( 8H ) -one :
  • Example 16 6- ( 2 , 6 -Dichlorophenyl ) -8-ethyl-2- ( ( 4- ( 4- methylpiperaz in- 1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 (8H) -one
  • Example 17 Ethyl 2- ( 6- ( 2 , 6 -dichlorophenyl ) -2- ( ( 4- ( 4- methylpiperaz in-l-yl ) phenyl ) amino ) -5-oxopyrido [2,3- d] pyrimidin-8 (5H -yl) acetate
  • Example 19 6- ( 2 , 6 -Dichlorophenyl ) -8- ( 3-hydroxypropyl ) -2- ( ( 4- ( 4-methylpiperaz in-l-yl ) phenyl ) amino ) pyrido [2,3- d] pyrimidin-5 ( 8H -one
  • Example 20 6- ( 2 , 5-Dimethyl-lH-pyrrol-l-yl ) -8- ( 4- methoxybenzyl ) -2- ( ( 4- ( 4-methylpiperazin-l-
  • Step 1 tert-Butyl ( 8- ( 4-methoxybenzyl ) -2- (methylthio) -5- oxo-5 , 8-dihydropyrido [ 2 , 3-d] pyrimidin-6 -yl ) carbamate : To a yellow solution of 8- ( 4-methoxybenzyl ) -2- (methylthio ) - 5-oxo-5, 8-dihydropyrido [ 2 , 3-d] pyrimidine-6 -carboxy1 ic acid (0.5 g, 1.40 mmol) in tert-butanol (10 mL) was added DIPEA (0.244 mL, 1.40 mmol) followed by diphenyl
  • Step 2 tert-Butyl ( 8- ( 4-methoxybenzyl ) -2- (( 4- ( 4- methylpiperaz in-l-yl ) phenyl ) amino ) -5-oxo-5, 8- dihydropyrido [ 2 , 3-d] pyrimidin-6-yl ) carbamate : Following the procedure for 8-methyl-2- ( ( 4- ( 4-methylpiperazin-l- yl ) phenyl ) amino ) -6 -phenylpyrido [ 2 , 3-d] pyrimidin-5 ( 8H) - one, tert-butyl ( 8- ( 4-methoxybenzyl ) -2- (methylthio ) -5- oxo-5 , 8-dihydropyrido [ 2 , 3-d] pyrimidin-6 -yl ) carbamate (0.084 mg, 0.19 mmol) was reacted to give the title compound (74 mg, 67%) as
  • Step 3 6-Amino-8- ( 4-methoxybenzyl ) -2- (( 4- ( 4- methylpiperaz in-l-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one: To a solution of tert-butyl (8-(4- methoxybenzyl ) -2- ( ( 4- ( 4-methylpiperaz in-1- yl ) phenyl ) amino ) -5-oxo-5, 8-dihydropyrido [ 2 , 3-d] pyrimidin- 6 -yl ) carbamate (74 mg, 0.131 mmol) in DCM (1 mL) was added TFA (1 mL) .
  • Step 4 6- ( 2 , 5-Dimethyl-lH-pyrrol-l-yl ) -8- ( 4- methoxybenzyl ) -2- ( ( 4- ( 4-methylpiperaz in-l- yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one : To a solution of 6-amino-8- ( 4-methoxybenzyl ) -2- (( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one (0.05 g, 0.106 mmol) in DMF (1 mL) was added 4- methylbenzenesulfonic acid (0.027 g, 0.159 mmol) and hexane-2 , 5-dione (0.012 g, 0.106 mmol) . The resulting mixture was heated for 30 min at 120° C under microwave conditions,
  • Example 21 6- ( 2 , 6 -Dichlorophenyl ) -2- ( ( 6- (piperazin-1- yl ) pyridin-3-yl ) amino pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one
  • Step 1 6- ( 2 , 6-Dichlorophenyl ) -8- ( 4-methoxybenzyl ) -2- ( ( 6- (piperaz in-1-yl ) pyridin-3-yl ) amino ) pyrido [2,3- d] pyrimidin-5 ( 8H) -one :
  • Step 2 6- ( 2 , 6-Dichlorophenyl ) -2- ( ( 6- (piperazin-1- yl ) pyridin-3-yl ) amino ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one : Following the procedure for 6- ( 2 , 6 -dichlorophenyl ) -2- ( ( 4- ( 4-methylpiperaz in-l-yl ) phenyl ) amino ) pyrido [2,3- d] pyrimidin-5 ( 8H) -one, 6- ( 2 , 6 -dichlorophenyl ) -8- ( 4- methoxybenzyl ) -2- ( (6- (piperaz in-l-yl ) pyridin-3- yl ) amino ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one was reacted to give crude material which was purified by preparative HPLC to afford the
  • Example 22 6- ( 2 , 6-Dichlorophenyl ) -2- (( 4- ( 2- (diethylamino ) ethoxy ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 (8H) -one
  • Step 1 6- (2, 6-Dichlorophenyl) -2- ( (4- (2- ( diethylamino ) ethoxy ) phenyl ) amino ) -8- ( 4- methoxybenzyl ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H) -one : Following the procedure for 8-methyl-2- ( ( 4- ( 4-methylpiperazin-l- yl ) phenyl ) amino ) -6 -phenylpyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) - one, 6- ( 2 , 6 -dichlorophenyl ) -8- ( 4-methoxybenzyl ) -2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one (55 mg, 0.116 mmol) was reacted with 4- (2- (2-
  • Step 2 6- ( 2 , 6-Dichlorophenyl ) -2- (( 4- ( 2-
  • Step 1 6- ( 2 , 6-Dichlorophenyl ) -8- ( 4-methoxybenzyl ) -2- ( (pyridin-4-ylmethyl ) amino ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H )
  • Step 2 6- ( 2 , 6-Dichlorophenyl ) -2- ( (pyridin-4- ylmethyl ) amino ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one :
  • Example 24 6- ( 2 , 6-Dichlorophenyl ) -2- (( 1-methylpiperidin- 4-yl ) amino ) pyrido [ 2 3-d] pyrimidin-5 ( 8H ) -one
  • Step 1 6- ( 2 , 6-Dichlorophenyl ) -8- ( 4-methoxybenzyl ) -2- (( 1 methylpiperidin-4-yl ) amino ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H) - one: Following the procedure for 8-methyl-2- ( ( 4- ( 4- methylpiperaz in-l-yl ) phenyl ) amino ) -6 -phenylpyrido [2,3- d] pyrimidin-5 ( 8H) -one, 6- ( 2 , 6 -dichlorophenyl ) -8- ( 4- methoxybenzyl ) -2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one (55 mg, 0.116 mmol) was reacted with 1- methylpiperidin-4-amine (13.2 mg, 0.116 mmol) instead of 4- ( 4-methylpiperazin-l-yl )
  • Step 2 6- ( 2 , 6-dichlorophenyl ) -2- (( l-methylpiperidin-4- yl ) amino ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one : Following the procedure for 6- ( 2 , 6-dichlorophenyl ) -2- (( 4- ( 4- methylpiperaz in- 1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one, 6- ( 2 , 6-dichlorophenyl ) -8- ( 4-methoxybenzy1 ) -2- ( ( l-methylpiperidin-4-yl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one was reacted to give crude material which was purified by HPLC to afford the title compound (5.1 mg, 17%) .
  • Example 25 7-Amino-6- ( 2-chlorophenyl ) -8-methyl-2- ( ( 4- ( 4- methylpiperaz in- 1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 (8H) -one
  • Step 1 Ethyl 4- (methylamino ) -2- (methylthio ) pyrimidine-5 carboxylate: To a solution of ethyl 4-chloro-2- (methylthio ) pyrimidine-5-carboxylate (10 g, 43 mmol) in anhydrous THF (100 mL) was added triethylamine (6.0 mL, 43 mmol) and monomethylamine (2M in THF, 22 mL, 43 mmol) The reaction mixture was heated to 50 °C overnight, then diluted with water (100 mL) and extracted into ethyl acetate (3 x 100 mL) .
  • Step 2 4- (Methylamino ) -2- (methylthio ) pyrimidine-5- carboxylic acid: Lithium hydroxide (211 mg, 8.8 mmol) was added to ethyl 4- (methylamino ) -2- (methylthio ) pyrimidine- 5-carboxylate (1.0 g, 4.4 mmol) in a mixture of methanol (8 mL) and water (4 mL) to give a white suspension. This was heated to 60 °C for 3 h and then concentrated to dryness under reduced pressure. The residue was re- dissolved in water (25 mL) and acidified with acetic acid to pH 5. The precipitated solid was isolated by
  • Step 3 4- (Methylamino ) -2- (methylthio ) pyrimidine-5- carbonyl fluoride: To a solution of 4- (methylamino ) -2- (methylthio ) pyrimidine-5-carboxylic acid (610 mg, 3.1 mmol) and triethylamine (470 pL, 3.4 mmol) in anhydrous dichloromethane (6 mL) was added dropwise a solution of cyanuric fluoride (525 pL, 6.1 mmol) in anhydrous
  • Step 4 7-Amino-6- ( 2-chlorophenyl ) -8-methyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one : A solution of 2- ( 2-chlorophenyl ) acetonitrile (34 mg, 0.22 mmol) in anhydrous DMF (1 mL) was cooled to 0 °C followed by the addition of sodium hydride (60% in mineral oil, 18 mg, 0.45 mmol) . The resulting mixture was stirred at RT for 10 min before re-cooling to 0 °C.
  • the reaction mixture was heated to 100 °C for 60 min then allowed to cool to RT, diluted with water (5 mL) and extracted into ethyl acetate (3 x 5 mL) .
  • the combined organic phases were washed with 1:1 water/brine (3 x 5 mL) , dried over Na 2 SC>4, filtered, and concentrated to dryness under reduced pressure.
  • the residue was purified by preparative HPLC to give the title compound (1.0 mg, 3%) as a pale yellow solid.
  • Example 26 6- ( 2-Chlorophenyl ) -7 , 8-dimethyl-2- ( ( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one
  • Step 1 2- ( 2-Chlorophenyl ) -1- ( 4- (methylamino ) -2- (methylthio ) pyrimidin-5-yl ) buta-1 , 3-diene-l , 3-diol :
  • a solution of 1- ( 2-chlorophenyl ) propan-2-one (170 mg, 0.99 mmol) in anhydrous DMF (2 mL) was cooled to 0 °C followed by the addition of sodium hydride (60% in mineral oil, 80 mg, 1.99 mmol) . The resulting mixture was stirred at RT for 10 min before re-cooling to 0 °C.
  • Step 2 6- ( 2-Chlorophenyl ) -7 , 8-dimethyl-2-
  • Step 3 6- ( 2-Chlorophenyl ) -7 , 8-dimethyl-2- ( ( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one: A suspension of 6 - ( 2-chlorophenyl ) -7 , 8- dimethyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one (80 mg, 0.24 mmol) in anhydrous DCM (1.5 mL) was cooled to 0 °C.
  • Step 1 2-Chloro-6- ( 2-chlorophenyl ) -7-methyl-5H- pyrano [ 2 , 3-d] pyrimidin-5-one :
  • a solution of l-(2- chlorophenyl ) propan-2-one (80 mg, 0.47 mmol) in anhydrous THF (1 mL) was cooled to 0 °C followed by the dropwise addition of tert-butylmagnesium chloride (2 M in diethyl ether, 0.24 mL, 0.47 mmol) and stirred at RT for 10 min.
  • Step 2 tert-Butyl 4- ( 4- (( 6- ( 2-chlorophenyl ) -7-methyl-5- oxo-5H-pyrano [ 2 , 3-d] pyrimidin-2- yl ) amino ) phenyl ) piperaz ine-l-carboxylate : A solution of 2-chloro-6- ( 2-chlorophenyl ) -7-methyl-5H-pyrano [2,3- d] pyrimidin-5-one (45 mg, 0.15 mmol), tert-butyl 4- (4- aminophenyl ) piperazine-l-carboxylate (41 mg, 0.15 mmol) and DIPEA (51 pL, 0.29 mmol) in anhydrous DMF (1 mL ) was heated to 100 °C under a nitrogen atmosphere for 60 min.
  • reaction mixture was allowed to cool to RT, diluted with water (5 mL) and extracted into ethyl acetate (3 x 5 mL) .
  • the combined organic phases were washed with 1:1 water/brine (3 x 5 mL) , dried over Na 2 SC>4, filtered, and concentrated to dryness under reduced pressure to give the title compound (42 mg, 52%) as a yellow solid.
  • Step 3 6- ( 2-Chlorophenyl ) -7-methyl-2- ( ( 4- (piperazin-1- yl ) phenyl ) amino ) -5H-pyrano [ 2 , 3-d] pyrimidin-5-one , 2HC1 : To a solution of tert-butyl 4- ( 4- (( 6- ( 2-chlorophenyl ) -7- methyl-5-oxo-5H-pyrano [ 2 , 3-d] pyrimidin-2- yl ) amino ) phenyl ) piperaz ine-l-carboxylate (42 mg, 0.077 mmol) in dichloromethane (1 mL) was added dropwise 4 M HC1 in 1,4-dioxane (1 mL) and the mixture stirred at RT for 90 min. The reaction mixture was then diluted with diethyl ether (5 mL) and slurried at RT for 15 min, before removal of the supernatant solvent by pipet
  • Example 28 6- ( 2-Chlorophenyl ) -2- (( 4- (piperazin-1- yl ) phenyl ) amino ) -5H-pyrano [ 2 , 3-d] pyrimidin-5-one .
  • Step 1 2- ( 2-Chlorophenyl ) acetaldehyde : A solution of methyl 2- ( 2-chlorophenyl ) acetate (3.0 g, 16 mmol) in anhydrous dichloromethane (30 mL) was cooled to -78 °C followed by the dropwise addition of DIBAL-H (1 M in toluene, 16.3 mL, 16 mmol) over a period of 30 min. The reaction mixture was stirred at -78 °C under a nitrogen atmosphere for a further 30 min, then quenched by the addition of methanol (15 mL) and poured into saturated sodium potassium tartrate (aq) solution (100 mL) .
  • Step 2 2-Chloro-6- ( 2-chlorophenyl ) -5H-pyrano [ 2 , 3- d] pyrimidin-5-one :
  • a solution of 2- (2- chlorophenyl ) acetaldehyde (730 mg, 4.7 mmol) in anhydrous THF (6 mL) was cooled to 0 °C followed by the dropwise addition of tert-butylmagnesium chloride (2 M in diethyl ether, 4.7 mL, 9.5 mmol) and stirring at RT for 10 min.
  • Step 3 tert-Butyl 4- ( 4- (( 6- ( 2-chlorophenyl ) -5-oxo-5H- pyrano [ 2 , 3-d] pyrimidin-2-yl ) amino ) phenyl ) piperaz ine-1- carboxylate: A solution of 2-chloro-6- ( 2-chlorophenyl) - 5H-pyrano [ 2 , 3-d] pyrimidin-5-one (42 mg, 0.14 mmol), tert- butyl 4- ( 4-aminophenyl ) piperazine-l-carboxylate (40 mg, 0.14 mmol) and DIPEA (50 pL, 0.29 mmol) in anhydrous DMF (1 mL) was heated to 100 °C under a nitrogen atmosphere for 60 min.
  • reaction mixture was allowed to cool to RT, diluted with water (5 mL) and extracted into ethyl acetate (3 x 5 mL) .
  • the combined organic phases were washed with 1:1 water/brine (3 x 5 mL) , dried over Na 2 S0 4 , filtered, and concentrated to dryness under reduced pressure. The residue was purified by flash
  • Step 4 6- ( 2-Chlorophenyl ) -2- ( ( 4- (piperazin-1- yl ) phenyl ) amino ) -5H-pyrano [ 2 , 3-d] pyrimidin-5-one , 2HC1 : To a solution of tert-butyl 4- ( 4- ( ( 6- ( 2-chlorophenyl) -5- oxo-5H-pyrano [ 2 , 3-d] pyrimidin-2- yl ) amino ) phenyl ) piperaz ine-l-carboxylate (50 mg, 0.094 mmol) in dichloromethane (1 mL) was added dropwise 4 M HC1 in 1,4-dioxane (1 mL) and the mixture stirred at RT for 2 h. The reaction mixture was then diluted with diethyl ether (5 mL) and slurried at RT for 15 min, before removal of the supernatant solvent by pipette.
  • Example 29 6- ( 2-Chlorophenyl ) -8-cyclopropyl-2- ( ( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one
  • Step 1 1- ( 4-Chloro-2- (methylthio ) pyrimidin-5-yl ) -2 -( 2- chlorophenyl ) ethanone : To a solution of 2- (2- chlorophenyl ) acetic acid (2 g, 11.7 mmol) in THF (30 mL) was added tert-butylmagnesium chloride (11.7 mL, 23.5 mmol) . The brown suspension formed was stirred at RT for 30 min and ethyl 4-chloro-2- (methylthio ) pyrimidine-5- carboxylate (2.46 g, 10.6 mmol) in 10 mL THF added. The resulting yellow suspension was stirred for 16 h.
  • Step 2 2- ( 2-Chlorophenyl ) -1- ( 4- ( cyclopropylamino ) -2- (methylthio ) pyrimidin-5-yl ) ethanone :
  • Step 3 6- ( 2-Chlorophenyl ) -8-cyclopropyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one :
  • Example 30 6- ( 4-Hydroxyphenyl ) -8-methyl-2- (( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin-
  • Step 1 6- ( 4-Hydroxyphenyl ) -8-methyl-2-
  • Step 2 4- ( 8-Methyl-2- (methylthio ) -5-oxo-5 , 8- dihydropyrido [ 2 , 3-d] pyrimidin-6-yl ) phenyl acetate: Acetic anhydride (0.014 mL, 0.147 mmol) was added to a stirred solution of 6- ( 4-hydroxyphenyl ) -8-methyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one (8.8 mg, 0.029 mmol) and triethylamine (0.5 mL, 3.59 mmol) in DCM (1.0 mL) at RT under nitrogen.
  • Step 3 6- ( 4-Hydroxyphenyl ) -8-methyl-2- ( ( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one: mCPBA ( ⁇ 77% pure) (7.4 mg, 0.033 mmol) in DCM (0.5 mL) was added to a stirred solution of 4- ( 8-methyl- 2- (methylthio ) -5-oxo-5, 8-dihydropyrido [ 2 , 3-d] pyrimidin-6 - yl)phenyl acetate (9.8 mg, 0.029 mmol) in toluene (1.0 mL) at RT under nitrogen. After 15 min, DIPEA (0.015 mL, 0.086 mmol) and 4- ( 4-methylpiperazin-l-yl ) aniline (6.0 mg, 0.032 mmol) were added, sequentially, and the
  • Example 31 6- ( 3-Hydroxyphenyl ) -8-methyl-2- ( ( 4- (piperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one
  • Step 1 6- ( 3-Hydroxyphenyl ) -8-methyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one :
  • Step 2 3- ( 8-Methyl-2- (methylthio ) -5-oxo-5 , 8- dihydropyrido [ 2 , 3-d] pyrimidin-6-yl ) phenyl acetate: Acetic anhydride (0.023 mL, 0.247 mmol) was added to a stirred solution of 6- ( 3-hydroxyphenyl ) -8-methyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one (14.8 mg,
  • Step 3 tert-Butyl 4- ( 4- (( 6- ( 3-hydroxyphenyl ) -8-methyl-5- oxo-5 , 8-dihydropyrido [ 2 , 3-d] pyrimidin-2- yl ) amino ) phenyl ) piperaz ine-l-carboxylate : mCPBA ( ⁇ 77% pure) (8.8 mg, 0.039 mmol) in DCM (0.5 ml) was added to a stirred solution of 3- ( 8-methyl-2- (methylthio ) -5-oxo-5 , 8- dihydropyrido [ 2 , 3-d] pyrimidin-6-yl ) phenyl acetate (11.6 mg, 0.034 mmol) in toluene (1.0 mL) at RT under nitrogen.
  • Step 4 6- ( 3-Hydroxyphenyl ) -8-methyl-2- ( ( 4- (piperaz in-1- yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one : TFA (1.0 mL, 13.0 mmol) was added to a stirred solution of tert-butyl 4- ( 4- ( ( 6 - ( 3-hydroxyphenyl ) -8-methyl-5-oxo-5 , 8- dihydropyrido [ 2 , 3-d] pyrimidin-2- yl ) amino ) phenyl ) piperaz ine-l-carboxylate (9.7 mg, 0.018 mmol) in DCM (1.0 mL) at RT under nitrogen.
  • Step 1 1- ( 4-chloro-2- (methylthio ) pyrimidin-5-yl ) -2 -( 2- chlorophenyl ) -2- ( 2- ( 4-methoxyphenyl ) hydrazono ) ethanone : To a solution of 4-methoxyaniline (0.170 g, 1.382 mmol) in cone. HC1 (0.433 ml, 5.20 mmol) with 1.5 ml of water at 0 °C was added sodium nitrite (0.095 g, 1.382 mmol) in 0.5 ml of water.
  • Step 2 3- ( 2-chlorophenyl ) -1- ( 4-methoxyphenyl ) -7- (methylthio) pyrimido [ 4, 5-c] pyridazin-4 ( IH) -one : To a solution of 1- ( 4-chloro-2- (methylthio ) pyrimidin-5-y1 ) -2- ( 2-chlorophenyl ) -2- ( 2- ( 4-methoxyphenyl ) hydrazono ) ethanone (0.4 g, 0.894 mmol) in acetonitrile (8 ml) was added potassium carbonate (0.148 g, 1.073 mmol) .
  • Step 3 3- ( 2-chlorophenyl ) -1- ( 4-methoxyphenyl ) -7- (( 4- ( 4- methylpiperaz in-l-yl ) phenyl ) amino ) pyrimido [4,5- c] pyridazin-4 ( IH) -one : Following the procedure for 8- methyl-2- ( ( 4- ( 4-methylpiperaz in-l-yl ) phenyl ) amino ) -6 - phenylpyrido [ 2 , 3-d] pyrimidin-5 ( 8H) -one, 3- (2- chlorophenyl ) -1- ( 4-methoxyphenyl ) -7-
  • Example 33 3- ( 2-chlorophenyl ) -l-methyl-7- ( ( 4- (piperazin- 1-y1 ) phenyl ) amino ) pyrimido [ 4 , 5-c ] pyridaz in-4 ( 1H ) -one
  • Step 1 1- ( 4-chloro-2- (methylthio) pyrimidin-5-yl ) -2- (2- chlorophenyl ) ethanone : To a solution of 2- (2- chlorophenyl ) acetic acid (10 g, 58.6 mmol) in THF (120 mL) under nitrogen, was added tert-butylmagnesium
  • Step 2 di-tert-butyl 3- ( 2-chlorophenyl ) -7- (methylthio ) - 4-oxo-3 , 4-dihydropyrimido [ 4 , 5-c ] pyridaz ine-1 , 2- dicarboxylate :
  • a solution of 1- ( 4-chloro-2- (methylthio ) pyrimidin-5-yl ) -2- ( 2-chlorophenyl ) ethanone (1.0 g, 3.19mmol) in anhydrous THF (20 mL) was cooled to -78°C.
  • Step 3 3- ( 2-chlorophenyl ) -7- (methylthio ) pyrimido [ 4 , 5- c] pyridazin-4-ol : To a solution of di-tert-butyl 3-(2- chlorophenyl ) -7- (methylthio) -4-oxo-3, 4- dihydropyrimido [ 4 , 5-c ] pyridaz ine-1 , 2-dicarboxylate
  • the reaction mixture was concentrated in vacuo.
  • the residue was dissolved in acetonitrile (20 mL) and 4, 5-dichloro-3 , 6-dioxocyclohexa-l , 4-diene-l, 2- dicarbonitrile (1.79 g, 7.89 mmol) was added in a microwave sealed tube.
  • the reaction mixture was heated by microwave irradiation at 100°C for 10 minutes.
  • the reaction mixture was concentrated in vacuo.
  • the residue was triturated with water and the precipitate was filtrated and washed with water and ethyl acetate to afford the title compound as a brown powder (1 g, 42%) .
  • Step 5 tert-butyl 4- ( 4- (( 3- ( 2-chlorophenyl ) -l-methyl-4- oxo-1 , 4-dihydropyrimido [ 4 , 5-c ] pyridaz in-7- yl ) amino ) phenyl ) piperaz ine-l-carboxylate : 3- (2- chlorophenyl ) -1-methyl-7- (methylthio ) pyrimido [4,5- c] pyridazin-4 ( 1H) -one (100 mg, 0.314 mmol) was dissolved in anhydrous dichloromethane . 3-Chloroperbenzoic acid 70% (93mg, 0.378 mmol) was added portionwise at 0°C. After stirring at room temperature for 30 minutes, all the starting material was converted into the
  • Step 1 tert-butyl 5- ( ( 3- ( 2-chlorophenyl ) -l-methyl-4-oxo- 1 , 4-dihydropyrimido [ 4 , 5-c ] pyridaz in- 7-yl ) amino ) -2- morpholinobenzyl (methyl ) carbamate : Following the
  • Step 2 3- ( 2-chlorophenyl ) -l-methyl-7- (( 3- ( (methylamino ) methyl ) -4- morpholinophenyl ) amino ) pyrimido [ 4 , 5-c ] pyridaz in-4 ( 1H) - one: tert-butyl 5- (( 3- ( 2-chlorophenyl ) -l-methyl-4-oxo- 1 , 4-dihydropyrimido [ 4 , 5-c ] pyridaz in- 7-yl ) amino ) -2- morpholinobenzyl (methyl ) carbamate (25 mg, 0.042 mmol) was dissolved in dichloromethane (1 mL) .
  • Step 1 tert-butyl 4- ( 4- (( 3- ( 2-chlorophenyl ) -4-oxo-l , 4- dihydropyrimido [ 4 , 5-c ] pyridaz in-7-yl ) amino ) -2- ( hydroxymethyl ) phenyl ) piperazine-l-carboxylate : Following the procedure of tert-butyl 4- ( 4- (( 3- ( 2-chlorophenyl ) -1- methyl- 4-oxo-l , 4-dihydropyrimido [ 4 , 5-c ] pyridaz in-7- yl ) amino ) phenyl ) piperaz ine-l-carboxylate , 3- ( 2- chlorophenyl ) -7- (methylthio ) pyrimido [ 4 , 5-c ] pyridaz in- 4(lH)-one (50 mg, 0.16 mmol) was reacted with tert-
  • Step 2 3- ( 2-chlorophenyl ) -7- (( 3- (hydroxymethyl ) -4- (piperaz in-l-yl ) phenyl ) amino ) -pyrimido [ 4 , 5-c ] pyridazin- 4(lH)-one: tert-butyl 4- ( 4- (( 3- ( 2-chlorophenyl ) -4-oxo- 1 , 4-dihydropyrimido [ 4 , 5-c ] pyridaz in-7-yl ) amino ) -2- (hydroxymethyl ) phenyl ) piperazine-l-carboxylate (30 mg, 0.053 mmol) was dissolved in dichloromethane ( 1 mL) . Then TFA (1 mL, 13 mmol) was added at 0°C. The resulting solution was stirred for 16 hours at room temperature until the starting material had been consumed (LC
  • Step 1 tert-butyl 4- ( 4- (( 3- ( 2-chlorophenyl ) -l-methyl-4- oxo-1 , 4-dihydropyrimido [ 4 , 5-c ] pyridaz in-7-yl ) amino ) -2- (hydroxymethyl ) phenyl ) piperazine-l-carboxylate : Following the procedure of tert-butyl 4- ( 4- (( 3- ( 2-chlorophenyl ) -1- methyl-4-oxo-l , 4-dihydropyrimido [ 4 , 5-c ] pyridaz in-7- yl ) amino ) phenyl ) piperaz ine-l-carboxylate , 3- ( 2- chlorophenyl ) -1-methyl-7- (methylthio ) pyrimido [4,5- c] pyridazin-4 ( 1H) -one (50 mg, 0.15 mmol) was reacted with
  • Step 2 3- ( 2-chlorophenyl ) -7- (( 3- (hydroxymethyl ) -4- (piperaz in-l-yl ) phenyl ) amino ) -1-methylpyrimido [4,5- c] pyridazin-4 ( 1H) -one : tert-butyl 4-(4-((3-(2- chlorophenyl ) -l-methyl-4-oxo-l , 4-dihydropyrimido [4, 5- c ] pyridaz in-7-yl ) amino ) -2- ( hydroxymethyl ) phenyl )
  • Step 1 tert-butyl 4- ( 4- (( 3- ( 2-chlorophenyl ) -l-methyl-4- oxo-1, 4-dihydropyrimido [ 4 , 5-c ] pyridaz in-7-yl ) amino ) -2- methylphenyl ) piperazine-l-carboxylate : Following the procedure of tert-butyl 4- ( 4- (( 3- ( 2-chlorophenyl ) -1- methyl-4-oxo-l , 4-dihydropyrimido [ 4 , 5-c ] pyridaz in-7- yl ) amino ) phenyl ) piperaz ine-l-carboxylate , 3- ( 2- chlorophenyl ) -l-methyl-7- (methylthio ) pyrimido [4,5- c] pyridazin-4 ( 1H) -one (100 mg, 0.314 mmol) was reacted with tert
  • Example 38 3- ( 2-chlorophenyl ) -7- ( ( 3-methoxy-4- (piperaz in-l-yl ) phenyl ) amino ) -1-methylpyrimido [4,5- c] pyridaz in-4 ( IH) -one
  • Step 1 tert-butyl 4- ( 4- (( 3- ( 2-chlorophenyl ) -l-methyl-4- oxo-1, 4-dihydropyrimido [ 4 , 5-c ] pyridaz in-7-yl ) amino ) -2- methoxyphenyl ) piperazine-l-carboxylate : Following the procedure of tert-butyl 4- ( 4- (( 3- ( 2-chlorophenyl ) -1- methyl-4-oxo-l , 4-dihydropyrimido [ 4 , 5-c ] pyridaz in-7- yl ) amino ) phenyl ) piperaz ine-l-carboxylate , 3- ( 2- chlorophenyl ) -1-methyl-7- (methylthio ) pyrimido [4,5- c] pyridazin-4 ( IH) -one (100 mg, 0.314 mmol) was reacted with tert
  • Step 2 3- ( 2-chlorophenyl ) -7- (( 3-methoxy-4- (piperaz in-1 yl ) phenyl ) amino ) -1-methylpyrimido [ 4 , 5-c ] pyridaz in-4 ( IH ) one: Following the procedure of 3- ( 2-chlorophenyl ) -1- methyl-7- ( ( 3- ( (methylamino ) methyl ) -4- morpholinophenyl ) amino ) pyrimido [ 4 , 5-c ] pyridaz in-4 ( IH) - one, tert-butyl 4- ( 4- ( ( 3- ( 2-chlorophenyl ) -l-methyl-4-oxo- 1 , 4-dihydropyrimido [ 4 , 5-c ] pyridaz in-7-yl ) amino ) -2- methoxyphenyl ) piperazine-l-carboxylate (49 mg, 0.085
  • Step 1 (R) -tert-butyl 4- ( 4- (( 3- ( 2-chlorophenyl ) -1- methyl-4-oxo-l , 4-dihydropyrimido [ 4 , 5-c ] pyridaz in-7- yl ) amino ) phenyl ) -2-methylpiperaz ine-l-carboxylate :
  • Step 2 (R) -3- ( 2-chlorophenyl ) -l-methyl-7- (( 4- ( 3- methylpiperaz in-l-yl ) phenyl ) amino ) pyrimido [4,5- c] pyridazin-4 ( 1H) -one : (R) -tert-butyl 4-(4-((3-(2- chlorophenyl ) -l-methyl-4-oxo-l , 4-dihydropyrimido [4, 5- c] pyridaz in-7-yl ) amino ) phenyl ) -2-methylpiperaz ine-1 - carboxylate (82 mg, 0.146 mmol) was reacted with HC1 (3 mL, 4 M in dioxane) in DCM (3mL) for 4h at RT .
  • Wee-1 activity In the measurement of Wee-1 activity, a commercial peptide Poly(Lys Tyr(4:l)) hydrobromide was purchased from Sigma Aldrich and used as the substrate. Activated Wee-1 kinase was purchased from Invitrogen (PV3817) and an ADP-Glo luminescent kit was purchased from Promega.
  • the plate was incubated at room temperature for 30 minutes before the addition of 2 pg/mL of substrate and 30 pM ATP in a 5 pL aliquot. The plate was centrifuged for one minute and incubated for 1 h at RT . 15 ⁇ 1 ⁇ L of ADP-Glo stop reagent was added to each well to quench the reaction and deplete unconverted ATP. The plate was incubated for a further 40 min in the dark at RT .
  • the plate was shaken for 1 min, and incubated in the dark for an additional hour.
  • Luminescence from each well was detected using the Biotek Synergy4 HD plate reader and the percentage inhibition of kinase activity calculated for each inhibitor tested. Positive (kinase only) and negative (no kinase) controls were added to each plate to ensure specific interaction of kinase and inhibitor. The IC 50 concentration for each inhibitor was calculated by plotting the percentage kinase inhibition against concentration of inhibitor and the curve generated by non-linear regression fitting.
  • the colorectal cancer cell lines HT-29 and HCT-116 were purchased from the ATCC and routinely maintained in
  • the cells were trypsinised from their growing vessel and counted, 100 ⁇ 1 ⁇ L of cell suspension containing 6000 cells was pipetted into black 96 well Co-star plates and incubated overnight to allow adherence to the surface at a temperature of 37°C and an atmosphere of 5% CO 2 .
  • Test compounds were formulated in DMSO and diluted in foetal calf serum supplemented medium. Incubating medium was removed by aspiration and diluted drug supplemented medium added to each well.
  • the plate was returned to the incubator for an additional eight hours at 37°C and an atmosphere of 5% CO 2 .
  • Post incubation, the drug supplemented medium was aspirated from each well and the cells were washed once in ice-cold phosphate buffered saline (PBS) .
  • PBS phosphate buffered saline
  • cell lysis buffer Cell Signalling Technologies #9803
  • 20 mM Tris 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton- X100, 2.5 mM sodium pyrophosphate, 1 mM glycerophosphate, 1 mM Na 3 VC>4 and 1 pg/mL leupeptin was added to each well of the 96 well plate and incubated at 4 °C for 30 min. The samples on the plate were snap frozen at -80 °C until required. Immediately before the continuation of the assay, the sample plate was thawed and centrifuged at 4 °C for 10 min and the supernatant transferred to
  • Cell supernatant was mixed in a ratio of 1:1 with sample dilutent buffer and vortexed for one minute. 100 pL of diluted sample was pipetted into pre-coated plates containing a rabbit polyclonal antibody for phospho-cdc2 (Tyrl5) (Cell Signalling Technologies PathScan kit
  • the plate was sealed and incubated overnight at 4 °C.
  • kit detection antibody was added to each well and the plate re-sealed and incubated at 37°C for 1 h. Post incubation the plate was washed and processed in a similar manner to that previously
  • TMB reagent tetramethylbenz idine
  • the percentage of phospho-cdc2 was calculated compared to DMSO control and plotted versus the concentration of inhibitor using GraphPad Prism. Data was fitted using non-linear regression analysis and IC 5 o values generated.
  • Method 3 Determining the anti-proliferative properties of Wee-1 inhibitors in the HT-29 cell line Cell Titre Glo (Promega) is a highly sensitive
  • the reagent uses a stable form of luciferase to measure ATP as an output of viability.
  • the luminescent values generated in the assay are directly proportional to the number of viable cells in your assay.
  • HT-29 cells were washed, detached and re-suspended in their respective fresh media. The cells were pelleted by centrifugation (Eppendorf 5414) and the spent supernatant was discarded. The cells were re-suspended by vortex mixing, counted and seeded into clear bottom white 96 well plates at a density of 5000 cells per well. The cells were incubated overnight at 37 °C (95% O 2 / 5% CO 2 ) , and next day treated with increasing concentrations of test compound formulated in fresh medium. The plates were returned to the incubator for 72 h.
  • Cell Titre Glo (Promega) was prepared by mixing the supplied reagents as per manufacturer's instructions and left to stand at RT .
  • the cell plates were removed from the incubator and 80 pL of the Cell Titre Glo solution added to each well. The plate was shaken for five minutes to ensure homogenous mixing of reagents and cells, then left to stand for 10 min at RT .
  • the cell viability post compound treatment was determined by the luminescent intensity emitted from the drug treated wells in the plate.
  • the assay plate was placed in the Biotex Synergy 4 Hybrid plate reader and the luminescence read in each well.
  • the compound treated wells were compared to vehicle treated wells and the % inhibition of cell viability calculated.
  • the data was analysed using GraphPad Prism, with IC 5 o values generated using non-linear regression of the data set .
  • Method 4 Determination of CLint estimates using human liver microsomes.
  • AKT1 (PKB alpha) - - - -
  • GSK3B (GSK3 beta) - - -
  • PDGFRB (PDGFR beta) - - +
  • PRKCB1 (PKC beta I) - - - -
  • RPS6KA3 (RSK2) - - - RPS6KB1 (p70S6K) — — —

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Abstract

The present invention relates to compounds that are useful as inhibitors of the activity of Wee-1 kinase. The present invention also relates to pharmaceutical compositions comprising these compounds and to methods of using these compounds in the treatment of cancer and methods of treating cancer.

Description

PHARMACEUTICAL COMPOUNDS
The present invention relates to compounds that are useful as inhibitors of the activity of Wee-1 kinase. The present invention also relates to pharmaceutical compositions comprising these compounds and to methods of using these compounds in the treatment of cancer and methods of treating cancer. BACKGROUND TO THE INVENTION
Cells are continually challenged on a daily basis, resulting in multiple lesions forming in DNA. The
lesions, if not repaired, can lead to mutations or cell death, thus complex signalling networks exist which ensure that lesions are detected, and repaired to
maintain the integrity of DNA.
Detection of DNA damage initiates a series of events which are key in maintaining the genome. Cell cycle checkpoints are designed to stop the cell cycle and allow repair of the lesion before allowing the cell to continue into mitosis . Two key checkpoints have been identified, one at the end of Gl phase and the second at G2, which work in tandem to ensure all lesions, are identified and repaired. In 50% of human cancers the Gl checkpoint is non-functional due to mutations in the tumour suppressor gene p53. However, the G2 check-point is seldomly mutated, and often found to be activated in cancer cells. Cancer cells exploit this to confer resistance to treatment modalities
including DNA damaging agents and radiation. Three kinases have been identified as key regulators of the G2 checkpoint, namely Chkl, Chk2 and Wee-1.
Inhibitors for the kinases are currently in clinical trials .
Wee-1 is a nuclear tyrosine kinase, which negatively regulates entry into mitosis, at the G2/M check-point by catalysing a phosphorylation of the cdc2 / cyclin B kinase complex. The phosphorylation occurs on the
Tyrosine 15 residue and leads to the inactivation of cdc2 / cyclin B complex ultimately preventing mitosis. Wee-1 function is intimately linked to that of Chkl and Chk2 due to their phosphorylation and inactivation of cdc25 on serine 216, as well as the reported activation of Wee-1 by Chk 1 & 2 (Ashwell et al 2012 DNA Repair in Cancer Therapy, DOI : 10.1016/B978-0-12-384999-1.10010-1) .
Wee-1 is downstream of the Chk family and is a crucial component of the checkpoint signalling cascade as it prevents cells from entering mitosis if lesions are detected .
Commonly administered anti-cancer compounds induce DNA damage; including anti-metabolites, platiniums,
topoisomerase inhibitors and alkylating agents. However their efficacy is limited due to excessive toxicity, resistance and lack of tumour selectivity. Compounds which work in combination with these agents to prevent DNA repair selectively in tumour cells would be extremely beneficial. The tumour suppressor gene p53, is commonly mutated in tumour cell lines, therefore the
administration of a Wee-1 kinase inhibitor, which will abrogate the G2 check point may lead to increased
sensitivity to DNA damaging agents. The potential for this has been reported, silencing of Wee-1 activity was sufficient to sensitize HeLa cells to doxorubicin due to abrogation of G2 arrest. In contrast, in normal breast epithelium which have a fully complement p53, the removal of Wee-1 function had little additional effect compared to doxorubicin alone (Wang et al 2004, Cancer Biology and Therapy 3:3; 305-313) .
It has been reported that cell lines harbouring mutations in the tumour suppressor gene p53 have increased
sensitivity to DNA damaging agents when co-administered with Wee-1 small molecule inhibitors. In vitro and in vivo efficacy has been reported when small molecule inhibitors are combined with gemcitabine, 5-fluorouracil, carboplatin, cisplatin (Hirai et al 2010, Cancer Biology & Therapy 9:7, 514-522), cytarabine (Tibes et al 2012, Blood. Mar 22 ; 119 ( 12 ): 2863-72 ) and Src inhibitors (Cozzi et al 2012, Cell Cycle 11:5, 1-11). Single agent
apoptotic efficacy, independent of p53 status, has been reported in sarcoma cell lines and in patient derived sarcoma samples (Kreahling et al 2012, Mol Cancer Ther . Jan; 11 (1) : 174-82) .
Irradiation is known to increase phosphorylation of the Tyrl5 and Thrl4 residues of cdc2, leading to a
radioresistant phenotype. Inhibition of Wee-1 activity by small molecules (Wang et al 2004, Cancer Biology and Therapy 3:3; 305-313) leads to a reduction in
phosphorylation and radiosensitization effect, with the effect more pronounced in p53 mutant cell lines.
Compounds having a kinase inhibitory effect, for example a Wee-1 kinase inhibitory effect, are described in WO2007/126122, US2010/0063024, EP2,213,673, W02008 / 133866 and US2007/0254892.
WO2010/067886, WO2010/067888, US2011/0135601 EP2,168,966, WO2005/090344, US2009/0048277 and Bioorg & Med Chem Lett Vol 15, pp 1931-1935 describe various compounds such as dihydropyrimidopyrimidine and pyridopyrimidinone
derivatives having a kinase inhibitory effect. In particular, the compounds of WO2005/090344 are said to show activity as protein kinase inhibitors, in particular Src family tyrosine kinase inhibitors. The compounds described in Bioorg & Med Chem Lett Vol 15, pp 1931-1935 are said to be 10-100-fold more potent inhibitors of c- Src than Weel, and variation of substituents on the 6- phenyl ring does not markedly alter this preference. It is said that solubilizing substituents off the 2-anilino ring in many cases increases Weel activity, lowering this preference to about 10-fold. 5-Alkyl substituted
analogues are said to be generally Weel selective, but at the expense of absolute potency.
It is one object of the present invention to overcome at least some of the disadvantages of the prior art or to provide a commercially useful alternative thereto.
It is a further object of the present invention to provide a compound having an improved selectivity towards Wee-1 kinase compared to known compounds or compositions. It is a further object of the present invention to provide a compound having an improved stability in human microsomes compared to known compounds or compositions. It is a further object of the present invention to provide a compound having an enhanced or similar kinase- inhibitory effect compared to known compounds or
compositions .
It is a further object of the present invention to provide a compound having an improved efficacy compared to known compounds or compositions.
It is a further object of the present invention to provide a compound having an improved efficacy and tolerability when administered in combination with other therapies compared to known compounds or compositions.
It is a further object of the present invention to provide a compound having an improved tolerability compared to known compounds or compositions.
SUMMARY OF THE INVENTION
In a first aspect the present invention provides a compound of Formula (I) :
Figure imgf000006_0001
(I) or a pharmaceutically acceptable salt or iV-oxide
derivative thereof, wherein:
X is an oxygen atom or a nitrogen atom;
Y is a carbon atom or a nitrogen atom; R1 is an optionally substituted aryl group;
R2 is a hydrogen atom, an optionally substituted alkyl group or an optionally substituted amino group when Y is a carbon atom and R2 is absent when Y is a nitrogen atom;
R3 is a hydrogen atom, an optionally substituted alkyl group or an optionally substituted aryl group when X is a nitrogen atom and R3 is absent when X is an oxygen atom;
R4 is an optionally substituted alkyl group or an optionally substituted aryl group.
Each aspect or embodiment as defined herein may be combined with any other aspect (s) or embodiment ( s ) unless clearly indicated to the contrary. In particular any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous. In a second aspect the present invention provides a pharmaceutical composition comprising the compound of formula (I), or a pharmaceutically acceptable salt or N- oxide derivative thereof, and at least one
pharmaceutically acceptable excipient .
In a third aspect the present invention provides the compound of formula (I), or a pharmaceutically acceptable salt or N-oxide derivative thereof, or a pharmaceutical composition comprising the compound of formula (I) for use in therapy.
In a fourth aspect the present invention provides the compound of formula (I) for use as a medicament. In a fifth aspect the present invention provides the compound of formula (I) for use in treating or preventing cancer . In a sixth aspect the present invention provides the compound of formula (I) for the manufacture of a
medicament for treating or preventing cancer.
In a seventh aspect the present invention provides the use of the compound of formula (I) for the manufacture of a medicament for treating or preventing cancer.
In an eighth aspect the present invention provides a method of treating or preventing cancer in a human or animal patient comprising administering to a patient in need thereof an effective amount of the compound of formula (I) or a pharmaceutical composition comprising the compound of formula (I) . Other preferred embodiments of the compounds according to the invention appear throughout the specification and in particular in the examples. Particularly preferred are those named compounds having greater activity as tested. Compounds having higher activity are more preferred over those having lower activity.
The present inventors have surprisingly found that the compounds of the present invention show an improved selectivity towards Wee-1 kinase. Preferably, in
particular, the compounds of the invention are selective over members of the Src family of kinases, for example LCK (Lymphocyte specific protein tyrosine kinase) and c- Src. Unexpectedly, the compounds of the present
invention also appear to show greater selectivity than a representative compound described in Bioorg & Med Chem Lett Vol 15, pp 1931-1935 (see Examples) .
Without wishing to be bound by theory it is thought that the compounds of the present invention tend to show an improved selectivity over other off-target kinases due to the position of the carbonyl (C=0) group as shown in formula (I), that is, the carbonyl group adjacent to the carbon atom to which R1 is attached.
Figure imgf000009_0001
(I)
The present inventors have surprisingly found that the compounds of the present invention show an improved or similar kinase-inhibitory effect compared to known compounds or compositions. In particular, the compounds of the present invention preferably show an improved or similar Wee-1 kinase-inhibitory effect compared to known compounds or compositions.
Preferably, the compounds of the present invention have an improved stability in human microsomes and/or an improved tolerability compared to known compounds or compositions .
DETAILED DESCRIPTION OF THE INVENTION
The term " alkyl group" refers to an aliphatic group containing at least carbon and hydrogen and containing 1 to 15 carbon atoms, such as 1 to 10 carbon atoms.
Attachment to the alkyl group occurs through a carbon atom . A " Cn alkyl" group refers to an aliphatic group containing n carbon atoms. For example, a Ci-Cio alkyl group
contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms.
An alkyl group may be straight chained or it may be branched.
An alkyl group may contain no ring structures or it may contain one or more rings (i.e. "a cycloalkyl group") . For example, a "cycloalkyl group" contains at least one ring. It is understood that attachment to a cycloalkyl group is via a ring atom of the cycloalkyl group. Each ring may contain 3 to 10 atoms, such as 4 to 8 or 5 to 7 atoms. Each ring may be independently selected to contain just carbon atoms or to contain both carbon atoms and from 1 to 4 heteroatoms selected from 0, N and S.
For cyclo-heteroalkyl groups (i.e. cycloalkyl groups that contain one or more heteroatoms), attachment to the cycloalkyl group may occur either through a carbon atom or, if one or more heteroatoms are contained in a ring, attachment may also occur through a heteroatom contained in a ring.
For example, a cycloalkyl group may be mono-cyclic or bi- cyclic. The bicyclic cycloalkyl group may have a spiro structure in which the two rings share one and the same ring atom, or may have a bicyclo structure in which the two rings share two or more ring atoms. Thus, a " Cn cycloalkyl" group contains n carbon atoms. All n carbon atoms may be contained in the ring(s) of the cycloalkyl group or one or more of the carbons may not be contained in the ring(s) and may instead form one or more chains branching from the ring.
If a Cn alkyl group is joined to a separate Cm alkyl group containing m carbon atoms to form, for example, a
heterocycle, the two alkyl groups contain a total number of m + n carbon atoms.
An alkyl group may be saturated or unsaturated. Thus, the alkyl group may be an alkenyl group (i.e. contain a carbon-carbon double bond) and / or an alkynyl group (i.e. contain a carbon-carbon triple bond) . If the alkyl group is unsaturated, it may contain at least 2 carbon atoms. It is understood that any unsaturated portions of an alkyl group are non-aromatic (aromatic groups fall within the scope of the definition of "aryl") . Any part of the alkyl group may be unsaturated, for example the straight, branched or cyclic portion of an alkyl group may contain a carbon-carbon double bond or a carbon- carbon triple bond. Attachment to an unsaturated alkyl group may occur through the unsaturated part of the alkyl group or may occur through the unsaturated part of the group .
For example, an unsaturated alkyl group may contain 1 to 4 carbon-carbon double bonds or 1 to 3 carbon-carbon triple bonds or 1 to 4 of a combination of carbon-carbon double bonds and carbon-carbon triple bonds.
An alkyl or cycloalkyl group can be optionally
substituted (a "substituted alkyl" or "substituted cycloalkyl") with one or more alkyl group substituents , which can be the same or different. An alkyl group substituent can be attached to the alkyl or cycloalkyl group through a carbon atom in the alkyl or cycloalkyl group, or, if one or more heteroatoms are contained in a cycloalkyl group, attachment may also occur through a heteroatom contained in the cycloalkyl group. The term "alkyl group substituent" includes but is not limited to alkyl, substituted alkyl, aralkyl, substituted aralkyl, halo, cyano, amino, alkylamino, arylamino, aryl,
substituted aryl, nitro, thio, acyl, hydroxyl, aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio, carboxyl, alkoxycarbonyl , oxo, alkylsulfonyl ,
arylsulfonyl and cycloalkyl. If a group, for example an alkyl group, is "optionally substituted" , it is understood that the group has one or more substituents attached (substituted) or does not have any substituents attached (unsubstituted) . Examples of unsubstituted saturated alkyl groups
containing no cyclic structures include methyl, ethyl, n- propyl, sec-propyl, n-butyl, sec-butyl, tert-butyl, pentyl (branched or unbranched) , hexyl (branched or unbranched) , heptyl (branched or unbranched) , octyl
(branched or unbranched) , nonyl (branched or unbranched) , and decyl (branched or unbranched) .
Examples of unsubstitued saturated cycloalkyl groups include cyclopropyl, cylcobutyl, cyclopentyl and
cyclohexyl.
Examples of unsaturated alkyl groups include ethenyl, trimethenyl, tetramethenyl , propenyl, butenyl, 2- methybutenyl , pentamethenyl , hexamethenyl and
cyclohexenyl . Examples of unsubstitued saturated cyclo-heteroalkyl groups include azetidinyl, pyrrolodinyl , piperidinyl, piperizinyl, tetrahydrofuranyl , tetrahydropyranyl , morpholine, thiomorpholine, homopiperaz ine , homo- piperidine, homomorpholine, homothiomorpholine, S,S- dioxythiomorpholine, S, S-dioxyhomothiomorpholine .
The term "aryl group" refers to a group containing at least one ring that is aromatic and containing 1 to 15 carbon atoms, such as 1 to 10 carbon atoms.
The aryl group can be optionally substituted (a
"substituted aryl") with one or more aryl group
substituents , which can be the same or different, wherein "aryl group substituent" includes but is not limited to alkyl, substituted alkyl aryl, substituted aryl, aralkyl, hydroxyl, alkoxyl, haloalkoxyl perhaloalkoxyl , aryloxyl, aralkyloxyl, carboxyl, alkanoyl, halo, nitro,
alkoxycarbonyl , aryloxycarbonyl , aralkoxycarbonyl , acyloxyl, cyano, acylamino, amido, carbamoyl,
alkylcarbamoyl , dialkylcarbamoyl , arylthio and alkylthio.
Where an aryl group is stated as being substituted at a particular position, attachment of the position to the aryl group is onto the aromatic ring of the aryl group itself rather than the position being joined to the aryl group through any non-aromatic side-chain of the aryl group. For example, when R1 is an aryl group in CR1, the C is attached to the aromatic part of the aryl group.
Each ring may be independently selected to contain only carbon atoms or to contain both carbon atoms and from 1 to 4 heteroatoms selected from 0, N and S. For
heteroaryl groups (i.e. aryl groups that contain one or more heteroatoms ) , attachment to the aryl group may occur either through a carbon atom or, if one or more
heteroatoms are contained in a ring, attachment may also occur through a heteroatom contained in a ring.
It is noted that the heteroatoms contained in a ring of a heteroaryl group may be substituted, for example forming an iV-oxide. For example, the aromatic group may be mono-cyclic or bi- cyclic, wherein one or both of the rings of a bi-cyclic system is aromatic. If one ring is non-aromatic, the non- aromatic ring may be further substituted with one or more substituents , which can be the same or different. The substituents include, but are not limited to alkyl, spirocyclic alkyl, aralkyl, substituted aralkyl, halo, cyano, amino, alkylamino, arylamino, aryl, substituted aryl, nitro, thio, acyl, hydroxyl, aryloxyl, alkoxyl, alkylthio, arylthio, aralkyloxyl, aralkylthio, carboxyl, alkoxycarbonyl , oxo, alkylsulfonyl , arylsulfonyl and cycloalkyl .
Examples of aryl groups include acridinyl, phenyl, carbazolyl, cinnolinyl, quinoxalinyl , pyrrazolyl, benzotriazolyl , furanyl, naphthyl, thienyl, thiazolyl, benzothienyl , benzofuranyl , quinolinyl, isoquinolinyl , oxazolyl, isoxazolyl, indolyl, pyrazinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrrolyl, tetrahydroquinoline, tetrahydroisoquinoline, benzimidazolyl and melaminyl.
It is noted that the term "heterocycle" includes within its scope both cycloalkyl groups containing one or more heteroatoms within the ring system and aryl groups containing one or more heteroatoms within the ring system. Heterocyclic groups may be, for example, pyrazoles, imidazoles and any of the various triazoles and may include the oxygen and/or sulphur containing analogues that is oxazoles, isoxazoles, thiazoles and isothiazoles and derivatives.
The term "nitrogen-containing heterocyclic group" refers to a monocyclic or bicyclic heterocyclic group containing at least one nitrogen atom, in which each ring comprises from 3 to 7 ring atoms and optionally contains, in addition to the nitrogen atom, zero or one or two or more, the same or different hetero atoms, but preferably one hetero atom selected from the group consisting of an oxygen atom, a nitrogen atom and a sulfur atom; and the heterocyclic group may be aromatic or aliphatic. The bicyclic heterocyclic group may have a spiro structure of which the two rings share one and the same ring atom, or may have a bicyclo structure of which the rings share two or more ring atoms. Examples of the nitrogen-containing heterocyclic group include, for example, a pyrrolyl group, an imidazolyl group, a pyrazolyl group, a
thiazolyl group, an isothiazolyl group, an oxazolyl group, an isoxazolyl group, a triazolyl group, a
tetrazolyl group, an oxadiazolyl group, a 1,2,3- thiadiazolyl group, a 1 , 2 , 4-thiadiazolyl group, a 1,3,4- thiadiazolyl group, a pyridyl group, a pyrazinyl group, a pyrimidinyl group, a pyridazinyl group, a 1 , 2 , 4-triazinyl group, a 1 , 3 , 5-triaz inyl group, an indolyl group, a benzimidazolyl group, a benzoxazolyl group, a
benzisoxazolyl group, a benzothiazolyl group, a
benzisothiazolyl group, an indazolyl group, a purinyl group, a quinolyl group, an isoquinolyl group, a
phthalazinyl group, a naphthyridinyl group, a quinoxalinyl group, a quinazolinyl group, a cinnolinyl group, a pteridinyl group, a pyrido [ 3 , 2-b] pyridyl group, an azetidinyl group, a pyrrolidinyl group, a dihydro- 1 , 2 , 4-triazolyl group, a dihydro-1 , 2 , 4-oxadiazolyl group, a dihydro-1 , 3 , 4-oxadiazolyl group, a dihydro-1 , 2 , 4- thiadiazolyl group, a dihydro-1 , 2 , 3 , 5-oxathiadiazolyl group, a piperidinyl group, a piperazinyl group, a dihydropyridyl group, a morpholinyl group, a
thiomorpholinyl group, a 2 , 6 -diazaspiro [ 3.5 ] nonyl group, a 2 , 7-diazaspiro [ 3.5 ] nonyl group, a 2,7- diazaspiro [ 4.5 ] decyl group, or a 2,7- diazabicyclo [ 3.3.0 ] octyl group, a 3,6- diazabicyclo [ 3.3.0 ] octyl group. The nitrogen-containing heterocyclic group can be
optionally substituted (a "substituted nitrogen- containing heterocyclic group") with one or more aryl group substituents or one or more alkyl group
substituents , which can be the same or different, wherein "aryl group substituent" and "alkyl group substituent" are as defined above.
The term "amino group" refers to the -NH2 group. The amino group can be optionally substituted (a "substituted amino") with one or more amino group substituents, which can be the same or different, wherein "amino group substituent" includes but is not limited to alkyl and aryl . The term " alkoxy group" refers to an alkyl-0 group. The alkoxy group can refer to linear, branched, or cyclic, saturated or unsaturated oxo-hydrocarbon chains,
including, for example, methoxyl, ethoxyl, propoxyl, isopropoxyl, butoxyl, f-butoxyl and pentoxyl. The alkoxy group can be optionally substituted (a "substituted alkoxy") with one or more alkoxy group substituents , which can be the same or different, wherein "alkoxy group substituent" includes but is not limited to alkyl, amino and aryl .
The term " alkanoyl group" refers to an organic acid group wherein the -OH of the carboxyl group has been replaced with another substituent. Thus, the alkanoyl group can be represented by the formula RC(=0)-, wherein R is an alkyl, aralkyl, or aryl group, optionally substituted by one or more alkyl or aryl group substituent. Examples of alkanoyl groups include an acetyl group, a propionyl group, a butyryl group, an isobutyryl group, a valeryl group, an isovaleryl group, a pivaloyl group.
The term " sulfonyl group" refers to a sulfonic acid group wherein the wherein the -OH of the sulfonyl group has been replaced with another substituent. For example, the substitutent may be an alkyl group ("an alkylsufonyl group") . An alkylsulfonyl group can be represented by the formula S {0) 2 ~ , wherein R is an alkyl group,
optionally substituted by one or more alkyl or aryl group substituent. Examples of alkylsulfonyl groups include a methylsulfonyl group, an ethylsulfonyl group, a
propylsulfonyl group, an isopropylsulfonyl group, a butylsulfonyl group, a sec-butylsulfonyl group, an isobutylsulfonyl group, a tert-butylsulfonyl group, a pentylsulfonyl group, an isopentylsulfonyl group, a hexylsulfonyl group and an isohexylsulfonyl group.
The term " sulfinyl group" refers to the bivalent -S(=0) group . The term " oxo group" refers to the -C(=0) group.
The term "halo" refers to a group selected from chlorine, fluorine, bromine and iodine.
For completeness, it is also noted that certain chemical formulae used herein define delocalized systems. This definition is known in the art as a definition of
aromaticity and may indicate the presence of, for
example, a mono-, di- or tri-cyclic system that contains (4n+2) electrons where n is an integer. In other words, these systems may display Hiickel aromaticity.
In whatever aspect, the compounds of the present
invention may possess some aspect of stereochemistry.
For example, the compounds may possess chiral centres and / or planes and / or axes of symmetry. As such, the compounds may be provided as single stereoisomers, single diastereomers , mixtures of stereoisomers or as racemic mixtures. Stereoisomers are known in the art to be molecules that have the same molecular formula and sequence of bonded atoms, but which differ in their spatial orientations of their atoms and / or groups. In addition, the compounds of the present invention may possess tautomerism. Each tautomeric form is intended to fall within the scope of the invention.
In addition, the compounds of the present invention may be provided as a pro-drug. Pro-drugs are transformed, generally in vivo, from one form to the active forms of the drugs described herein. For example, a prodrug may be formed by protecting the -N-H group to which R4 is attached with a hydrolysable group that gives -NH on hydrolysis. Alternatively or additionally, when X, Y or Z and/or any moieties appended thereto taken separately or together are -N-H, one or more of these may be
protected as a physiological hydrolyzable amide. In addition, the elements of the compounds of the present invention may be provided as isotopes. For example, hydrogen may also be deuterium.
In addition, the compounds of the present invention may be provided in the form of their pharmaceutically
acceptable salts or as co-crystals. For example, the compounds may be provided having protonated amine groups.
The term "pharmaceutically acceptable salt" refers to ionic compounds formed by the addition of an acid to a base. The term refers to such salts that are considered in the art as being suitable for use in contact with a patient, for example in vivo and pharmaceutically
acceptable salts are generally chosen for their non- toxic, non-irritant characteristics.
The term "co-crystal" refers to a multi- component molecular crystal, which may comprise non-ionic
interactions .
Pharmaceutically acceptable salts and co-crystals may be prepared by ion exchange chromatography or by reacting the free base or acidic form of a compound with
stoichiometric amounts or with an excess of the desired salt-forming inorganic or organic acid or base in one or more suitable solvents, or by mixing the compound with another pharmaceutically acceptable compound capable of forming a co-crystal. Salts known in the art to be generally suitable for use in contact with a patient include salts derived from inorganic and / or organic acids, including the
hydrobromide , hydrochloride, sulphate, bisulphate, nitrate, acetate, oxalate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate and tartrate. These may include cations based on the alkali and alkaline earth metals, such as sodium, potassium, calcium and magnesium, as well as ammonium, tetramethylammonium,
tetraethylammonium . Further reference is made to the number of literature sources that survey suitable
pharmaceutically acceptable salts, for example the
Handbook of pharmaceutical salts published by IUPAC.
In addition, the compounds of the present invention may sometimes exist as zwitterions, which are considered as part of the invention. The present inventors have discovered that the compounds of the present invention are useful in the treatment of medical conditions associated with disordered cell growth, including, but not restricted to, cancer, in particular cancers associated with mutations in the tumour suppressor gene p53.
For example, cancers include cardiac cancers, lung cancers, gastrointestinal cancers, genitourinary tract cancers, liver cancers, bone cancers, nervous system cancers, gynecological cancers, hematologic cancers, skin cancers and adrenal gland cancers.
For example, cancers include adrenal tumors, bile duct, bladder, blood, bone and connective tissue, brain and central nervous system, breast, cervical, colon and rectal (colorectal), endometrial, esophageal, gallbladder, head and neck, Hodgkin's Lymphoma,
hypopharangeal , kidney, laryngeal, leukemias, liver, lung, lymphoma, mediastinal tumors, melanoma (malignant melanoma) , mesothelioma, multiple myeloma, nasal cavity, nasopharyngeal, neuroendocrine tumors, non-Hodgkin ' s lymphoma, oral, oesophagus, oropharyngeal, ovarian, pancreas, paranasal sinus, parathyroid, penis, pituitary tumors, prostate, salivary gland, sarcoma, skin, spine, stomach, testicular, thyroid, urethra, uterine, vaginal and vulvar.
The compounds of the present invention are also useful in preparing a medicament that is useful in treating the diseases described above, in particular cancer.
The present invention is further directed to a method of inhibiting Wee-1 activity which comprises administering to a mammal in need thereof a pharmaceutically effective amount of the compound of the present invention.
The compounds of this invention may be administered to mammals, including humans, either alone or, in
combination with pharmaceutically acceptable carriers, excipients or diluents, in a pharmaceutical composition, according to standard pharmaceutical practice. The compounds can be administered orally or parenterally, including the intravenous, intramuscular,
intraperitoneal, subcutaneous, rectal and topical routes of administration.
The present invention also includes within its scope the use of the compounds of the present invention in
combination with a second or further drug in the treatment of cancer. The second or further drug may be a drug that is already known in the art in the treatment of cancer . The present invention also includes the use of the compounds of the invention in a regime including the step of radiotherapy. The radiotherapy maybe an ordinary method of treatment by x-ray, γ-ray, neutron, proton or electron beam irradiation. The co-administration of compounds contained in this invention may lead to the potentiation of the radiation therapy, thus classifying them as radio-sensitizers .
In particular, cancers often become resistant to therapy. The development of resistance may be delayed or overcome by the administration of a combination of drugs that includes the compounds of the present invention for example in cancers which are known to be resistant to DNA damaging agents or radiotherapy.
For example, drugs that may be used in combination with the compounds of the present invention may target the same or a similar biological pathway to that targeted by the compounds of the present invention or may act on a different or unrelated pathway.
Depending on the disease to be treated, a variety of combination partners may be co-administered with the compounds of the present invention. The second active ingredient may include, but is not restricted to:
alkylating agents, including cyclophosphamide,
ifosfamide, thiotepa, melphalan, chloroethylnitrosourea and bendamustine ; platinum derivatives, including
cisplatin, oxaliplatin, carboplatin and satraplatin ; antimitotic agents, including vinca alkaloids (vincristine, vinorelbine and vinblastine), taxanes
(paclitaxel, docetaxel), epothilones and inhibitors of mitotic kinases including aurora and polo kinases;
topoisomerase inhibitors, including anthracyclines , epipodophyllotoxins , camptothecin and analogues of camptothecin ; antimetabolites, including 5-fluorouracil, capecitabine , cytarabine, gemcitabine, 6-mercaptopurine, 6 -thioguanine , fludarabine, methotrexate and premetrexed; protein kinase inhibitors, including imatinib, gefitinib, sorafenib, sunitinib, erlotinib, dasatinib, and
lapatinib; proteosome inhibitors, including bortezomib; histone deacetylase inhibitors, including valproate and SAHA; antiangiogenic drugs, including bevacizumab;
monoclonal antibodies, including trastuzumab, rituximab, alemtuzumab, tositumomab, cetuximab, panitumumab;
conjugates of myoclonal antibodies, including Gemtuzumab ozogamicin, Ibritumomab tiuxetan; hormonal therapies, including antiestrogens (tamoxifen, raloxifen,
anastrazole, letrozole, examestane) antiandrogens
(Flutamide, Bicalutamide ) and Luteinisng Hormone
Analogues or antagonists.
With regard to combination therapy the compounds of the present invention may be administered separately,
sequentially, simultaneously, concurrently or may be chronologically staggered with one or more standard therapeutics such as any of those mentioned above. Preferably, the present invention provides a compound of Formula ( I ) :
Figure imgf000024_0001
or a pharmaceutically acceptable salt or iV-oxide
derivative thereof, wherein:
X is an oxygen atom or a nitrogen atom;
Y is a carbon atom or a nitrogen atom;
R1 is an optionally substituted aryl group;
R2 is a hydrogen atom, an optionally substituted alkyl group or an optionally substituted amino group when Y is a carbon atom and R2 is absent when Y is a nitrogen atom;
R3 is a hydrogen atom, an optionally substituted alkyl group or an optionally substituted aryl group when X is a nitrogen atom and R3 is absent when X is an oxygen atom;
R4 is an optionally substituted alkyl group or an optionally substituted aryl group.
In a preferred embodiment Y is a carbon atom.
Preferably, R2 is a hydrogen atom, an amino group or an alkyl group when Y is a carbon atom. More preferably, R2 is a hydrogen atom, an NH2 group or a methyl group when Y is a carbon atom. Most preferably, R2 is a hydrogen atom when Y is a carbon atom.
In an alternative preferred embodiment, Y is a nitrogen atom. R2 is absent when Y is a nitrogen atom. When Y is a nitrogen atom, it is particularly preferred that R1 is a mono-halogenated aryl group, for example a 2-chlorophenyl group .
In a preferred embodiment X is a nitrogen atom.
Preferably, R3 is a hydrogen atom or an optionally
substituted alkyl group when X is a nitrogen atom.
Preferably when X is a nitrogen atom, R3 is a hydrogen atom or a substituted alkyl group wherein the alkyl group substituent is selected from the group consisting of an alkoxy-subst ituted aryl group, a carboxyl group, an alkoxycarbonyl group and a hydroxyl group. More
preferably, when X is a nitrogen atom, R3 is a hydrogen atom, a methyl group, an ethyl group, a propenyl group, a hydroxyl-subst ituted propyl group or a cyclopropyl group. More preferably still, R3 is a hydrogen atom or a methyl group. Most preferably, R3 is a methyl group.
In a preferred embodiment R4 is a group represented by the formula a ) :
Figure imgf000025_0001
(a)
Z is a nitrogen atom or an optionally substituted methine group;
R4a is a hydrogen atom, a halo group, an optionally substituted C1-C6 alkyl group, an optionally substituted C1-C6 alkoxy group or is a nitrogen-containing
heterocyclic group optionally substituted with a
substituent selected from the group consisting of a halo group, an optionally substituted C1-C6 alkyl group, an oxo group and an optionally substituted amino group; R4 is a hydrogen atom, a halo group, an optionally substituted C1-C6 alkyl group or an optionally substituted C1-C6 alkoxy group;
or, when R4a and R4 exist on adjacent ring atoms of the group of the formula (b) :
Figure imgf000026_0001
(b)
R a and R and the ring atoms to which they are attached may form, as taken together, a C3-C7 alkyl group, in which one or two methylene groups constituting the C3- C7 alkyl group may be each independently replaced by an oxygen atom or a group of -N(Rle)-, and the C3-C7 alkyl group may be substituted with one or more substituents selected from the group consisting of a halo group and a C1-C6 alkyl group;
or R4a and R4 and the ring atoms to which they are attached may form, as taken together, a spiro ring or a bicyclo ring to be formed of a 5-membered to 7-membered aliphatic ring and any other 3-membered to 7-membered aliphatic ring, in which one or two or more methylene groups constituting the spiro ring or the bicyclo ring may be each independently replaced by an oxygen atom, a sulfur atom, a sulfinyl group, a sulfonyl group, an oxo group or a group of -N(Rlf)-, and the spiro ring or the bicyclo ring may be each independently substituted with a substituent selected from the group consisting of a halo group, a hydroxyl group or a C1-C6 alkyl group; and
Rle and Rlf are each independently a hydrogen atom or a Ci- C6 alkyl group optionally substituted with a substituent selected from the group consisting of a halo group, a hydroxyl group, a cyano group, an oxo group, a C1-C6 alkyl group, a C 1-C6 alkoxy group, an amino group, a substituted amino group and a nitrogen-containing heterocyclic group.
In a preferred embodiment R4 is a group represented by the formula ( c ) :
Figure imgf000027_0001
(c)
wherein R4a is a hydrogen atom, a halo group, a C 1-C6 alkyl group or an optionally substituted C 1-C6 alkoxy group, or is a nitrogen-containing heterocyclic group optionally substituted with a substituent selected from the group consisting of a halo group, a C 1-C6 alkyl group, an oxo group and a group of -Q1-N (Rla) Rl ;
R4 is a hydrogen atom, a halo group, an optionally substituted C 1-C6 alkyl group or an optionally substituted C1-C6 alkoxy group;
Rla and Rl each independently is a hydrogen atom, a C1-C6 alkyl group, or as taken together, they may form an optionally substituted C2-C6 alkyl group; and
Q1 is a single bond or a C 1-C3 alkyl group.
In a preferred embodiment R4a is a substituted C 1-C6 alkoxy group or a nitrogen-containing heterocyclic group
substituted with a substituent selected from the group consisting of a halo group and a C 1-C6 alkyl group; and R4 is a hydrogen atom.
Preferably, when R4a is a substituted C 1-C6 alkoxy group, preferably a substituted C i_C2 alkoxy group, the
substituent is an amino group. In a preferred embodiment R a is a nitrogen-containing heterocyclic group optionally substituted with a C1-C6 alkyl group; and
R4 is a hydrogen atom, a methyl group, a methoxy group or a methyl group substituted by a hydroxy group.
In a preferred embodiment R4a is a nitrogen-containing heterocyclic group optionally substituted with a methyl group; and
R4 is a hydrogen atom, a methyl group, a methoxy group or a methyl group substituted by a hydroxy group.
In a preferred embodiment R4 is a group represented by the formula d) :
Figure imgf000028_0001
(d) wherein R m is selected from the group consisting of a hydrogen atom and an optionally substituted C1-C6 alkyl group. More preferably Rlm is a hydrogen atom or a methyl group .
In a preferred embodiment R4 is a substituted alkyl group wherein the alkyl group contains no ring structures or is a cycloalkyl group, said cycloalkyl group containing carbon atoms and 0 or 1 or 2 heteroatoms elected from 0, N and S, more preferably containing carbon atoms and 1 nitrogen atom. Preferably, when R4 is a substituted alkyl group, at least one of the alkyl group substituents is an optionally substituted Ci-6 alkyl group or a substituted aryl group containing carbon atoms and 0 or 1 or 2 heteroatoms elected from 0, N and S, more preferably containing carbon atoms and 1 nitrogen atom.
In a preferred embodiment R1 is a group represented by the formula (e) :
Figure imgf000029_0001
(e)
wherein R2a is a hydrogen atom, a halo group, a hydroxyl group, a cyano group, a C 1-C6 alkyl group, a C 1-C6 alkoxy group or a C 1-C6 alkoxy-Ci-C6 alkyl group;
R3a and R3 are each independently selected from the group consisting of a hydrogen atom, a hydroxyl group, an amino group, an alkoxy group and a C 1-C6 alkyl group;
T is a nitrogen atom, or a methine group which may be substituted with a halo group, a hydroxyl group, a cyano group, a C 1-C6 alkyl group, a C 1-C6 alkoxy group or a C1-C6 alkoxy-Ci-C6 alkyl group.
In a preferred embodiment, R a and R are each
independently a hydrogen atom, T is a methine group and R3a is a hydroxyl group.
Preferably, R1 is a group represented by the formula (f ) :
Figure imgf000030_0001
(f )
wherein R a and R are each independently selected from the group consisting of a hydrogen atom and a
hydroxyl group and T is a nitrogen atom or a methine group. Preferably, one of R3a and R3 is a hydrogen atom and the other is a hydroxyl group. More preferably R3a is a hydroxyl group and R3 is a hydrogen atom. In a preferred embodiment R1 is a group represented by the formula (g) :
Figure imgf000030_0002
(g)
wherein R2a is a hydrogen atom, a halo group, a hydroxyl group, a cyano group, a C 1-C6 alkyl group, a C 1-C6 alkoxy group or a C 1-C6 alkoxy-Ci-C6 alkyl group; and
T is a nitrogen atom, or a methine group which may be substituted with a halo group, a hydroxyl group, a cyano group, a C 1-C6 alkyl group, a C 1-C6 alkoxy group or a C1-C6 alkoxy-Ci-C6 alkyl group.
In a preferred embodiment R a is a halo group, a C 1-C6 alkyl group or a C 1-C6 alkoxy group, and
T is a methine group or is a methine group
substituted with a halo group or a C 1-C6 alkyl group. In a preferred embodiment R2a is a halo group; and
T is a methine group substituted with a halo group.
In a preferred embodiment R1 is a 2 , 6-dichlorophenyl group, that is wherein R1 wherein is a group represented by the formula (g) and R2a is a chlorine atom and T is a methine group substituted with a chlorine atom.
In a preferred embodiment of the compound of formula (I), Y is a nitrogen atom and R1 is a group represented by the formula h) :
Figure imgf000031_0001
(h)
wherein R2a is a hydrogen atom, a hydroxyl group, a cyano group, a C1-C6 alkyl group, a C1-C6 alkoxy group or a C1-C6 alkoxy-Ci-C6 alkyl group;
R3a and R3 are each independently selected from the group consisting of a hydrogen atom, a hydroxyl group, an amino group, an alkoxy group and a C1-C6 alkyl group; and T' is a nitrogen atom, or a methine group which may be substituted with a halo group, a hydroxyl group, a cyano group, a Ci~C6 alkyl group, a Ci~C6 alkoxy group or a Ci~C6 alkoxy-Ci-C6 alkyl group. More preferably, R2a , R3a and R3 are independently a hydrogen atom, and T' is a methine group substituted with a halo group. Most preferably, Y is a nitrogen atom and R1 is a 2- chlorophenyl group.
In a preferred embodiment the present invention provides a compound of Formula (II) :
Figure imgf000032_0001
(ID
or a pharmaceutically acceptable salt or iV-oxide
derivative thereof, wherein:
R1 is an optionally substituted aryl group;
R2 is a hydrogen atom;
R3 is a hydrogen atom or an optionally substituted alkyl group;
R4 is an optionally substituted alkyl group or an optionally substituted aryl group.
Preferably, in the compound of Formula (II), R1 is a group represented by the formula (e) as defined above and/or R4 is a group represented by the formula (a) as defined above. More preferably, R1 is a group represented by the formula (f) or the formula (g) as defined above and/or R4 is a group represented by the formula (c) or formula (d) as defined above. More preferably still, R1 is a group represented by the formula (g) as defined above and/or R4 is a group represented by the formula (d) as defined above. Most preferably, R1 is a 2 , 6-dichlorophenyl group or a 4-hydroxyphenyl group and/or R2 is a hydrogen atom and/or R3 is a hydrogen atom, a methyl group, an ethyl group, a propenyl group, a hydroxyl-substituted propyl group or a cyclopropyl group and/or R4 is a group
represented by the formula (d) as defined above.
In a preferred embodiment the present invention provides a compound of Formula (III) :
Figure imgf000033_0001
(iir
or a pharmaceutically acceptable salt or iV-oxide
derivative thereof, wherein:
R1 is an optionally substituted aryl group;
R3 is a hydrogen atom or an optionally substituted alkyl group;
R4 is an optionally substituted aryl group. Preferably, in the compound of Formula (III), R1 is a group represented by the formula (h) as defined above and/or R4 is a group represented by the formula (a) as defined above. More preferably, R1 is a group represented by the formula (h) as defined above and/or R4 is a group represented by the formula (c) as defined above. More preferably still, R1 is a group represented by the formula (h) as defined above and/or R3 is a hydrogen atom or a methyl group and/or R4 is a group represented by the formula (c) wherein R4a is a nitrogen-containing
heterocyclic group optionally substituted with a C1-C6 alkyl group; and R4 is a hydrogen atom, a methyl group, a methoxy group or a methyl group substituted by a hydroxy group. More preferably still, R1 is a 2-chlorophenyl group and/or R3 is a methyl group and/or R4 is a group re resented by the formula (i) :
Figure imgf000034_0001
(i)
wherein R5a is a hydrogen atom, a methyl group, a methoxy group or a methyl group substituted by a hydroxy group; and R6a is a hydrogen atom or a methyl group. Most preferably, R1 is a 2-chlorophenyl group and/or R3 is a methyl group and/or R4 is a group represented by the formula (i) as defined above, wherein R5a is a hydrogen atom, a methyl group, a methoxy group or a methyl group substituted by a hydroxy group; and R6a is a hydrogen atom . In a preferred embodiment the compound of formula (I) or formula (II) is selected from the following:
( 1 ) 8-Methy1-2- ( ( 4- ( 4-methylpiperaz in-1-yl ) phenyl ) amino ) - 6 -phenylpyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one ;
(2) 6- (2, 6-Dichlorophenyl) -8-methyl-2- ( (4- (4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one;
( 3 ) 6- ( 2-Chlorophenyl ) -8-methyl-2- ( ( 4- ( 4-methylpiperaz in- 1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one ;
(4) 6- (2-Methoxyphenyl) -8-methyl-2- ( (4- (4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one;
( 5 ) 8-Methy1-2- ( ( 4- ( 4-methylpiperaz in-1-yl ) phenyl ) amino ) - 6- (o-tolyl) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one ; (6) 6- (2, 4-Dichlorophenyl) -8-methyl-2- ( (4- (4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one;
(7) 6- (2, 5-Dichlorophenyl) -8-methyl-2- ( (4- (4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 (8H) -one;
(8) 6- (2, 3-Dichlorophenyl) -8-methyl-2- ( (4- (4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 (8H) -one;
(9) 8-Methyl-2- (( 4- ( 4-methylpiperaz in-l-yl ) phenyl ) amino ) - 6 - ( thiophen-3-yl ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one ;
(10) 8-Methyl-6- (5-methyl-l, 3, 4-oxadiazol-2-yl ) -2- ( (4- (piperaz in-l-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one;
(11) 6- (3-Chloropyridin-2-yl) -8-methyl-2- ( ( 4- ( 4- methylpiperaz in-l-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one;
(12) 6 - ( 2 , 6 -Dichlorophenyl ) -8-methy1-2- ( ( 4- (piperaz in-l- yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one ;
(13) 8-Allyl-6- (2, 6-dichlorophenyl) -2- ( (4- (4- methylpiperaz in-l-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one;
(14) 6- (2, 6 -Dichlorophenyl) -8- ( 4-methoxybenzyl ) -2- ( ( 4- ( 4- methylpiperaz in-l-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one;
(15) 6 - ( 2 , 6 -Dichlorophenyl ) -2- ( ( 4- ( 4-methylpiperaz in-l- yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one ;
(16) 6- (2, 6 -Dichlorophenyl) -8-ethyl-2- ( (4- (4- methylpiperaz in-l-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one;
(17) Ethyl 2- (6- (2, 6-dichlorophenyl) -2- ( (4- (4- methylpiperaz in-l-yl ) phenyl ) amino ) -5-oxopyrido [2,3- d] pyrimidin-8 ( 5H) -yl ) acetate ; (18) 2-(6-(2,6 -Dichlorophenyl ) -2- ( ( 4- ( 4-methylpiperaz in-
1-yl ) phenyl ) amino ) -5-oxopyrido [ 2 , 3-d] pyrimidin-8 ( 5H ) - yl) acetic acid.2HCl;
( 19 ) 6- ( 2 , 6 -Dichlorophenyl ) -8- ( 3-hydroxypropyl ) -2- ( ( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin-
5 (8H) -one;
( 20 ) 6- ( 2 , 5-Dimethyl-lH-pyrrol-l-yl ) -8- ( 4-methoxybenzyl ) -
2- ( ( 4- ( 4-methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [2,3- d] pyrimidin-5 ( 8H) -one;
(21) 6- ( 2 , 6 -Dichlorophenyl ) -2- ( ( 6- (piperazin-1- yl ) pyridin-3-yl ) amino ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one; (22) 6- (2, 6 -Dichlorophenyl) -2- ( (4- (2-
( diethylamino ) ethoxy ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 (8H) -one;
(23) 6- ( 2 , 6 -Dichlorophenyl ) -2- ( (pyridin-4- ylmethyl ) amino ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one ;
( 24 ) 6- ( 2 , 6 -Dichlorophenyl ) -2- ( ( l-methylpiperidin-4- yl ) amino ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one ;
(25) 7-Amino-6- ( 2-chlorophenyl ) -8-methyl-2- ( (4- (4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one;
(26) 6- (2-Chlorophenyl) -7, 8-dimethyl-2- ( (4- (4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one;
(27) 6- ( 2-Chlorophenyl ) -7-methyl-2- (( 4- (piperazin-1- yl ) phenyl ) amino ) -5H-pyrano [ 2 , 3-d] pyrimidin-5-one ;
(28) 6 - ( 2-Chlorophenyl ) -2- ( (4- (piperaz in-1- yl ) phenyl ) amino ) -5H-pyrano [ 2 , 3-d] pyrimidin-5-one .2HC1 ;
(29) 6- (2-Chlorophenyl) -8-cyclopropyl-2- ( (4- (4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one;
(30) 6- ( 4-Hydroxyphenyl) -8-methyl-2- ( ( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one; (31) 6 - ( 3-Hydroxyphenyl ) -8 -methy1-2- ( ( 4- (piperaz in- 1- yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one .
In a preferred embodiment the compound of formula (I) or formula (III) is selected from the following:
(32) 3- ( 2-chlorophenyl ) -1- ( 4-methoxyphenyl ) -7- ( ( 4- ( 4- methylpiperaz in-l-yl ) phenyl ) amino ) pyrimido [4,5- c] pyridaz in- 4 ( 1H) -one;
( 33 ) 3- ( 2-chlorophenyl ) -1 -methyl- 7- ( ( 4- (piperaz in- 1 - yl ) phenyl ) amino ) pyrimido [ 4 , 5-c ] pyridaz in-4 ( 1H ) -one ;
(34) 3- (2-chlorophenyl) -1 -methyl- 7- ( (3- ( (methylamino ) methyl ) -4- morpholinophenyl ) amino ) pyrimido [ 4 , 5-c ] pyridaz in-4 ( 1H) - one ;
(35) 3- ( 2-chlorophenyl ) -7- (( 3- (hydroxymethyl ) -4-
( piperaz in-l-yl ) phenyl ) amino ) -pyrimido [ 4 , 5-c ] pyridaz in- 4 ( 1H) -one;
( 36 ) 3- ( 2-chlorophenyl ) -7- ( (3- (hydroxymethyl ) -4-
( piperaz in-l-yl ) phenyl ) amino ) - 1 -methylpyrimido [4,5- c] pyridazin-4 ( 1H) -one;
(37) 3- (2-chlorophenyl) -l-methyl-7- ( (3-methyl-4-
( piperaz in-l-yl ) phenyl ) amino ) pyrimido [ 4 , 5-c ] pyridaz in- 4 ( 1H) -one;
(38) 3- ( 2-chlorophenyl ) -7- ( ( 3-methoxy-4- (piperaz in- 1- yl ) phenyl ) amino ) -1 -methylpyrimido [ 4 , 5-c ] pyridaz in-4 ( 1H) - one ;
(39) 3- (2-chlorophenyl) -7- ( (3- (hydroxymethyl) -4- (4- i sopropylpiperaz in-l-yl ) phenyl ) amino ) -1- methylpyrimido [4, 5-c] pyridazin-4 ( 1H) -one; and
(40) (R) -3- (2-chlorophenyl) -l-methyl-7- ( (4- (3- methylpiperaz in-l-yl ) phenyl ) amino ) pyrimido [4,5- c] pyridazin-4 ( 1H) -one . In a preferred embodiment there is provided the compound of formula (I), or a pharmaceutically acceptable salt or N-oxide derivative thereof, and at least one
pharmaceutically acceptable excipient .
Suitable pharmaceutically acceptable excipients would be known by the person skilled in the art, for example, fats, water, physiological saline, alcohol (e.g.,
ethanol), glycerol, polyols, aqueous glucose solution, extending agent, disintegrating agent, binder, lubricant, wetting agent, stabilizer, emulsifier, dispersant, preservative, sweetener, colorant, seasoning agent or aromatizer, concentrating agent, diluent, buffer
substance, solvent or solubilizing agent, chemical for achieving storage effect, salt for modifying osmotic pressure, coating agent or antioxidant, saccharides such as lactose or glucose; starch of corn,
wheat or rice; fatty acids such as stearic acid;
inorganic salts such as magnesium metasilicate aluminate or anhydrous calcium phosphate; synthetic polymers such as polyvinylpyrrolidone or polyalkylene glycol; alcohols such as stearyl alcohol or benzyl alcohol; synthetic cellulose derivatives such as methylcellulose,
carboxymethylcellulose, ethylcellulose or
hydroxypropylmethylcellulose ; and other conventionally used additives such as gelatin, talc, plant oil and gum arabic .
In a preferred embodiment there is provided a
pharmaceutical composition comprising the compound of formula (I), or a pharmaceutically acceptable salt or N- oxide derivative thereof, and at least one
pharmaceutically acceptable excipient. In a preferred embodiment there is provided a pharmaceutical composition comprising the compound of formula (I) comprising one or more further
pharmaceutically active agents.
In a preferred embodiment there is provided the compound of formula (I), or a pharmaceutically acceptable salt or N-oxide derivative thereof, or a pharmaceutical
composition comprising the compound of formula (I) for use in therapy.
In a preferred embodiment there is provided the compound of formula (I) for use as a medicament. In a preferred embodiment there is provided the compound of formula (I) for use in treating or preventing cancer.
In a preferred embodiment there is provided the compound of formula (I), or a pharmaceutically acceptable salt or N-oxide derivative thereof, or a pharmaceutical
composition comprising the compound of formula (I) for use as a medicament and/or for use in treating or preventing cancer. In a preferred embodiment there is provided the use of the compound of formula (I) for the manufacture of a medicament for treating or preventing cancer.
In a preferred embodiment there is provided a method of treating or preventing cancer in a human or animal patient comprising administering to a patient in need thereof an effective amount of a compound of formula {1} or a pharmaceutical composition comprising formula (I) . Preferably, the compounds of the present invention have an IC5o value for Wee-1 kinase of about 1 nM to about 7000 nM, more preferably from about 1 nM to about 1500 nM, or from about 1 nM to about 500 nM, or from about 1 nM to about 250 nM, or from about 1 nM to about 100 nM, or from about 1 nM to about 50 nM, or from about 1 nM to about 10 nM, most preferably less than ΙΟηΜ. A method for
determining the IC50 value of a compound for Wee-1 kinase is described below (see examples) .
When introducing elements of the present disclosure or the preferred embodiments ( s ) thereof, the articles "a", "an", "the" and "said" are intended to mean that there are one or more of the elements. The terms "comprising", "including" and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.
The foregoing detailed description has been provided by way of explanation and illustration, and is not intended to limit the scope of the appended claims. Many
variations in the presently preferred embodiments
illustrated herein will be apparent to one of ordinary skill in the art, and remain within the scope of the appended claims and their equivalents.
The following non-limiting examples further illustrate the present invention. EXAMPLES
The present invention will now be described in relation to several examples. Examples 1 to 31 were synthesised according to the methods described subsequently. IC5o values were determined as described below and are represented in the following table.
5
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
9 ++ ++ +++
1 H 0 ++ ++ +++
1 H
*Weel activity is classified as the following: ' + '
corresponds to an IC5o in the range of 100 nM to 10 μΜ and '++' corresponds to an IC5o of <100 nM.
** For representative examples, HT29 pCDC2 activity is classified as the following: '+' corresponds to an IC50 >500 nM and '++' corresponds to an IC50 ≤500 nM.
*** For representative examples, HT29 proliferation
activity is classified as the following: '+' corresponds to an IC50 >4 μΜ, '++' corresponds to an IC50 in the range of 1 μΜ to 4 μΜ and '+++' corresponds to an IC50 <1 μΜ.
Representative compounds (e.g. Example 2) caused dose dependent inhibition of phosphorylation of cdc2 in HT29 and HCT-116 cells, and inhibition of proliferation of
HT29 cells at concentrations < 10μΜ.
Representative examples (e.g. 33-38 & 40) have CLint
values <25uL/min/mg in human liver microsome incubations (method 4) . In the same assay, the clinical Weel
inhibitor MK1775 has a CLint value of 28uL/min/mg .
Abbreviations
aq: aqueous; dba : dibenzylideneacetone ; DCM:
dichloromethane ; DIPEA: diisopropylethylamine ; DMA: N,N- dimethyl acetamide; DMF : N, W-dimethylformamide ; DMSO: dimethylsulfoxide ; dppf : 1,1'- bis ( diphenylphosphino ) ferrocene ; EtOAc: ethyl acetate; ESI: electrospray ionisation; h: hour; HPLC: high
pressure liquid chromatography; LC: liquid
chromatography; LCMS : liquid chromatography mass
spectrometry; M: molar; m/z : mass-to-charge ratio; mCPBA: 3-chloroperbenzoic acid; MeOH: methanol; min: minutes; MS: mass spectrometry; NMR: nuclear magnetic resonance; RT : retention time; RB : round-bottomed; RT : room
temperature; SM: starting material; SPhos: 2- dicyclohexylphosphino-2 ' , 6 ' -dimethoxybiphenyl ; TFA :
trifluoroacetic acid; THF : tetrahydrofuran; TLC: thin layer chromatography.
General Experimental Conditions Solvents and reagents
Common organic solvents that were used in reactions (e.g. THF, DMF, DCM, and methanol) were purchased anhydrous from Sigma-Aldrich® in Sure/Seal™ bottles and were handled appropriately under nitrogen. Water was
deionised using an Elga PURELAB Option-Q. All other solvents used (i.e. for work-up procedures and
purification) were generally HPLC grade and were used as supplied from various commercial sources. Unless
otherwise stated, all starting materials used were purchased from commercial suppliers and used as supplied. Microwave synthesis
Unless quoted otherwise, microwave experiments were carried out using a CEM Discover™/Explorer24™ system controlled by Synergy 1.5 software. In other cases a Biotage Initiator™ Eight was used. Both machines give good reproducibility and control at temperature ranges from 60-250°C and pressures of up to maximum of 20 bar.
Flash chromatography
Purification of compounds by flash chromatography was achieved using a Biotage Isolera Four system. Unless otherwise stated, Biotage KP-Sil SNAP cartridge columns (10-340 g) were used along with the stated solvent system and an appropriate solvent gradient depending on compound polarity. In the case of more polar and basic compounds, Biotage KP-NH SNAP cartridge columns (11 g) were used.
NMR spectroscopy
1H NMR spectra were recorded at ambient temperature using a Bruker Avance (400 or 500 MHz) spectrometer. All chemical shifts (δ) are expressed in ppm. Residual solvent signals were used as an internal standard and the characteristic solvent peaks were corrected to the reference data outlined in J. Org. Chem., 1997, 62, p7512-7515; in other cases, NMR solvents contained tetramethylsilane, which was used as an internal
standard .
High Pressure Liquid Chromatography
Liquid Chromatography Mass Spectrometry (LCMS)
experiments to determine retention times (RT) and
associated mass ions were performed using one of the following methods: Method A: The system consists of an Agilent Technologies 6140 single quadrupole mass spectrometer linked to an Agilent Technologies 1290 Infinity LC system with UV diode array detector and autosampler. The spectrometer consists of a multimode ionization source ( electrospray and atmospheric pressure chemical ionizations) operating in positive and negative ion mode. LCMS experiments were performed on each sample submitted using the following conditions: LC Column: Zorbax Eclipse Plus C18 RRHD 1.8 micron 50 x 2.1 mm maintained at 40°C. Mobile phases: A) 0.1% (v/v) formic acid in water; B) 0.1% (v/v) formic acid in acetonitrile .
Gradient Time Flow
%A %B
(min) (mL/min)
0.00 1.0 95 5
1.80 1.0 0 100
2.20 1.0 0 100
2.21 1.0 95 5
2.50 1.0 95 5 Method B: The system consisted of a ThermoFinnigan LCQ Advantage Mass Spectrometer with Surveyor LC system and 200 position autosampler. The LC system was coupled to an inline Surveyor DAD detector and ESI source operating in positive and negative ion mode. LCMS experiments were performed on each sample submitted using the following conditions: LC Column: Luna 3 micron C18 50 x 2 mm.
Mobile phases: A) 0.1% (v/v) formic acid in water; B) 0.1% (v/v) formic acid in acetonitrile.
Gradient Time Flow
(min) (mL/min)
0.00 0 6 95 5
7.00 0 6 5 95
8.00 0 6 5 95
8.20 0 6 95 5
11.00 0 6 95 5 Preparative High Pressure Liquid Chromatography
The system consisted of an Agilent Technologies 6120 single quadrupole mass spectrometer linked to an Agilent Technologies 1200 Preparative LC system with Multiple Wavelength detector and autosampler. The mass
spectrometer used a multimode ionization source
(electrospray and atmospheric pressure chemical
ionizations) operating in positive and negative ion mode. Fraction collection was mass-triggered (multimode
positive and negative ion) . Purification experiments, unless otherwise stated, were performed under basic conditions at an appropriate solvent gradient that was typically determined by the retention time found using HPLC Method A. In cases were the basic conditions were unsuccessful, acidic conditions were employed.
Basic conditions: LC Column: Waters XBridge™ Prep C18 5 μπι OBDTM 19 x 50 mm column at RT . Mobile phase: A) 0.1% (v/v) ammonium hydroxide in water; B) 0.1% (v/v) ammonium hydroxide in 95:5, acetonitrile/water . Total experiment time was ca. 10 min and an example method is given:
Gradient Time Flow
%A %B
(min) (mL/min)
0.00 20.0 50 50
3.00 20.0 12 88
5.00 20.0 12 88
7.00 20.0 0 100
8.0 20.0 0 100
8.20 20.0 50 50
Acidic conditions: LC Column: Waters XBridge™ Prep C18 5μπι OBDTM 19 x 50 mm column at RT . Mobile phase: A) Water 0.1% (v/v) formic acid in water; B) 0.1% (v/v) formic acid in 95:5, acetonitrile/water . Total experiment time was ca. 10 min and an example method is given:
Gradient Time Flow
%A %B
(min) (mL/min)
0.00 20.0 95 5
7.00 20.0 0 100
9.00 20.0 0 100
9.20 20.0 95 5 The pure fractions were combined and concentrated using a Genevac EZ-2 Elite, unless stated otherwise.
Nomenclature
Unless otherwise indicated, the nomenclature of
structures was determined using the 'Convert Structure to Name' function of ChemBioDraw Ultra 12.0.2
( CambridgeSoft /PerkinElmer ) .
Example 1: 8-Methyl-2- ( ( 4- ( 4-methylpiperazin-l- yl ) phenyl ) amino ) -6 -phenylpyrido [ 2 , 3-d] pyrimidin-5 ( 8H) -one
Figure imgf000051_0001
Step 1: Ethyl 4- ( ( 3-ethoxy-3-oxopropyl ) (methyl ) amino) -2- (methylthio ) pyrimidine-5-carboxylate : Ethyl 4-chloro-2- (methylthio ) pyrimidine-5-carboxylate (426 mg, 1.833 mmol) [commercially available] was added to a stirred solution of ethyl 3- (methylamino ) propanoate (240 mg, 1.83 mmol) [prepared according to the literature procedure: J. Org. Chem., 1985, 50, p3979-3982; J. Med. Chem., 2005, 48, p4100-4110] in THF (5.0 mL) at RT under nitrogen. The temperature was increased to 50 °C. After 1 h, the solvents were removed in vacuo and the remaining residue was purified by flash chromatography (0-100%, EtOAc in cyclohexane) to give the title compound (256 mg, 43%) as a colourless oil. LCMS (Method A): RT = 1.37 min, m/z = 328 [M+H]+.
Step 2: Ethyl 5-hydroxy-8-methyl-2- (methylthio) -7, 8- dihydropyrido [ 2 , 3-d] pyrimidine-6 -carboxylate : Potassium tert-butoxide solution (1.0 M in THF) (0.767 mL, 0.767 mmol) was added to a stirred solution of ethyl 4-((3- ethoxy-3-oxopropyl ) (methyl) amino) -2-
(methylthio ) pyrimidine-5-carboxylate (251 mg, 0.767 mmol) in toluene (2.5 mL) at RT under nitrogen. Further toluene (2.5 mL) was added to allow agitation due to the formed precipitation. After 2 h, acetic acid (50 μΐ) and water (4.0 mL) were added. The resulting biphasic mixture was separated, the aqueous layer was extracted (DCM x 3), the combined organic phase was washed with water, dried (Phase Separator), and the solvents were removed in vacuo to give the title compound (200 mg, 93%) as a yellow solid. LCMS (Method A): RT = 1.33 min, m/z = 282 [M+H]+.
Step 3: Ethyl 8-methyl-2- (methylthio ) -5-oxo-5 , 8- dihydropyrido [ 2 , 3-d] pyrimidine-6-carboxylate : Bromine
(0.036 mL, 0.703 mmol) in chloroform (1.0 mL) was added to a stirred solution of ethyl 5-hydroxy-8-methyl-2- (methylthio ) -7 , 8-dihydropyrido [ 2 , 3-d] pyrimidine-6 - carboxylate (198 mg, 0.703 mmol) and triethylamine (0.294 mL, 2.11 mmol) in chloroform (3.5 mL) at 0 °C under nitrogen. The temperature was allowed to increase to RT . After 18 h, LCMS showed no residual SM. The reaction mixture was partitioned between saturated sodium
thiosulfate (aq) solution and DCM, separated, extracted (DCM x 2), dried (Phase Separator), and the solvents were removed in vacuo to give the title compound (181 mg, 92%) that was carried through to the next step without further purification. LCMS (Method A): RT = 0.88 min, m/z = 280 [M+H]+.
Step 4: 8-Methyl-2- (methylthio ) -5-oxo-5 , 8- dihydropyrido [ 2 , 3-d] pyrimidine-6-carboxylic acid: 1M HC1 (aq) solution (17.4 mL, 17.4 mmol) was added to a stirred solution of ethyl 8-methyl-2- (methylthio) -5-oxo-5, 8- dihydropyrido [ 2 , 3-d] pyrimidine-6-carboxylate (1.62 g, 5.79 mmol) in 1,4-dioxane (16.0 mL) at RT under nitrogen. The temperature was increased to 95 °C. After 1 h, the reaction mixture was cooled to RT, the precipitate that formed was filtered off and washed sequentially by water and EtOAc, and was dried under vacuum to give the title compound (1.07 g, 74%) as an off-white solid. LCMS
(Method A): RT = 0.84 min, m/z = 252 [M+H]+. Step 5: 6-Iodo-8-methyl-2- (methylthio) pyrido [2, 3- d] pyrimidin-5 ( 8H ) -one : Lead (IV) acetate (582 mg, 1.31 mmol), iodine (333 mg, 1.31 mmol) and ( E ) -1 , 1 ' - ( diazene- 1 , 2-diyl ) dicyclohexanecarbonitrile (14.6 mg, 0.06 mmol) were added sequentially to a pre-degassed solution of 8- methyl-2- (methylthio) -5-oxo-5, 8-dihydropyrido [2,3- d] pyrimidine-6-carboxylic acid (300 mg, 1.19 mmol) in carbon tetrachloride (6.0 mL) at RT in a 10 mL vial. The vessel was sealed and heated at 80 °C. After 20 hours, the reaction mixture was partitioned between DCM and saturated sodium thiosulfate (aq) solution, separated, extracted (DCM x 3), filtered/dried (Phase Separator), the solvents were removed in vacuo and the remaining residue was purified by flash chromatography (0-100%, EtOAc in cyclohexane) to give the title compound (159 mg, 40%) as a pale yellow solid. LCMS (Method A): RT = 0.94 min, m/z = 334 [M+H]+.
Step 6: 8-Methyl-2- (methylthio) -6 -phenylpyrido [2, 3- d] pyrimidin-5 ( 8H) -one : PdCl2 (dppf ) -DCM adduct (10.4 mg, 0.013 mmol) was added to a pre-degassed solution of 6- iodo-8-methyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) - one (42.3 mg, 0.127 mmol), phenylboronic acid (20.1 mg, 0.165 mmol) and sodium carbonate (40.4 mg, 0.381 mmol) in DMF (2.0 mL) in a 10 mL vial. The reaction mixture was heated to 80 °C with stirring (conventional) . After 3 h, the reaction mixture was concentrated in vacuo (Genevac EZ-2 Elite) . The remaining residue was partitioned between DCM and 1:1 water/brine, separated, extracted (2 x DCM), dried (Phase Separator), the solvents were removed in vacuo, and the remaining residue was purified by flash chromagraphy (0-50%, EtOAc in cyclohexane) to give the title compound (18.9 mg, 53%) as a yellow solid. LCMS (Method A): RT = 1.13 min, m/z = 284 [M+H]+.
Step 7: 8-Methyl-2- ( ( 4- ( 4-methylpiperazin-l- yl ) phenyl ) amino ) -6 -phenylpyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) - one: mCPBA (<77% pure) (17.3 mg, 0.077 mmol) in DCM (1.0 mL) was added to a stirred solution of 8-methyl-2- (methylthio ) -6 -phenylpyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one
(18.9 mg, 0.067 mmol) in toluene (2.0 mL) at 0 °C under nitrogen. After 10 min, LCMS showed that the sulfoxide was the major peak (RT = 0.55 min) . DIPEA (0.035 mL, 0.200 mmol) was added dropwise, followed by 4- (4- methylpiperazin-l-yl ) aniline (14.0 mg, 0.073 mmol) in toluene (0.5 mL) . After 16 hours, the reaction mixture was concentrated in vacuo and purified by flash
chromatography using a KP-NH column (0-100% EtOAc in cyclohexane) to give product material that needed further purification by preparative HPLC. The pure fractions were concentrated to give the title compound (4.8 mg, 17%) as a yellow solid. Ή NMR (500 MHz, CDC13): δ 9.35 (s, 1H), 7.63-7.58 (m, 5H) , 7.45-7.38 (m, 3H) , 7.34 (t, 1H) , 6.98 (d, 2H), 3.82 (s, 3H) , 3.59-3.45 (m, 4H) , 3.30- 2.29 (m, 4H) , 2.81-2.68 (m, 3H) . LCMS (Method A): RT = 0.77 min, m/z = 427 [M+H]+.
Example 2: 6- ( 2 , 6 -Dichlorophenyl ) -8-methyl-2- ( ( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin-
5 ( 8H) -one
Figure imgf000055_0001
Step 1: 6- ( 2 , 6-Dichlorophenyl ) -8-methyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one : Pd2 (dba ) 3 (4.2 mg, 4.6 pmol) was added to a pre-degassed solution of 6 -iodo-8-methyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one (153 mg, 0.460 mmol), (2,6- dichlorophenyl ) boronic acid (114 mg, 0.599 mmol), SPhos (7.6 mg, 0.018 mmol) and potassium phosphate, tribasic (293 mg, 1.38 mmol) in a, , -trifluorotoluene (2.0 mL) in a 10 mL vial. The vessel was sealed and the reaction mixture was stirred at 80 °C. After 16 h, analysis by LCMS showed incomplete reaction. On increasing the temperature to 90 °C, LCMS showed no further change.
Further Pd(OAc) 2 (4 mg) and SPhos (8 mg) were added.
After a further 16 h, the solvents were removed in vacuo (Genevac EZ-2), the remaining residue was partitioned between EtOAc and sodium bicarbonate (aq) solution, separated, extracted (EtOAc x 2), and dried (Phase
Separator) . The solvents were removed in vacuo and the remaining residue was purified by flash chromatography (0-100%, EtOAc in cyclohexane) to give the title compound
(15.5 mg, 10%) as a white solid. LCMS (Method A): RT = 1.21 min, m/z = 352 [M+H]+. Step 2: 6- ( 2 , 6-Dichlorophenyl ) -8-methyl-2- ( ( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one: mCPBA (<77% pure) (11.4 mg, 0.051 mmol) in DCM (1.0 mL) was added to a stirred solution of 6-(2,6- dichlorophenyl ) -8-methyl-2- (methylthio ) pyrido [2,3- d] pyrimidin-5 ( 8H) -one (15.5 mg, 0.044 mmol) in toluene (2.0 mL) at 0 °C under nitrogen. After 20 min, LCMS showed sulfoxide as the major peak (RT = 0.83 min), along with some sulfone (RT = 0.97 min). DIPEA (0.023 mL, 0.132 mmol) was added, followed by dropwise addition of 4- (4- methylpiperazin-l-yl ) aniline (9.3 mg, 0.048 mmol) in toluene (0.5 mL) and the temperature was increased to 60 °C. After 16 h, the solvents were removed in vacuo and the remaining residue was purified by preparative HPLC. The pure fractions were concentrated to give the title compound (4.5 mg, 20%) as a yellow solid. Ή NMR (500 MHz, CDC13) : δ 9.32 (s, 1H) , 8.23 (s, 1H) , 7.60 (d, 2H) , 7.46 (s, 1H) , 7.41 (d, 2H) , 7.25 (t, 1H, overlapping with solvent), 6.98 (d, 2H) , 3.81 (s, 3H) , 3.36 (t, 4H) , 2.94 (t, 4H) , 2.57 (s, 3H) . LCMS (Method A): RT = 0.84 min, m/z = 495 [M+H]+.
Example 3: 6- ( 2-Chlorophenyl ) -8-methyl-2- ( ( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin-
5 ( 8H) -one
Figure imgf000056_0001
Step 1: 6- ( 2-Chlorophenyl ) -8-methyl-2-
(methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one : Pd2 (dba ) 3 (3.3 mg, 3.6 pmol) was added to a pre-degassed solution of 6 -iodo-8-methyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one (30.0 mg, 0.090 mmol), ( 2-chlorophenyl ) boronic acid (42.2 mg, 0.270 mmol), SPhos (3.0 mg, 7.2 pmol) and potassium phosphate, tribasic (24.9 mg, 0.117 mmol) in toluene (1.0 mL) in a 4 mL vial. The vessel was sealed and the reaction mixture was heated to 90 °C, with stirring. After 1 h, the reaction mixture was loaded directly onto a column and purified by flash
chromatography (0-100%, EtOAc in cyclohexane) to give the title compound (20.1 mg, 70%) as a white solid. LCMS (Method A): RT = 1.16 min, m/z = 318 [M+H]+.
Step 2: 6- ( 2-Chlorophenyl ) -8-methyl-2- ( ( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one: mCPBA (<77% pure) (16.4 mg, 0.073 mmol) in DCM (0.5 mL) was added to a stirred solution of 6-(2- chlorophenyl ) -8-methyl-2- (methylthio ) pyrido [2,3- d] pyrimidin-5 ( 8H) -one (20.1 mg, 0.063 mmol) in DCM (2.0 mL) at 0 °C under nitrogen. After 15 min, DIPEA (0.033 mL, 0.190 mmol) was added, followed by dropwise addition of 4- ( 4-methylpiperazin-l-yl ) aniline (13.3 mg, 0.070 mmol) in DCM (0.5 mL) . The temperature was allowed to increase to RT . After 16 h, the solvents were removed in vacuo and the residue was dissolved in DMF (2.0 mL) and transferred to a 10 mL vial and heated to 120 °C for 20 min (CEM Discover/Explorer24 ) . The solvents were removed in vacuo (Genevac EZ-2 Elite) and the remaining residue was purified by preparative HPLC (basic conditions) to give the title compound (8.1 mg, 28%) as a yellow solid. Ή NMR (500 MHz, CDC13): δ 9.32 (s, 1H) , 7.62-7.51 (m, 4H) , 7.50-7.45 (m, 1H) , 7.40-7.35 (m, 1H) , 7.33-7.28 (m, 2H), 6.97 (d, 2H), 3.80 (s, 3H) , 3.28 (t, 4H) , 2.77 (t, 4H) , 2.46 (s, 3H) . LCMS (Method A): RT = 0.82 min, m/z = 461 [M+H]+. Example 4: 6- ( 2-Methoxyphenyl ) -8-methyl-2- ( ( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin-
5 ( 8H) -one
Figure imgf000058_0001
Step 1: 6- ( 2-Methoxyphenyl ) -8-methyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one : Pd2(dba)3 (3.3 mg, 3.6 pmol) was added to a pre-degassed solution of 6 -iodo-8-methyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one (30.0 mg, 0.090 mmol), ( 2-methoxyphenyl ) boronic acid (41.1 mg, 0.270 mmol), SPhos (3.0 mg, 7.2 pmol) and potassium phosphate, tribasic (57.3 mg, 0.270 mmol) in toluene (1.0 mL) in a 4 mL vial. The vessel was sealed and the reaction mixture was heated to 90 °C, with stirring. After 1 h, the reaction mixture was allowed to cool to RT . The material was loaded directly onto a column and was purified by flash chromatography (0-100%, EtOAc/cyclohexane ) to give the title compound (20.3 mg, 72%) as a yellow solid. LCMS (Method A): RT = 1.09 min, m/z = 314 [M+H]+. Step 2: 6- ( 2-Methoxyphenyl ) -8-methyl-2- (( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one: mCPBA (<77% pure) (16.8 mg, 0.075 mmol) in DCM (0.5 mL) was added to a stirred solution of 6-(2- methoxyphenyl ) -8-methyl-2- (methylthio ) pyrido [2,3- d] pyrimidin-5 ( 8H) -one (20.3 mg, 0.065 mmol) in DCM (2.0 mL) at 0 °C under nitrogen. After 15 min, DIPEA (0.034 mL, 0.194 mmol) was added, followed by dropwise addition of 4- ( 4-methylpiperazin-l-yl ) aniline (13.6 mg, 0.071 mmol) in DCM (0.5 mL) . The temperature was allowed to increase to RT . After 16 h, analysis by LCMS showed no reaction. The solvents were removed in vacuo and the residue was dissolved in DMF (2.0 mL) and transferred to a 10 mL vial and heated to 120°C for 20 min (CEM
Discover/Explorer24 ) . The solvents were removed in vacuo (Genevac EZ-2) and the remaining residue was purified by preparative HPLC to give the title compound (13.0 mg,
44%) as a yellow solid. Ή NMR (500 MHz, CDCI3 ) : δ 9.31 (s, 1H), 7.60-7.55 (m, 3H) , 7.39-7.31 (m, 3H) , 7.05-6.95 (m, 4H), 3.81 (s, 3H) , 3.77 (s, 3H) , 3.24 (t, 4H) , 2.64 (t, 4H) , 2.39 (s, 3H) . LCMS (Method A): RT = 0.76 min, m/z = 457 [M+H]+.
Example 5: 8-Methyl-2- (( 4- ( 4-methylpiperazin-l- yl ) phenyl ) amino ) -6- (o-tolyl) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) - one
Figure imgf000059_0001
Step 1: 8-Methyl-2- (methylthio) -6- (o-tolyl) pyrido [2, 3- d] pyrimidin-5 ( 8H) -one : Pd2(dba)3 (3.3 mg, 3.6 pmol) was added to a pre-degassed solution of 6-iodo-8-methyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one (30.0 mg, 0.090 mmol), o-tolylboronic acid (36.7 mg, 0.270 mmol), SPhos (3.0 mg, 7.2 pmol) and potassium phosphate, tribasic (57.3 mg, 0.270 mmol) in toluene (1.0 mL) in a 4 mL vial. The vessel was sealed and the reaction mixture was heated to 90 °C, with stirring. After 16 h, the reaction mixture was loaded directly onto a column and purified by flash chromatography (0-100%, EtOAc in cyclohexane) to give the title compound (16.9 mg, 63%) as a yellow solid. LCMS (Method A): RT = 1.17 min, m/z = 298 [M+H] +. Step 2: 8-Methyl-2- (methylsulfonyl ) -6- ( o- tolyl ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H) -one : mCPBA (<77% pure) (49.0 mg, 0.219 mmol) was added to a stirred solution of 8-methyl-2- (methylthio) -6- (o-tolyl) pyrido [2,3- d] pyrimidin-5 ( 8H) -one (16.9 mg, 0.057 mmol) in DCM (1.5 mL) at RT under nitrogen. After 16 h, the reaction mixture was partitioned between DCM and sodium
bicarbonate (aq) solution, separated, extracted (DCM x 2), dried (Phase Separator), the solvents were removed in vacuo, and the remaining residue was purified by flash chromatography (0-100%, EtOAc in cyclohexane) to give the title compound (17.9 mg, 96%) as a yellow solid. LCMS (Method A): RT = 0.92 min, m/z = 330 [M+H]+.
Step 3: 8-Methyl-2- ( ( 4- ( 4-methylpiperazin-l- yl ) phenyl ) amino ) -6- (o-tolyl) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) - one: TFA (0.021 mL, 0.272 mmol) was added to a stirred solution of 8-methyl-2- (methylsulfonyl ) -6- ( o- tolyl ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H) -one (17.9 mg, 0.054 mmol) and 4- ( 4-methylpiperazin-l-yl ) aniline (11.4 mg, 0.060 mmol) in 2-propanol (1.0 mL) in a 10 mL vial. The vessel was sealed and irradiated at 120 °C for 20 min (CEM Discover/Explorer24 ) . LCMS analysis showed residual starting material. The reaction was rerun under the same conditions. LCMS analysis showed further conversion, but still some starting material. The reaction was rerun at 150 °C for 20 min. The solvents were removed in vacuo and the remaining residue was partitioned between
saturated sodium bicarbonate (aq) solution and DCM. The biphasic mixture was separated, extracted (DCM x 2), dried (Phase Separator), and the solvents were removed in vacuo. The remaining residue was purified by flash chromatography using a KP-NH column (0-100%, EtOAc in cyclohexane) to give the title compound (4.1 mg, 17%) as a yellow solid. Ή NMR (500 MHz, CDC13): δ 9.32 (s, 1H) , 7.59 (d, 2H), 7.44 (s, 1H) , 7.39 (br s, 1H) , 7.29-7.25 (m, 2H, overlapping solvent), 7.24-7.19 (m, 1H) , 7.16 (d, 1H), 6.98 (d, 2H), 3.79 (s, 3H) , 3.36 (br s, 4H) , 2.85 (br s, 4H) , 2.55 (s, 3H) , 2.27 (s, 3H) . LCMS (Method A): RT = 0.84 min, m/z = 441 [M+H]+.
Example 6: 6- ( 2 , 4-Dichlorophenyl ) -8-methyl-2- (( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one
Figure imgf000061_0001
Step 1: 6- ( 2 , 4-Dichlorophenyl ) -8-methyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one : Pd2 (dba ) 3 (3.3 mg, 3.6 pmol) was added to a pre-degassed solution of 6 -iodo-8-methyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one (30.0 mg, 0.090 mmol), (2,4- dichlorophenyl ) boronic acid (172 mg, 0.900 mmol), SPhos (3.0 mg, 7.2 pmol) and potassium phosphate, tribasic (57.3 mg, 0.270 mmol) in toluene (1.0 mL) in a 4 mL vial. The vessel was sealed and the reaction mixture was heated to 90 °C, with stirring. After 18 h, the reaction mixture was loaded directly onto a column and purified by flash chromatography (0-100%, EtOAc in cyclohexane) to give the title compound (22.1 mg, 70%) as a pale yellow solid. LCMS (Method A): RT = 1.29 min, m/z = 352 [M+H]+. Step 2: 6- ( 2 , 4-Dichlorophenyl ) -8-methyl-2- ( ( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one: mCPBA (<77% pure) (19.0 mg, 0.085 mmol) in DCM (0.5 mL) was added to a stirred solution of 6-(2,4- dichlorophenyl ) -8-methyl-2- (methylthio ) pyrido [2,3- d] pyrimidin-5 ( 8H) -one (22.1 mg, 0.063 mmol) in DCM (2.0 mL) at 0 °C under nitrogen. After 30 min, the
temperature was allowed to increase to RT (ice bath removed). After 2 h, DIPEA (0.033 mL, 0.188 mmol) was added and the reaction mixture was concentrated in vacuo. The residue was dissolved in DMF (2.0 mL) and transferred to a 10 mL vial and further DIPEA (0.033 mL, 0.188 mmol) and 4- ( 4-methylpiperazin-l-yl ) aniline (13.2 mg, 0.069 mmol) was added. The vessel was sealed and irradiated at 120 °C for 20 min (CEM Discover/Explorer24 ) . The
solvents were removed in vacuo (Genevac EZ-2) and the remaining residue was purified by flash chromatography using a KP-NH column (0-100%, EtOAc in cyclohexane) and further purified using a Biotage® ISOLUTE® SCX-2 column to give the title compound (2.9 mg, 9%) as a yellow solid. Ή NMR (500 MHz, CDC13): δ 9.31 (s, 1H) , 7.59-7.53 (m, 3H) , 7.50 (d, 1H), 7.37 (br s, 1H) , 7.34 (d, 1H) , 7.30 (dd, 1H), 6.97 (d, 2H) , 3.80 (s, 3H) , 3.26 (br s, 4H) , 2.66 (br s, 4H) , 2.42 (br s, 3H) . LCMS (Method A): RT = 0.94 min, m/z = 495 [M+H]+.
Example 7: 6- ( 2 , 5-Dichlorophenyl ) -8-methyl-2- ( ( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one
Figure imgf000062_0001
Step 1: 6- ( 2 , 5-Dichlorophenyl ) -8-methyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one : Pd2 (dba ) 3 (3.3 mg, 3.6 pmol) was added to a pre-degassed solution of 6 -iodo-8-methyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one (30.0 mg, 0.090 mmol), (2,5- dichlorophenyl ) boronic acid (172 mg, 0.900 mmol), SPhos (3.0 mg, 7.2 pmol) and potassium phosphate, tribasic (57.3 mg, 0.270 mmol) in toluene (1.0 mL) in a 4 mL vial. The vessel was sealed and the reaction mixture was heated to 90 °C, with stirring. After 18 h, the reaction mixture was loaded directly onto a column and purified by flash chromatography (0-100%, EtOAc in cyclohexane) to give the title compound (29.1 mg, 92%) as a white solid. LCMS (Method A): RT = 1.28 min, m/z = 352 [M+H]+.
Step 2: 6- ( 2 , 5-Dichlorophenyl ) -8-methyl-2- (( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one: mCPBA (<77% pure) (25.0 mg, 0.112 mmol) in DCM (0.5 mL) was added to a stirred solution of 6-(2,5- dichlorophenyl ) -8-methyl-2- (methylthio ) pyrido [2,3- d]pyrimidin-5 (8H) -one (29.1 mg, 0.083 mmol) in DCM (2.0 mL) at 0 °C under nitrogen. After 2 h, DIPEA (0.043 mL, 0.248 mmol) was added and the reaction mixture was concentrated in vacuo. The residue was dissolved in DMF (2.0 mL) and transferred to a 10 mL vial and DIPEA (0.043 mL, 0.248 mmol) and 4- ( 4-methylpiperazin-l-yl ) aniline (17.4 mg, 0.091 mmol) were added. The vessel was sealed and irradiated at 120 °C for 20 min (CEM
Discover/Explorer24 ) . The solvents were removed in vacuo (Genevac EZ-2 Elite) and the remaining residue was purified by flash chromatography using a KP-NH column (0- 100%, EtOAc in cyclohexane) to give the title compound (2.0 mg, 5%) as a yellow solid. Ή NMR (500 MHz, CDC13): δ 9.31 (s, 1H), 7.59-7.53 (m, 3H) , 7.43-7.34 (m, 3H) , 7.27 (dd, 1H), 6.98 (d, 2H) , 3.80 (s, 3H) , 3.23 (br s, 4H) , 2.62 (br s, 4H) , 2.38 (br s, 3H) . LCMS (Method A): RT = 0.93 min, m/z = 495 [M+H]+. Example 8: 6- ( 2 , 3-Dichlorophenyl ) -8-methyl-2- ( ( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin-
5 ( 8H) -one
Figure imgf000064_0001
Step 1: 6- ( 2 , 3-Dichlorophenyl ) -8-methyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one : Pd2(dba)3 (3.3 mg, 3.6 pmol) was added to a pre-degassed solution of 6 -iodo-8-methyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one (30.0 mg, 0.090 mmol), (2,3- dichlorophenyl ) boronic acid (172 mg, 0.900 mmol), SPhos (3.0 mg, 7.2 pmol) and potassium phosphate, tribasic
(57.3 mg, 0.270 mmol) in toluene (1.0 mL) in a 4 mL vial. The vessel was sealed and the reaction mixture was heated to 90 °C, with stirring. After 16 h, the reaction mixture was cooled and loaded directly onto a column and purified by flash chromatography (0-100%, EtOAc in cyclohexane) to give the title compound (20.8 mg, 66%) as a white solid. LCMS (Method A): RT = 1.26 min, m/z = 352 [M+H] +. Step 2: 6- ( 2 , 3-Dichlorophenyl ) -8-methyl-2- (( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one: mCPBA (<77% pure) (20.4 mg, 0.091 mmol) in DCM (0.5 mL) was added to a stirred solution of 6-(2,3- dichlorophenyl ) -8-methyl-2- (methylthio ) pyrido [2,3- d] pyrimidin-5 ( 8H) -one (20.8 mg, 0.059 mmol) in DCM (2.0 mL) at 0 °C under nitrogen. After 30 min, DIPEA (0.031 mL, 0.177 mmol) was added and the reaction mixture was concentrated in vacuo. The residue was dissolved in DMF (2.0 mL) and transferred to a 10 mL vial and 4- (4- methylpiperazin-l-yl ) aniline (12.4 mg, 0.065 mmol) was added. The vessel was sealed and irradiated at 120 °C for 20 min (CEM Discover/Explorer24 ) . The solvents were removed in vacuo (Genevac EZ-2) and the remaining residue was purified by flash chromatography using a KP-NH column (0-100%, EtOAc in cyclohexane) to give the title compound (2.4 mg, 8%) as a yellow solid. Ή NMR (500 MHz, CDCI3 ) : δ 9.32 (s, 1H), 7.57 (d, 2H) , 7.53 (s, 1H) , 7.48 (dd, 1H) , 7.39 (br s, 1H) , 7.27 (dd, 1H, overlapping with solvent), 7.24 (t, 1H, overlapping with solvent), 6.98 (d, 2H), 3.80 (s, 3H), 3.29 (t, 4H) , 2.71 (t, 4H) , 2.45 (s, 3H) . LCMS (Method A): RT = 0.92 min, m/z = 495
[M+H] +.
Example 9: 8-Methyl-2- ( ( 4- ( 4-methylpiperazin-l- yl ) phenyl ) amino ) -6 - ( thiophen-3-yl ) pyrido [ 2 , 3-d] pyrimidin- 5 (8H) -one
Figure imgf000065_0001
Step 1: 8-Methyl-2- (methylthio) -6- (thiophen-3- yl) pyrido [2, 3-d] pyrimidin-5 ( 8H) -one : Pd2(dba)3 (3.3 mg,
3.6 pmol) was added to a pre-degassed solution of 6-iodo- 8-methy1-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one (30.0 mg, 0.090 mmol), thiophen-3-ylboronic acid (34.6 mg, 0.270 mmol), SPhos (3.0 mg, 7.2 pmol) and thiophen-3- ylboronic acid (34.6 mg, 0.270 mmol) in toluene (1.0 mL) in a 4 mL vial. The vessel was sealed and the reaction mixture was heated to 90 °C, with stirring. After 16 h, the reaction mixture was cooled and loaded directly onto a column and purified by flash chromatography (0-100%, EtOAc in cyclohexane) to give the title compound (17.6 mg, 68%) as a yellow solid. LCMS (Method A) : RT = 1.14 min, m/z = 290 [M+H]+.
Step 2: 8-Methyl-2- ( ( 4- ( 4-methylpiperazin-l- yl ) phenyl ) amino ) -6 - ( thiophen-3-yl ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one: mCPBA (<77% pure) (21.0 mg, 0.094 mmol) was added to a stirred solution of 8-methyl-2- (methylthio) -6- (thiophen-3-yl ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H) -one (17.6 mg, 0.061 mmol) in toluene (2.0 mL) at RT under nitrogen. After 15 min, DIPEA (0.032 mL, 0.182 mmol) and 4- (4- methylpiperazin-l-yl ) aniline (12.8 mg, 0.067 mmol) in DMF (2.0 mL) were added. After 18 h at RT, LCMS showed no reaction with mostly the sulfoxide intermediate observed. The reaction mixture was heated at 40°C. After a further 2 h, LCMS showed almost no conversion. The reaction mixture was heated to 60 °C. After a further 18 h, the solvents were removed in vacuo (Genevac EZ-2 Elite) and the remaining residue was purified by preparative HPLC to give the title compound (1.7 mg, 6%) as a yellow solid. 1E NMR (500 MHz, CDC13) : δ 9.35 (s, 1H) , 8.10 (dd, 1H) , 7.79 (s, 1H), 7.56 (d, 2H) , 7.43-7.38 (m, 2H) , 7.36 (dd, 1H) , 6.97 (d, 2H), 3.82 (s, 3H) , 3.22 (t, 4H) , 2.61 (t, 4H) , 2.37 (s, 3H) . LCMS (Method A) : RT = 0.83 min, m/z = 433 [M+H]+.
Example 10: 8-Methyl-6- ( 5-methyl-l , 3 , 4-oxadiazol-2-yl ) -2- ( ( 4- ( piperaz in- 1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 (8H) -one
Figure imgf000066_0001
Step 1: Ethyl 2 , 4-dichloropyrimidine-5-carboxylate : Ethyl 4-chloro-2- (methylthio ) pyrimidine-5-carboxylate (50.0 g, 215 mmol) in sulfuryl chloride (50.0 mL, 615 mmol) was heated at 50 °C under an atmosphere of nitrogen. After 2 hours, the reaction mixture was cooled and carefully poured onto saturated sodium bicarbonate (aq) solution and was extracted using DCM (x 3) . The combined organic phase was dried (Phase Separator) and the solvents were removed in vacuo to give title compound (crude, ca. 50 g) that carried through to the next step without further purification. Ή NMR (500 MHz, CDCI3 ) : δ 9.02 (s, 1H) , 4.46 (dd, 2H) , 1.43 (t, 3H) .
Step 2: Ethyl 2-chloro-4- ( ( 3-ethoxy-3- oxopropyl) (methyl ) amino ) pyrimidine-5-carboxylate : Ethyl 3- (methylamino ) propanoate (17.2 g, 131 mmol) in DCM (100 mL) was added to a stirred solution of ethyl 2,4- dichloropyrimidine-5-carboxylate (crude, 25.2 g, 114 mmol) and DIPEA (43.8 mL, 251 mmol) in DCM (100 mL) in a 500 mL RB flask at 0 °C under nitrogen. After 15 min, the solvents were removed in vacuo to give the title compound (crude, 36.0 g) that was carried through to the next step without further purification. LCMS (Method A) : RT = 1.22 min, m/z = 316 [M+H]+.
Step 3: Ethyl 2-chloro-5-hydroxy-8-methyl-7 , 8- dihydropyrido [ 2 , 3-d] pyrimidine-6-carboxylate : Potassium tert-butoxide (1 M in THF) solution (114 mL, 114 mmol) was added to a stirred solution of ethyl 2-chloro-4- ( ( 3- ethoxy-3-oxopropyl ) (methyl ) amino ) pyrimidine-5-carboxylate (36.0 g, 114 mmol) in toluene (360 mL) at RT under nitrogen. After 1 h, further potassium tert-butoxide (1 M in THF) solution (228 mL, 2 eq) was required to attain complete reaction. After a further 2 h, acetic acid (6.53 mL, 114 mmol) and water (500 mL) were added. The resulting biphasic mixture was extracted (DCM x 3) and dried (Phase Separator) . The solvents were removed in vacuo to give the title compound (crude, 30.7 g, 114 mmol) as a yellow solid that was carried through to the next step without further purification. LCMS (Method A) : RT = 1.29 min, m/z = 268 [M-H]~.
Step 4: Ethyl 2-chloro-8-methyl-5-oxo-5 , 8- dihydropyrido [ 2 , 3-d] pyrimidine-6 -carboxylate : Bromine (5.87 mL, 114 mmol) in chloroform (200 mL) was added to a stirred solution of ethyl 2-chloro-5-hydroxy-8-methyl- 7 , 8-dihydropyrido [ 2 , 3-d] pyrimidine-6 -carboxylate (30.7 g, 114 mmol) and triethylamine (47.7 mL, 342 mmol) in chloroform (400 mL) at 0 °C under nitrogen. After 16 h, the reaction mixture was partitioned between saturated sodium thiosulfate (aq) solution and DCM, separated, extracted (DCM x 2), and dried (Phase Separator) . On standing, a precipitate formed and was collected to give the title compound (6.86 g, 23% over 4 steps) as a pale yellow solid. LCMS (Method A): RT = 0.79 min, m/z = 268 [M+H] +.
Step 5: Ethyl 2- ( ( 4- ( 4- ( tert-butoxycarbonyl ) piperaz in-1- yl ) phenyl ) amino ) -8-methyl-5-oxo-5 , 8-dihydropyrido [2,3- d] pyrimidine-6 -carboxylate : tert-Butyl 4- (4- aminophenyl ) piperazine-l-carboxylate (1.12 g, 4.03 mmol) was added to a stirred solution of ethyl 2-chloro-8- methyl-5-oxo-5 , 8-dihydropyrido [ 2 , 3-d] pyrimidine-6 - carboxylate (1.08 g, 4.03 mmol) in DMF (10.0 mL) in a 50 mL RB flask. The reaction mixture was heated at 100 °C. After 1 h, the reaction mixture was allowed to cool to RT and a precipitate formed that was collected by filtration and washed with methanol to give the title compound (1.75 g, 85%) as a bright yellow solid. (Method A) : RT = 1.25 min, m/z = 509 [M+H]+.
Step 6: Sodium 2- (( 4- ( 4- (tert-butoxycarbonyl) piperazin-1- yl ) phenyl ) amino ) -8-methyl-5-oxo-5 , 8-dihydropyrido [2,3- d] pyrimidine-6 -carboxylate : 2 M sodium hydroxide (aq) solution (4.92 mL, 9.83 mmol) was added to a stirred solution of ethyl 2- (( 4- ( 4- ( tert- butoxycarbonyl ) piperaz in-1-yl ) phenyl ) amino ) -8-methy1-5- oxo-5 , 8-dihydropyrido [ 2 , 3-d] pyrimidine-6 -carboxylate (1.0 g, 1.966 mmol) in 1,4-dioxane (5.0 mL) in a 50 mL RB flask. The reaction mixture was heated at 50 °C. After 16 h, a precipitate had formed and the resulting
suspension was allowed to cool to RT . Methanol (10 mL) was added and the solid material was collected by
filtration and washed using water, methanol and
diisopropyl ether, successively, to give the title compound (939 mg, 95%) as a pale yellow solid. LCMS (Method A): RT = 1.26 min, m/z = 481 [M+H]+ (N.B. under the acidic conditions of the method this data represents the corresponding free acid) .
Step 7: 8-Methyl-6- ( 5-methyl-l , 3 , 4-oxadiazol-2-yl ) -2- ( ( 4- (piperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one: Acetohydrazide (8.1 mg, 0.109 mmol) was added to sodium 2- (( 4- ( 4- (tert-butoxycarbonyl) piperazin-1- yl ) phenyl ) amino ) -8-methyl-5-oxo-5 , 8-dihydropyrido [2,3- d] pyrimidine-6 -carboxylate (50.0 mg, 0.100 mmol) in POCI3 (1.0 mL, 10.7 mmol) at RT in a 10 mL vial. The vessel was sealed and the temperature was increased to 100 °C with stirring. After 2 h, the reaction mixture was poured onto ice water, neutralised using 2 M NaOH (aq) solution, extracted (DCM x 3) and dried (Phase
Separator) . The solvents were removed in vacuo and the crude material was purified using preparative HPLC to give the title compound (0.4 mg, 0.8%) as a yellow solid. LCMS (Method A): RT = 0.55 min, m/z = 419 [M+H]+. Example 11: 6- ( 3-Chloropyridin-2-yl ) -8-methyl-2- ( ( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin-
Figure imgf000070_0001
Step 1: 6- ( 3-Chloropyridin-2-yl ) -8-methyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one :
Palladium ( I I ) bromide (12.1 mg, 0.045 mmol) was added to a pre-degassed solution of 8-methyl-2- (methylthio ) -5-oxo- 5, 8-dihydropyrido [ 2 , 3-d] pyrimidine-6-carboxylic acid (228 mg, 0.907 mmol), 3-chloro-2-iodopyridine (435 mg, 1.82 mmol), silver carbonate (250 mg, 0.907 mmol) and
( oxybis ( 2 , 1-phenylene )) bis ( diphenylphosphine ) (48.9 mg, 0.091 mmol) in toluene (2.7 mL ) /DMA (0.3 mL) in a 10 mL vial. The vessel was sealed and irradiated at 150 °C for 1 h (CEM Discover/Explorer24 ) . The reaction mixture was loaded directly onto a column and purified by flash chromatography (0-100%, EtOAc in cyclohexane) to give the title compound (29.1 mg, 10%) as an off-white solid.
LCMS (Method A): RT = 0.91 min, m/z = 319 [M+H]+. Step 2: 6- ( 3-Chloropyridin-2-yl ) -8-methyl-2- ( ( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one: mCPBA (<77%) (31.5 mg, 0.141 mmol) in DCM (0.5 mL) was added to a stirred solution of 6- (3- chloropyridin-2-yl ) -8-methyl-2- (methylthio ) pyrido [2,3- d]pyrimidin-5 (8H) -one (29.1 mg, 0.091 mmol) in DCM (2.0 mL) at RT under nitrogen. After 15 min, a solution of 4- ( 4-methylpiperazin-l-yl ) aniline (19.2 mg, 0.100 mmol) and DIPEA (0.048 mL, 0.274 mmol) in DMF (2.0 mL) was added and the reaction mixture was transferred to a 10 mL vial. The vessel was sealed and irradiated at 120 °C for 20 min (CEM Discover/Explorer24 ) . The solvents were removed in vacuo (Genevac EZ-2) and the remaining residue required purification by preparative HPLC under basic conditions and acidic conditions, successively, to give the title compound (0.5 mg, 1.1% yield) as a yellow solid. 1 H NMR (500 MHz, CDC13) : δ 9.34 (s, 1H) , 8.55 (dd, 1H) , 7.80 (dd, 1H) , 7.76 (s, 1H), 7.57 (d, 2H) , 7.47 (br s, 1H) , 7.27 (dd, 1H, overlapping solvent), 6.97 (d, 2H) , 3.81 (s, 3H) , 3.28 (t, 4H), 2.75 (t, 4H) , 2.45 (s, 3H) . LCMS (Method A): RT = 0.68 min, m/z = 462 [M+H]+.
Example 12: 6- ( 2 , 6 -Dichlorophenyl ) -8-methyl-2- ( ( 4- (piperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one :
Figure imgf000071_0001
Step 1: tert-Butyl 4- ( 4- ( ( 6-iodo-8-methyl-5-oxo-5, 8- dihydropyrido [ 2 , 3-d] pyrimidin-2- yl ) amino ) phenyl ) piperaz ine-l-carboxylate : To a stirred solution of 6 -iodo-8-methyl-2- (methylthio ) pyrido [ 2 , 3- d] pyrimidin-5 ( 8H) -one (100 mg, 0.30 mmol) in toluene (2 mL) at 0 °C under nitrogen was added mCPBA (<77% pure) (78 mg, 0.35 mmol) in DCM (2 mL). After 30 min, DIPEA (0.157 mL, 0.90 mmol) was added, followed by the addition of tert-butyl 4- ( 4-aminophenyl ) piperazine-l-carboxylate (92 mg, 0.33 mmol) in toluene (1.0 mL) . The reaction mixture was stirred at 60 °C until deemed complete by
LCMS analysis. The reaction mixture was cooled to RT and diluted with DCM (15 mL) and brine (10 mL) and extracted. The organic portion was dried (Phase Separator) and concentrated in vacuo. The residue obtained was purified by flash chromatography (0-100%, EtOAc in cyclohexane) to afford the title compound (44.0 mg, 26%) as a yellow solid. LCMS (Method A): RT = 1.33 min, m/z = 563 [M+H]+.
Step 2: tert-Butyl 4- ( 4- (( 6- ( 2 , 6-dichlorophenyl ) -8- methyl-5-oxo-5 , 8-dihydropyrido [ 2 , 3-d] pyrimidin-2- yl ) amino ) phenyl ) piperaz ine-l-carboxylate : Pd2(dba)3 (3.9 mg, 3.91 pmol) was added to a pre-degassed solution of 6- iodo-8-methyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) - one (44 mg, 0.078 mmol), ( 2 , 6-dichlorophenyl ) boronic acid (149 mg, 0.782 mmol), SPhos (2.6 mg, 6.26 pmol) and potassium phosphate, tribasic (50 mg, 0.235 mmol) in a, , -trifluorotoluene (2.0 mL) in a 10 mL vial. The vessel was sealed and the reaction mixture was stirred at 80 °C for 18 h. The reaction mixture was cooled to RT and diluted with DCM and brine and extracted. The organic extracts were dried (Phase Separator) and concentrated before being purified by flash chromatography (0-100%, EtOAc in cyclohexane) to afford the title compound (30 mg, 66%) as an off-white solid. [N.B. the product
contained -50% starting material but was used in the final step regardless]. LCMS (Method A): RT = 1.47 min, m/z = 581, 583 [M+H]+.
Step 3: 6- ( 2 , 6-Dichlorophenyl ) -8-methyl-2- ( ( 4- (piperazin 1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one : To a solution of tert-butyl 4- ( 4- (( 6- ( 2 , 6-dichlorophenyl ) -8- methyl-5-oxo-5 , 8-dihydropyrido [ 2 , 3-d] pyrimidin-2- yl ) amino ) phenyl ) piperaz ine-l-carboxylate (20 mg, 0.034 mmol) in DCM (2 mL) at 0 °C was added TFA (1 mL, 12.98 mmol) . The reaction mixture was then stirred at 0 °C for 2 min before being concentrated in vacuo. The residue was partitioned between DCM (25 mL) and saturated NaHCC>3 (aq) solution (15 mL) . The organic extracts were separated and dried (Phase Separator) and concentrated affording a brown residue. The residue was purified by preparative HPLC to afford the title compound (7.2 mg, 44%) as a yellow solid. Ή NMR (500 MHz, methanol-d4) : δ 9.19 (s, 1H) , 7.95 (s, 1H), 7.75 (br s, 2H) , 7.5 (d, 2H) , 7.35 (m, 1H) , 7.03 (d, 2H), 3.89 (s, 3H) , 3.18 (m, 4H) , 3.01 (m, 4H) . LCMS (Method A): RT = 0.82 min, m/z = 481, 483
[M+H] +.
Example 13: 8-Allyl-6- ( 2 , 6 -dichlorophenyl ) -2- ( ( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one
Figure imgf000073_0001
Step 1: Ethyl 3- ( allylamino ) propanoate : Prop-2-en-l-amine (15.1 mL, 202 mmol) was added dropwise to a solution of ethyl acrylate (20 mL, 184 mmol) in EtOH (100 mL) and the resulting solution was stirred for 18 h at RT . The reaction mixture was concentrated in vacuo to afford the title compound (28 g, 97%) which was used without further purification. Ή NMR (500 MHz, CDC13): δ 5.80-5.88 (m, 1H), 5.17 (dd, 2H) , 4.16 (q, 2H) , 3.28 (d, 2H) , 2.91 (t, 2H), 2.54 (t, 2H), 1.70 (bs, 1H) , 1.30 (s, 3H) .
Step 2: Ethyl 4- ( allyl ( 3-ethoxy-3-oxopropyl ) amino ) -2- (methylthio ) pyrimidine-5-carboxylate : Following the procedure for ethyl 4- ( ( 3-ethoxy-3- oxopropyl ) (methyl ) amino ) -2- (methylthio ) pyrimidine-5- carboxylate, ethyl 3- ( allylamino ) propanoate (28 g, 178 mmol) was reacted to afford the title compound as a yellow oil which was used without further purification (61 g, 97%). LCMS (Method A): RT = 1.41 min, m/z = 354 [M+H] +.
Step 3: Ethyl 8-allyl-5-hydroxy-2- (methylthio ) -7 , 8- dihydropyrido [ 2 , 3-d] pyrimidine-6 -carboxylate : Following the procedure for ethyl 5-hydroxy-8-methyl-2-
(methylthio ) -7 , 8-dihydropyrido [ 2 , 3-d] pyrimidine-6 - carboxylate, ethyl 4- ( allyl ( 3-ethoxy-3-oxopropyl ) amino ) - 2- (methylthio ) pyrimidine-5-carboxylate (30.0 g, 85 mmol) was reacted to afford the title compound (25 g, 96%) as a yellow solid which was used without further purification. LCMS (Method A): RT = 1.28 min, m/z = 308 [M+H]+.
Step 4: Ethyl 8-allyl-2- (methylthio ) -5-oxo-5 , 8- dihydropyrido [ 2 , 3-d] pyrimidine-6 -carboxylate : Following the procedure for ethyl 8-methyl-2- (methylthio ) -5-oxo- 5 , 8-dihydropyrido [ 2 , 3-d] pyrimidine-6 -carboxylate , ethyl 8-allyl-5-hydroxy-2- (methylthio ) -7 , 8-dihydropyrido [2,3- d] pyrimidine-6 -carboxylate (20.0 g, 65 mmol) was reacted to afford the title compound (19 g, 96%) as a yellow solid which was used without further purification. LCMS (Method A): RT = 1.04 min, m/z = 306 [M+H]+.
Step 5: 8-Allyl-2- (methylthio ) -5-oxo-5 , 8- dihydropyrido [ 2 , 3-d] pyrimidine-6 -carboxylie acid :
Following the procedure for 8-methyl-2- (methylthio ) -5- oxo-5 , 8-dihydropyrido [ 2 , 3-d] pyrimidine-6 -carboxylie acid, ethyl 8-allyl-2- (methylthio) -5-oxo-5, 8-dihydropyrido [2, 3- d] pyrimidine-6 -carboxylate (19.0 g, 65 mmol) was reacted to afford the title compound (10.4 g, 60%) as a white solid which was used without further purification. 1H NMR (500 MHz, DMSO-d6): δ 14.23 (br s, 1H) , 9.57 (s, 1H) , 9.27 (s, 1H), 6.05-6.13 (m, 1H) , 5.25-5.31 (m, 2H) , 5.13 (d, 2H) , 2.64 (s, 3H) .
Step 6: 8-Allyl-6- ( 2 , 6 -dichlorophenyl ) -2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one : To a solution of 8-allyl-2- (methylthio ) -5-OXO-5 , 8- dihydropyrido [ 2 , 3-d] pyrimidine-6-carboxylic acid (50 mg, 0.18 mmol) in toluene (2 mL) and DMA (0.22 mL) was added 1 , 3-dichloro-2-iodobenzene (98 mg, 0.36 mmol) and silver carbonate (49.7 mg, 0.18 mmol) . The suspension was degassed for 5 min and (oxybis(2,l- phenylene) ) bis (diphenylphosphine) (9.7 mg, 0.018 mmol) and palladium (II) chloride (1.6 mg, 9.0 pmol) added. The reaction mixture was then heated in a microwave
(Biotage Initiator) at 150 °C for 1 h. The reaction mixture was concentrated in vacuo and purified by flash chromatography (0-100%, EtOAc in cyclohexane) to afford the title compound (12.4 mg, 18%) as a yellow solid.
LCMS (Method A): RT = 1.55 min, m/z = 378 [M+H]+.
Step 7: 8-Allyl-6- ( 2 , 6 -dichlorophenyl ) -2- ( ( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one: Following the procedure for 8-methyl-2- ( ( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) -6 -phenylpyrido [2,3- d] pyrimidin-5 ( 8H) -one, 8-allyl-6- (2, 6 -dichlorophenyl ) -2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one (50.0 mg, 0.13 mmol) was reacted to give crude material which was purified by flash chromatography using a KP-NH column (0- 100% EtOAc in cyclohexane) to afford the title compound (4.5 mg, 6%) as a yellow solid. Ή NMR (500 MHz, DMSO- d6) : δ 10.21 (br s, 1H) , 9.07 (s, 1H) , 8.06 (s, 1H) , 7.63 (d, 2H), 7.56 (d, 2H) , 7.44 (dd, 1H) , 6.95 (d, 2H) , 6.07- 6.13 (m, 1H), 5.18 (dd, 2H) , 4.86 (d, 2H) , 3.10-3.11 (m, 4H) , 2.45-2.47 (m, 4H) , 2.23 (s, 3H) . LCMS (Method A): R. = 0.91 min, m/z = 522 [M+H]+. Example 14: 6- ( 2 , 6 -Dichlorophenyl ) -8- ( 4-methoxybenzyl ) -2- ( ( 4- ( 4-methylpiperaz in-l-yl ) phenyl ) amino ) pyrido [2,3- d] pyrimidin-5 ( 8H -one
Figure imgf000076_0001
Step 1: Ethyl 3- (( 4-methoxybenzyl ) amino ) propanoate :
Following the procedure for ethyl 3-
( ally1amino ) propanoate , ( 4-methoxyphenyl ) methanamine (13.8 g, 101 mmol) was reacted to afford the title
compound (21.8 g, 99%) as a yellow oil which was used without further purification. Ή NMR (500 MHz, CDC13) : δ 7.27 (d, 2H), 6.87 (d, 2H) , 4.16 (q, 2H) , 3.83 (s, 3H) , 3.74 (s, 2H), 3.28 (d, 2H) , 2.91 (t, 2H) , 2.54 (t, 2H) , 1.78 (bs, 1H) , 1.26 (s, 3H) .
Step 2: Ethyl 4- ( ( 3-ethoxy-3-oxopropyl ) (4- methoxybenzyl ) amino ) -2- (methylthio ) pyrimidine-5- carboxylate :
Following the procedure for ethyl 4- ( ( 3-ethoxy-3- oxopropyl ) (methyl ) amino ) -2- (methylthio ) pyrimidine-5- carboxylate, ethyl 3- (( 4-methoxybenzyl ) amino ) propanoate (21.8 g, 92 mmol) was reacted to afford the title
compound (37 g, 93%) as a yellow oil which was used without further purification. LCMS (Method A): RT = 1.50 min, m/z = 434 [M+H]+. Step 3: Ethyl 5-hydroxy-8- ( 4-methoxybenzyl ) -2- (methylthio ) -7 , 8-dihydropyrido [ 2 , 3-d] pyrimidine-6 - carboxylate: Following the procedure for ethyl 5-hydroxy- 8-methyl-2- (methylthio) -7, 8-dihydropyrido [2,3- d] pyrimidine-6 -carboxylate , ethyl 4- ( ( 3-ethoxy-3- oxopropyl ) ( 4-methoxybenzyl ) amino ) -2-
(methylthio ) pyrimidine-5-carboxylate (37.1 g, 85.6 mmol) was reacted to afford the title compound (36 g, 93%) as a yellow solid which was used without further purification. LCMS (Method A): RT = 1.33 min, m/z = 388 [M+H]+.
Step 4: Ethyl 8- ( 4-methoxybenzyl ) -2- (methylthio ) -5-oxo- 5, 8-dihydropyrido [ 2 , 3-d] pyrimidine-6 -carboxylate :
Following the procedure for ethyl 8-methyl-2- (methylthio ) -5-oxo-5, 8-dihydropyrido [ 2 , 3-d] pyrimidine-6 - carboxylate, ethyl 5-hydroxy-8- ( 4-methoxybenzyl ) -2- (methylthio ) -7 , 8-dihydropyrido [ 2 , 3-d] pyrimidine-6 - carboxylate (20.0 g, 51.6 mmol) was reacted to afford the title compound (19 g, 95%) as a yellow solid which was used without further purification. LCMS (Method A) : RT = 1.23 min, m/z = 386 [M+H]+.
Step 5: 8- ( 4-Methoxybenzyl ) -2- (methylthio ) -5-oxo-5 , 8- dihydropyrido [ 2 , 3-d] pyrimidine-6 -carboxylie acid :
Following the procedure for 8-methyl-2- (methylthio ) -5- oxo-5 , 8-dihydropyrido [ 2 , 3-d] pyrimidine-6 -carboxylie acid, ethyl 8- ( 4-methoxybenzyl ) -2- (methylthio ) -5-oxo-5, 8- dihydropyrido [ 2 , 3-d] pyrimidine-6-carboxylate (19.0 g, 49.3 mmol) was reacted to afford the title compound (10.4 g, 59%) as a white solid which was used without further purification. Ή NMR (500 MHz, DMSO-d6): δ 14.21 (br s, 1H) , 9.37 (s, 1H), 9.24 (s, 1H) , 7.36 (d, 2H) , 6.93 (d, 2H) , 5.67 (s, 1H), 3.72 (s, 3H) , 2.62 (s, 3H) . Step 6: 6- ( 2 , 6-Dichlorophenyl ) -8- ( 4-methoxybenzyl ) -2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one :
Following the procedure for 8-allyl-6- ( 2 , 6- dichlorophenyl ) -2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one, 8- ( 4-methoxybenzyl ) -2- (methylthio ) -5-oxo-5 , 8- dihydropyrido [ 2 , 3-d] pyrimidine-6-carboxylic acid (560 mg, 1.56 mmol) was reacted to afford the title compound (0.31 g, 42%) as a yellow solid which was used without further purification. LCMS (Method A): RT = 1.46 min, m/z = 458 [M+H]+.
Step 7: 6- ( 2 , 6-Dichlorophenyl ) -8- ( 4-methoxybenzyl ) -2- (( 4- ( 4-methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [2,3- d] pyrimidin-5 ( 8H ) -one :
Following the procedure for 8-methyl-2- ( ( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) -6 -phenylpyrido [2,3- d] pyrimidin-5 ( 8H) -one, 6- ( 2 , 6 -dichlorophenyl ) -8- ( 4- methoxybenzyl ) -2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one (0.3 g, 0.65 mmol) was reacted to give crude material which was purified by flash chromatography using a KP-NH column (0-100% EtOAc in cyclohexane) to afford the title compound (0.2 g, 51%) as a yellow solid. 1H NMR (500 MHz, DMSO-d6): δ 10.11 (br s, 1H) , 9.05 (s, 1H) , 8.26 (s, 1H), 7.57 (d, 2H) , 7.52 (d, 2H) , 7.44 (dd, 1H) , 7.23 (m, 2H) , 6.93 (d, 2H) , 6.87 (d, 2H) , 5.36 (s, 2H) , 3.70 (s, 3H), 3.10-3.12 (m, 4H) , 2.45-2.47 (m, 4H) , 2.23 (s, 3H) . LCMS (Method A): RT = 0.97 min, m/z = 601
[M+H] +. Example 15: 6- ( 2 , 6-Dichlorophenyl ) -2- (( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one
Figure imgf000079_0001
A solution of 6- ( 2 , 6 -dichlorophenyl ) -8- ( 4-methoxybenzyl ) - 2- ( ( 4- ( 4-methylpiperaz in- 1-yl ) phenyl ) amino ) pyrido [2,3- d] pyrimidin-5 ( 8H ) -one (15.6 mg, 0.026 mmol) in TFA (2 mL, 26.0 mmol) was heated at 150 °C in a microwave for 30 min. The solution was cooled to RT, concentrated in vacuo, suspended in ethyl acetate and washed with
saturated NaHCC>3 (aq) solution (100 mL), dried over magnesium sulphate, filtered, and concentrated in vacuo. The resultant residue was subjected to flash
chromatography using a KP-NH column (0-100% EtOAc in cyclohexane) to afford the title compound (4.5 mg, 36%) . 1E NMR (500 MHz, DMSO-d6) : δ 8.98 (s, 1H) , 7.79 (d, 2H) , 7.73 (s, 1H), 7.45 (d, 2H) , 7.26 (dd, 1H) , 6.88 (d, 2H) , 3.04-3.08 (m, 4H) , 2.43-2.45 (m, 4H) , 2.22 (s, 3H) . LCMS (Method A) : RT = 0.76 min, m/z = 481 [M+H]+.
Example 16: 6- ( 2 , 6 -Dichlorophenyl ) -8-ethyl-2- ( ( 4- ( 4- methylpiperaz in- 1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 (8H) -one
Figure imgf000079_0002
To a solution of 6- ( 2 , 6-dichlorophenyl ) -2- ( ( 4- ( 4- methylpiperaz in- 1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin 5(8H)-one (50.0 mg, 0.104 mmol) in DMF (1 mL) was added sodium hydride (4.2 mg, 0.104 mmol), and the resulting solution was stirred for 1 h at RT . Iodoethane (10 pL, 0.125 mmol) was added and the solution stirred for 2 h. The reaction mixture was suspended in ethyl acetate and washed with water (3 x 100 mL) , dried over magnesium sulphate, filtered, and concentrated in vacuo. The resultant residue was subjected to preparative HPLC to afford the title compound (7.9 mg, 15%) . Ή NMR (500 MHz, CDC13) : δ 9.34 (s, 1H) , 7.65 (bs, 1H) , 7.58 (d, 2H) , 7.50 (s, 1H), 7.44 (d, 2H) , 7.26 (d, 1H) , 7.00 (d, 2H) , 4.28 (q, 2H), 3.27-3.29 (m, 4H) , 2.71-2.73 (m, 4H) , 2.45 (s, 3H) , 1.51 (t, 3H) . LCMS (Method A) : RT = 0.87 min, m/z = 509 [M+H]+.
Example 17: Ethyl 2- ( 6- ( 2 , 6 -dichlorophenyl ) -2- ( ( 4- ( 4- methylpiperaz in-l-yl ) phenyl ) amino ) -5-oxopyrido [2,3- d] pyrimidin-8 (5H -yl) acetate
Figure imgf000080_0001
Following the procedure for 6 -( 2 , 6 -dichlorophenyl )- 8- ethyl-2- ( ( 4- ( 4-methylpiperaz in-l- yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one , 6 - ( 2 , 6 - dichlorophenyl ) -2- ( ( 4- ( 4-methylpiperaz in-l- yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one (50.0 mg, 0.104 mmol) was reacted with ethyl 2-bromoacetate (0.012 mL, 0.104 mmol) to afford the crude compound. The resultant residue was subjected to preparative HPLC to afford the title compound (35.1 mg, 59%) . Ή NMR (500 MHz, CDCI3) : δ 9.29 (s, 1H) , 7.53 (bs, 1H) , 7.48 (d, 2H) , 7.41 (s, 1H), 7.40 (d, 2H) , 7.25 (dd, 1H) , 6.95 (d, 2H) , 4.88, (s, 2H), 4.26, (q, 2H) , 3.33, (m, 4H) , 2.80, (m, 4H), 2.50 (s, 3H) , 1.25 (t, 3H) . LCMS (Method A) : RT = 0.88 min, m/z = 568 [M+H]+. Example 18: 2- ( 6- ( 2 , 6 -Dichlorophenyl ) -2- ( ( 4- ( 4- methylpiperaz in-l-yl ) phenyl ) amino ) -5-oxopyrido [2,3- d] pyrimidin-8 ( 5H) -yl ) acetic acid
Figure imgf000081_0001
To a solution of ethyl 2- ( 6- ( 2 , 6-dichlorophenyl ) -2- ( ( 4- ( 4-methylpiperaz in-l-yl ) phenyl ) amino ) -5-oxopyrido [2,3- d] pyrimidin-8 ( 5H) -yl ) acetate (30 mg, 0.053 mmol)
dissolved in ethanol (1 mL) was added potassium hydroxide (14.8 mg, 0.264 mmol) . The solution was refluxed until the starting material had been consumed (TLC) . Ethanol was removed in vacuo and 2 M HC1 (aq) was added. The resulting solid was filtered off and washed with water to give the title compound (5 mg, 18%) . Ή NMR (500 MHz, methanol-d4) : δ 9.07 (s, 1H) , 8.06 (s, 1H) , 7.64 (d, 2H) , 7.57 (d, 2H), 7.45 (dd, 1H) , 6.95 (d, 2H) , 4.94 (s, 2H) , 3.36 (s, 4H), 2.94 (s, 4H) , 2.57 (s, 3H) . LCMS (Method B) : RT = 3.65 min, m/z = 540 [M+H]+.
Example 19: 6- ( 2 , 6 -Dichlorophenyl ) -8- ( 3-hydroxypropyl ) -2- ( ( 4- ( 4-methylpiperaz in-l-yl ) phenyl ) amino ) pyrido [2,3- d] pyrimidin-5 ( 8H -one
Figure imgf000081_0002
Following the procedure for 6 -( 2 , 6 -dichlorophenyl )- 8- ethy1-2- ( ( 4- ( 4-methylpiperazin-l- yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one , 6-(2,6- dichlorophenyl ) -2- ( ( 4- ( 4-methylpiperazin-l- yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one (50.0 mg, 0.104 mmol) was reacted with 3-bromopropan-l-ol (13.0 mg, 0.093 mmol) to afford the crude compound. The resultant residue was subjected to preparative HPLC to afford the title compound (12 mg, 20%) . Ή NMR (500 MHz, methanol-d4) : δ 9.22 (s, 1H) , 7.96 (s, 1H) , 7.69 (m, 2H) , 7.50 (d, 2H), 7.39 (dd, 1H) , 7.05 (d, 2H) , 4.46 (t, 2H) , 3.68 (t, 2H), 3.22 (m, 4H) , 2.66 (m, 4H) , 2.38 (s, 3H) , 2.10-2.14 (m, 2H) . LCMS (Method A): RT = 0.77 min, m/z = 540 [M+H]+.
Example 20: 6- ( 2 , 5-Dimethyl-lH-pyrrol-l-yl ) -8- ( 4- methoxybenzyl ) -2- ( ( 4- ( 4-methylpiperazin-l-
Figure imgf000082_0001
Step 1: tert-Butyl ( 8- ( 4-methoxybenzyl ) -2- (methylthio) -5- oxo-5 , 8-dihydropyrido [ 2 , 3-d] pyrimidin-6 -yl ) carbamate : To a yellow solution of 8- ( 4-methoxybenzyl ) -2- (methylthio ) - 5-oxo-5, 8-dihydropyrido [ 2 , 3-d] pyrimidine-6 -carboxy1 ic acid (0.5 g, 1.40 mmol) in tert-butanol (10 mL) was added DIPEA (0.244 mL, 1.40 mmol) followed by diphenyl
phosphoryl azide (0.30 mL, 1.40 mmol), and the resulting mixture was heated to 75 °C under a nitrogen atmosphere. After 16 h, the reaction mixture was allowed to cool to RT, added to saturated sodium bicarbonate (aq) solution and extracted into ethyl acetate. The solvents were removed under reduced pressure to afford the title compound (84 mg, 14% crude) as a yellow solid that was used in the next step without further purification. LCMS (Method A): RT = 1.53 min, m/z = 429 [M+H]+.
Step 2: tert-Butyl ( 8- ( 4-methoxybenzyl ) -2- (( 4- ( 4- methylpiperaz in-l-yl ) phenyl ) amino ) -5-oxo-5, 8- dihydropyrido [ 2 , 3-d] pyrimidin-6-yl ) carbamate : Following the procedure for 8-methyl-2- ( ( 4- ( 4-methylpiperazin-l- yl ) phenyl ) amino ) -6 -phenylpyrido [ 2 , 3-d] pyrimidin-5 ( 8H) - one, tert-butyl ( 8- ( 4-methoxybenzyl ) -2- (methylthio ) -5- oxo-5 , 8-dihydropyrido [ 2 , 3-d] pyrimidin-6 -yl ) carbamate (0.084 mg, 0.19 mmol) was reacted to give the title compound (74 mg, 67%) as a yellow solid which was used without further purification. LCMS (Method B) : RT = 2.62 min, m/z = 572 [M+H]+.
Step 3: 6-Amino-8- ( 4-methoxybenzyl ) -2- (( 4- ( 4- methylpiperaz in-l-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one: To a solution of tert-butyl (8-(4- methoxybenzyl ) -2- ( ( 4- ( 4-methylpiperaz in-1- yl ) phenyl ) amino ) -5-oxo-5, 8-dihydropyrido [ 2 , 3-d] pyrimidin- 6 -yl ) carbamate (74 mg, 0.131 mmol) in DCM (1 mL) was added TFA (1 mL) . The solution was stirred at RT for 5 min and the solvents were removed under reduced pressure. The residue was suspended in ethyl acetate and washed with NaHCC>3 (100 mL), dried over magnesium sulphate, filtered and concentrated in vacuo to give the title compound (50 mg, 81%) as a yellow solid which was used without further purification. LCMS (Method B) : RT = 1.76 min, m/z = 472 [M+H]+.
Step 4: 6- ( 2 , 5-Dimethyl-lH-pyrrol-l-yl ) -8- ( 4- methoxybenzyl ) -2- ( ( 4- ( 4-methylpiperaz in-l- yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one : To a solution of 6-amino-8- ( 4-methoxybenzyl ) -2- (( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one (0.05 g, 0.106 mmol) in DMF (1 mL) was added 4- methylbenzenesulfonic acid (0.027 g, 0.159 mmol) and hexane-2 , 5-dione (0.012 g, 0.106 mmol) . The resulting mixture was heated for 30 min at 120° C under microwave conditions, cooled to RT, suspended in ethyl acetate and washed with brine (100 mL) , dried over magnesium
sulphate, filtered, and concentrated in vacuo. The resultant residue was subjected to preparative HPLC to afford the title compound (12 mg, 21%) . Ή NMR (500 MHz, CDC13) : δ 9.26 (s, 1H) , 7.75 (bs, 1H) , 7.47 (s, 1H) , 7.41 (d, 2H), 7.11 (d, 2H), 6.86 (d, 2H) , 6.80 (d, 2H) , 5.80 (s, 2H), 5.25 (s, 2H), 3.70 (s, 3H) , 3.13-3.15 (m, 4H) , 2.51-2.53 (m, 4H) , 2.29 (s, 3H) , 1.89 (s, 6H) . LCMS (Method B) : RT = 2.53 min, m/z = 550 [M+H]+.
Example 21: 6- ( 2 , 6 -Dichlorophenyl ) -2- ( ( 6- (piperazin-1- yl ) pyridin-3-yl ) amino pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one
Figure imgf000084_0001
Step 1: 6- ( 2 , 6-Dichlorophenyl ) -8- ( 4-methoxybenzyl ) -2- ( ( 6- (piperaz in-1-yl ) pyridin-3-yl ) amino ) pyrido [2,3- d] pyrimidin-5 ( 8H) -one :
Following the procedure for 8-methyl-2- ( ( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) -6 -phenylpyrido [2,3- d] pyrimidin-5 ( 8H) -one, 6- ( 2 , 6-dichlorophenyl ) -8- ( 4- methoxybenzyl ) -2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one (55 mg, 0.116 mmol) was reacted with tert-butyl 4- ( 5-aminopyridin-2-yl ) piperazine-l-carboxylate (32.3 mg, 0.116 mmol) instead of 4- ( 4-methylpiperazin-l-yl ) aniline to give crude material which was purified by flash chromatography (0-100%, EtOAc in cyclohexane) to afford the title compound (50 mg, 63%) as a solid. LCMS (Method B) : RT = 6.06 min, m/z = 688 [M+H]+.
Step 2: 6- ( 2 , 6-Dichlorophenyl ) -2- ( ( 6- (piperazin-1- yl ) pyridin-3-yl ) amino ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one : Following the procedure for 6- ( 2 , 6 -dichlorophenyl ) -2- ( ( 4- ( 4-methylpiperaz in-l-yl ) phenyl ) amino ) pyrido [2,3- d] pyrimidin-5 ( 8H) -one, 6- ( 2 , 6 -dichlorophenyl ) -8- ( 4- methoxybenzyl ) -2- ( (6- (piperaz in-l-yl ) pyridin-3- yl ) amino ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one was reacted to give crude material which was purified by preparative HPLC to afford the title compound (4.0 mg, 13%). XH NMR (500 MHz, DMSO-d6) : δ 9.98 (br s, 1H) , 9.05 (s, 1H) , 8.66 (br s, 1H), 7.92 (br s, 1H) , 7.80 (s, 1H) , 7.55 (d, 2H) , 7.42 (t, 1H), 6.85 (d, 1H) , 3.45 (br s, 4H) , 2.90 (br s, 4H) . LCMS (Method A): RT = 0.71 min, m/z = 468 [M+H]+.
Example 22: 6- ( 2 , 6-Dichlorophenyl ) -2- (( 4- ( 2- (diethylamino ) ethoxy ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 (8H) -one
Figure imgf000085_0001
Step 1: 6- (2, 6-Dichlorophenyl) -2- ( (4- (2- ( diethylamino ) ethoxy ) phenyl ) amino ) -8- ( 4- methoxybenzyl ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H) -one : Following the procedure for 8-methyl-2- ( ( 4- ( 4-methylpiperazin-l- yl ) phenyl ) amino ) -6 -phenylpyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) - one, 6- ( 2 , 6 -dichlorophenyl ) -8- ( 4-methoxybenzyl ) -2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one (55 mg, 0.116 mmol) was reacted with 4- (2-
(diethylamino) ethoxy) aniline (24.2 mg, 0.116 mmol) instead of 4- ( 4-methylpiperazin-l-yl ) aniline to give crude material which was purified by flash chromatography using a KP-NH column (0-20% MeOH in CH2C12) to afford the title compound (31 mg, 43%) as a solid. LCMS (Method B) : RT = 4.61 min, m/z = 618 [M+H]+.
Step 2: 6- ( 2 , 6-Dichlorophenyl ) -2- (( 4- ( 2-
( diethylamino ) ethoxy ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one: Following the procedure for 6- (2, 6- dichlorophenyl ) -2- ( ( 4- ( 4-methylpiperazin-l- yl) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one , 6 - ( 2 , 6 - dichlorophenyl ) -2- ( ( 4- ( 2-
( diethylamino ) ethoxy ) phenyl ) amino ) -8- ( 4- methoxybenzyl ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H) -one was reacted to give crude material which was purified by HPLC to afford the title compound (4.3 mg, 18%). XH NMR (500 MHz, DMSO-d6) : δ 10.03 (br s, 1H) , 9.05 (s, 1H) , 7.80 (s, 1H), 7.75 (br s, 2H) , 7.54 (d, 2H) , 7.41 (t, 1H) , 6.92 (d, 2H), 4.05 (t, 2H), 2.85 (br t, 2H) , 2.63 (q, 4H), 1.01 (t, 6H) . LCMS (Method A): RT = 0.84 min, m/z = 498 [M+H] +.
Example 23: 6- ( 2 , 6-Dichlorophenyl ) -2- ( (pyridin-4- ylmethyl ) amino ) pyrido 2 , 3-d] pyrimidin-5 ( 8H ) -one
Figure imgf000086_0001
Step 1: 6- ( 2 , 6-Dichlorophenyl ) -8- ( 4-methoxybenzyl ) -2- ( (pyridin-4-ylmethyl ) amino ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) one: Following the procedure for 8-methyl-2- ( ( 4- ( 4- methylpiperaz in-l-yl ) phenyl ) amino ) -6 -phenylpyrido [2,3- d] pyrimidin-5 ( 8H) -one, 6- ( 2 , 6 -dichlorophenyl ) -8- ( 4- methoxybenzyl ) -2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one (55 mg, 0.116 mmol) was reacted with pyridin-4- ylmethanamine (12.5 mg, 0.116 mmol) instead of 4- (4- methylpiperazin-l-yl ) aniline to give crude material which was purified by flash chromatography using a KP-NH column (0-20% MeOH in CH2CI2) to afford the title compound as a solid (36 mg, 60%) . LCMS (Method B) : RT = 4.21 min, m/z = 518 [M+H]+.
Step 2: 6- ( 2 , 6-Dichlorophenyl ) -2- ( (pyridin-4- ylmethyl ) amino ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one :
Following the procedure for 6- ( 2 , 6 -dichlorophenyl ) -2- ( ( 4- ( 4-methylpiperaz in-l-yl ) phenyl ) amino ) pyrido [2,3- d] pyrimidin-5 ( 8H) -one, 6- ( 2 , 6 -dichlorophenyl ) -8- ( 4- methoxybenzyl ) -2- ( (pyridin- 4-ylmethyl ) amino ) pyrido [2,3- d] pyrimidin-5 ( 8H ) -one was reacted to give crude material which was purified by HPLC to afford the title compound (7.2 mg, 12%) . 1E NMR (500 MHz, DMSO-d6) : δ 11.73 (br s, 1H) , 8.90 (s, 1H), 8.49 (br t, 1H) , 8.42 (br s, 2H) , 7.60 (s, 1H), 7.44 (d, 2H), 7.31 (t, 1H) , 7.27 (m, 2H) , 4.58 (br s, 1H) , 4.51 (d, 1H) . LCMS (Method A) : RT = 0.65 min, m/z = 398 [M+H]+.
Example 24: 6- ( 2 , 6-Dichlorophenyl ) -2- (( 1-methylpiperidin- 4-yl ) amino ) pyrido [ 2 3-d] pyrimidin-5 ( 8H ) -one
Figure imgf000087_0001
Step 1: 6- ( 2 , 6-Dichlorophenyl ) -8- ( 4-methoxybenzyl ) -2- (( 1 methylpiperidin-4-yl ) amino ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H) - one: Following the procedure for 8-methyl-2- ( ( 4- ( 4- methylpiperaz in-l-yl ) phenyl ) amino ) -6 -phenylpyrido [2,3- d] pyrimidin-5 ( 8H) -one, 6- ( 2 , 6 -dichlorophenyl ) -8- ( 4- methoxybenzyl ) -2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one (55 mg, 0.116 mmol) was reacted with 1- methylpiperidin-4-amine (13.2 mg, 0.116 mmol) instead of 4- ( 4-methylpiperazin-l-yl ) aniline to give crude material which was purified by flash chromatography using a KP-NH column (0-20% MeOH in CH2CI2) to afford the title compound as a solid (33 mg, 54%) . LCMS (Method B) : RT = 4.26 min, m/z = 524 [M+H]+.
Step 2: 6- ( 2 , 6-dichlorophenyl ) -2- (( l-methylpiperidin-4- yl ) amino ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one : Following the procedure for 6- ( 2 , 6-dichlorophenyl ) -2- (( 4- ( 4- methylpiperaz in- 1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one, 6- ( 2 , 6-dichlorophenyl ) -8- ( 4-methoxybenzy1 ) -2- ( ( l-methylpiperidin-4-yl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one was reacted to give crude material which was purified by HPLC to afford the title compound (5.1 mg, 17%) . 1E NMR (500 MHz, DMSO-d6) : δ 11.86 (br s, 1H) , 8.94 (m, 1H), 7.92 (d, 1H) , 7.68 (m, 1H) , 7.51 (d, 2H) , 7.40 (t, 1H), 3.81 (m, 1H), 2.79 (d, 2H) , 2.18 (s, 3H) , 1.96 (t, 2H) , 1.85 (m, 2H) , 1.59 (q, 2H) . LCMS (Method A) : RT = 0.65 min, m/z = 404 [M+H]+.
Example 25: 7-Amino-6- ( 2-chlorophenyl ) -8-methyl-2- ( ( 4- ( 4- methylpiperaz in- 1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 (8H) -one
Figure imgf000088_0001
Step 1: Ethyl 4- (methylamino ) -2- (methylthio ) pyrimidine-5 carboxylate: To a solution of ethyl 4-chloro-2- (methylthio ) pyrimidine-5-carboxylate (10 g, 43 mmol) in anhydrous THF (100 mL) was added triethylamine (6.0 mL, 43 mmol) and monomethylamine (2M in THF, 22 mL, 43 mmol) The reaction mixture was heated to 50 °C overnight, then diluted with water (100 mL) and extracted into ethyl acetate (3 x 100 mL) . The combined organic phases were dried over Na2S04, filtered and concentrated to dryness under reduced pressure to give the title compound (10 g, quantitative yield) as an off-white solid. 1H NMR (500 MHz, CDC13) : δ 8.60 (s, 1H) , 8.16 (br s, 1H) , 4.31 (q, 2H), 3.07 (d, 3H), 2.54 (s, 3H) , 1.36 (t, 3H) . LCMS (Method A): RT = 1.14 min, m/z = 228 [M+H]+.
Step 2: 4- (Methylamino ) -2- (methylthio ) pyrimidine-5- carboxylic acid: Lithium hydroxide (211 mg, 8.8 mmol) was added to ethyl 4- (methylamino ) -2- (methylthio ) pyrimidine- 5-carboxylate (1.0 g, 4.4 mmol) in a mixture of methanol (8 mL) and water (4 mL) to give a white suspension. This was heated to 60 °C for 3 h and then concentrated to dryness under reduced pressure. The residue was re- dissolved in water (25 mL) and acidified with acetic acid to pH 5. The precipitated solid was isolated by
filtration, washed with water (2 x 10 mL) and freeze- dried to give the title compound (610 mg, 70%) as an off- white solid. XH NMR (500 MHz, DMSO-d6): δ 13.10 (br s, 1H), 8.49 (s, 1H), 8.36 (br s, 1H) , 2.98 (d, 3H) , 2.49 (s, 3H) . LCMS (Method A): RT = 0.52 min, m/z = 200
[M+H]+.
Step 3: 4- (Methylamino ) -2- (methylthio ) pyrimidine-5- carbonyl fluoride: To a solution of 4- (methylamino ) -2- (methylthio ) pyrimidine-5-carboxylic acid (610 mg, 3.1 mmol) and triethylamine (470 pL, 3.4 mmol) in anhydrous dichloromethane (6 mL) was added dropwise a solution of cyanuric fluoride (525 pL, 6.1 mmol) in anhydrous
dichloromethane (3 mL) and the mixture stirred at RT for 2 h. The reaction mixture was diluted with dichloromethane (20 mL) , washed with ice-cold water (2 x 20 mL), dried over Na2S04, filtered, and concentrated to dryness under reduced pressure to give the title compound (555 mg, 90%) as an off-white solid. XH NMR (500 MHz, CDC13) : δ 8.56 (s, 1H) , 7.60 (br s, 1H) , 3.14 (d, 3H) , 2.57 (s, 3H) . LCMS (Method A): RT = 1.05 min, m/z = 202.15 [M+H]+.
Step 4: 7-Amino-6- ( 2-chlorophenyl ) -8-methyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one : A solution of 2- ( 2-chlorophenyl ) acetonitrile (34 mg, 0.22 mmol) in anhydrous DMF (1 mL) was cooled to 0 °C followed by the addition of sodium hydride (60% in mineral oil, 18 mg, 0.45 mmol) . The resulting mixture was stirred at RT for 10 min before re-cooling to 0 °C. To the suspension was added dropwise a solution of 4- (methylamino ) -2- (methylthio ) pyrimidine-5-carbonyl fluoride (45 mg, 0.22 mmol) in anhydrous DMF (0.5 mL), the mixture was allowed to warm to RT and stirred for 60 min. Additional sodium hydride, 60% in mineral oil (9 mg, 0.22 mmol) was added and the mixture stirred at RT for a further 60 min.
Further sodium hydride, 60% in mineral oil (9 mg, 0.22 mmol) was added and the mixture stirred at RT for 60 min. The reaction mixture was diluted with water (8 mL) and extracted into ethyl acetate (3 x 5 mL) . The combined organic phases were washed with 1:1 water/brine (3 x 5 mL), dried over Na2S04, filtered, and concentrated to dryness under reduced pressure to give the title compound (23 mg, 31%) as a beige solid. XH NMR (500 MHz, DMSO-d6) : δ 8.94 (s, 1H), 7.54 (t, 1H) , 7.38 (t, 2H) , 7.25 (t, 1H) , 6.53 (br s, 2H) , 3.78 (s, 3H) , 2.62 (s, 3H) . LCMS
(Method A): RT = 0.83 min, m/z = 333/335 [M+H]+. Step 5: 7-Amino-6- ( 2-chlorophenyl ) -8-methyl-2- ( ( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one: To a suspension of 7-amino-6- ( 2-chlorophenyl ) - 8-methy1-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one (23 mg, 0.069 mmol) in anhydrous toluene (1 mL) was added mCPBA (<77% pure) (14 mg, 0.064 mmol) and the mixture stirred at RT for 30 min. The reaction mixture was concentrated to dryness under reduced pressure and re- dissolved in anhydrous dichloromethane (1 mL) . Further mCPBA (<77% pure) (14 mg, 0.064 mmol) was added and the mixture stirred at RT for a further 30 min. To this was added a solution of 4- ( 4-methylpiperazin-l-yl ) aniline (15 mg, 0.076 mmol) and DIPEA (36 pL, 0.21 mmol) in anhydrous THF (0.5 mL) and stirred at RT overnight. The mixture was concentrated to dryness under reduced pressure and re- dissolved in anhydrous DMF (1 mL) . The reaction mixture was heated to 100 °C for 60 min then allowed to cool to RT, diluted with water (5 mL) and extracted into ethyl acetate (3 x 5 mL) . The combined organic phases were washed with 1:1 water/brine (3 x 5 mL) , dried over Na2SC>4, filtered, and concentrated to dryness under reduced pressure. The residue was purified by preparative HPLC to give the title compound (1.0 mg, 3%) as a pale yellow solid. XH NMR (500 MHz, methanol-d4) : δ 9.04 (s, 1H) , 8.56 (s, 1H), 7.61 (d, 2H) , 7.55-7.58 (m, 1H) , 7.38-7.42 (m, 2H), 7.31-7.34 (m, 1H) , 7.02 (d, 2H) , 3.84 (s, 3H) , 3.20 (t, 4H), 2.64 (t, 4H) , 2.36 (s, 3H) . LCMS (Method A): RT = 0.63 min, m/z = 476 [M+H]+. Example 26: 6- ( 2-Chlorophenyl ) -7 , 8-dimethyl-2- ( ( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one
Figure imgf000092_0001
Step 1: 2- ( 2-Chlorophenyl ) -1- ( 4- (methylamino ) -2- (methylthio ) pyrimidin-5-yl ) buta-1 , 3-diene-l , 3-diol : A solution of 1- ( 2-chlorophenyl ) propan-2-one (170 mg, 0.99 mmol) in anhydrous DMF (2 mL) was cooled to 0 °C followed by the addition of sodium hydride (60% in mineral oil, 80 mg, 1.99 mmol) . The resulting mixture was stirred at RT for 10 min before re-cooling to 0 °C. To the suspension was added dropwise a solution of 4- (methylamino ) -2- (methylthio ) pyrimidine-5-carbonyl fluoride (200 mg, 0.99 mmol) in anhydrous DMF (1 mL), the mixture was allowed to warm to RT and stirred for 60 minutes. The reaction mixture was diluted with water (20 mL) and washed with ethyl acetate (3 x 10 mL) . The aqueous phase was
acidified by the dropwise addition of citric acid
solution and extracted into ethyl acetate (3 x 10 mL) . The combined organic phases were washed with 1 : 1
water/brine (3 x 10 mL) , dried over Na2SC>4, filtered, and concentrated to dryness under reduced pressure to give the title compound (210 mg, 60%) as a white solid. 1ti NMR (500 MHz, CDC13) : δ 8.51 (s, 1H) , 7.27-7.33 (m, 2H) , 7.18- 7.22 (m, 2H), 3.42 (d, 1H) , 3.34 (d, 1H) , 3.22 (s, 3H) , 2.56 (s, 3H) . LCMS (Method A): RT = 1.35 min, m/z = 350, 352 [M+H]+.
Step 2: 6- ( 2-Chlorophenyl ) -7 , 8-dimethyl-2-
(methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one : A solution of 2- ( 2-chlorophenyl ) -1- ( 4- (methylamino ) -2- (methylthio ) pyrimidin-5-yl ) buta-1 , 3-diene-l , 3-diol (160 mg, 0.46 mmol) in polyphosphoric acid (1.6 g, 16 mmol) was heated to 140 °C under a nitrogen atmosphere for 3 h. The reaction mixture was allowed to cool to RT, basified by the addition of 2 M NaOH (aq) solution and extracted into ethyl acetate (3 x 20 mL) . The combined organic phases were dried over Na2S04, filtered and concentrated to dryness under reduced pressure. The residue was purified by flash chromatography using a KP-NH cartridge (10-100%, EtOAc in cyclohexane) to give the title
compound (80 mg, 53%) as a tan solid. 1ti NMR (500 MHz, CDC13) : δ 9.35 (s, 1H) , 7.49-7.52 (m, 1H) , 7.31-7.36 (m, 2H) , 7.21-7.24 (m, 1H) , 3.95 (s, 3H) , 2.66 (s, 3H) , 2.29 (s, 3H) . LCMS (Method A): RT = 1.16 min, m/z = 332, 334 [M+H] +.
Step 3: 6- ( 2-Chlorophenyl ) -7 , 8-dimethyl-2- ( ( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one: A suspension of 6 - ( 2-chlorophenyl ) -7 , 8- dimethyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one (80 mg, 0.24 mmol) in anhydrous DCM (1.5 mL) was cooled to 0 °C. To the mixture was added mCPBA (<77% pure) (50 mg, 0.22 mmol) and the resulting solution stirred at 0 °C for 30 min. To the reaction mixture was added DIPEA (126 pL, 0.72 mmol), 4- ( 4-methylpiperazin-l-yl ) aniline (51 mg, 0.27 mmol) and anhydrous toluene (1.5 mL) . The reaction mixture was heated to 50 °C overnight, additional DIPEA (126 pL, 0.72 mmol) and 4- ( 4-methylpiperazin-l-yl ) aniline (51 mg, 0.27 mmol) were added and the mixture was heated to 50°C for a further 6 h. The reaction mixture was allowed to cool to RT and was concentrated to dryness under reduced pressure. The residue was purified by preparative HPLC to give the title compound (14 mg, 12%) as a yellow solid. XH NMR (500 MHz, DMSO-d6): δ 10.03 (br s, 1H), 9.01 (s, 1H), 7.65 (br s, 2H) , 7.55 (dd, 1H), 7.37-7.42 (m, 2H) , 7.23 (dd, 1H) , 6.96 (d, 2H) , 3.82 (s, 3H) , 3.11 (t, 4H), 2.46 (t, 4H) , 2.22 (s, 3H) , 2.20 (s, 3H) . LCMS (Method A): RT = 0.81 min, m/z = 475, 477
[M+H] +.
Example 27: 6- ( 2-Chlorophenyl ) -7-methyl-2- ( ( 4- (piperazin- 1-yl ) phenyl ) amin -5H-pyrano [ 2 , 3-d] pyrimidin-5-one
Figure imgf000094_0001
Step 1: 2-Chloro-6- ( 2-chlorophenyl ) -7-methyl-5H- pyrano [ 2 , 3-d] pyrimidin-5-one : A solution of l-(2- chlorophenyl ) propan-2-one (80 mg, 0.47 mmol) in anhydrous THF (1 mL) was cooled to 0 °C followed by the dropwise addition of tert-butylmagnesium chloride (2 M in diethyl ether, 0.24 mL, 0.47 mmol) and stirred at RT for 10 min. The suspension was re-cooled to 0 °C before the dropwise addition of a solution of 2 , 4-dichloropyrimidine-5- carbonyl chloride (100 mg, 0.47 mmol) in anhydrous THF (0.5 mL) . The reaction mixture was stirred at RT for 60 min then quenched by the addition of water (5 mL), neutralised with a few drops of citric acid solution and extracted into ethyl acetate (3 x 5 mL) . The combined organic phases were dried over Na2S04, filtered and concentrated to dryness under reduced pressure. The residue was purified by flash chromatography (10-60%, EtOAc in cyclohexane) to give the title compound (45 mg, 31%) as an orange solid. XH NMR (500 MHz, CDC13): δ 9.39 (s, 1H), 7.52 (dd, 1H) , 7.34-7.42 (m, 2H) , 7.22 (dd, 1H) , 2.30 (s, 3H) . LCMS (Method A): RT = 1.27 min, m/z = 307, 309 [M+H]+.
Step 2: tert-Butyl 4- ( 4- (( 6- ( 2-chlorophenyl ) -7-methyl-5- oxo-5H-pyrano [ 2 , 3-d] pyrimidin-2- yl ) amino ) phenyl ) piperaz ine-l-carboxylate : A solution of 2-chloro-6- ( 2-chlorophenyl ) -7-methyl-5H-pyrano [2,3- d] pyrimidin-5-one (45 mg, 0.15 mmol), tert-butyl 4- (4- aminophenyl ) piperazine-l-carboxylate (41 mg, 0.15 mmol) and DIPEA (51 pL, 0.29 mmol) in anhydrous DMF (1 mL ) was heated to 100 °C under a nitrogen atmosphere for 60 min. The reaction mixture was allowed to cool to RT, diluted with water (5 mL) and extracted into ethyl acetate (3 x 5 mL) . The combined organic phases were washed with 1:1 water/brine (3 x 5 mL) , dried over Na2SC>4, filtered, and concentrated to dryness under reduced pressure to give the title compound (42 mg, 52%) as a yellow solid. 1H NMR (500 MHz, CDC13) : δ 9.18 (s, 1H) , 8.07 (br s, 1H) , 7.53 (br d, 2H), 7.47-7.51 (m, 1H) , 7.31-7.37 (m, 2H) , 7.21- 7.25 (m, 1H), 6.96 (d, 2H) , 3.59 (t, 4H) , 3.12 (t, 4H) , 2.19 (s, 3H), 1.49 (s, 9H) . LCMS (Method A): RT = 1.56 min, m/z = 548, 550 [M+H]+.
Step 3: 6- ( 2-Chlorophenyl ) -7-methyl-2- ( ( 4- (piperazin-1- yl ) phenyl ) amino ) -5H-pyrano [ 2 , 3-d] pyrimidin-5-one , 2HC1 : To a solution of tert-butyl 4- ( 4- (( 6- ( 2-chlorophenyl ) -7- methyl-5-oxo-5H-pyrano [ 2 , 3-d] pyrimidin-2- yl ) amino ) phenyl ) piperaz ine-l-carboxylate (42 mg, 0.077 mmol) in dichloromethane (1 mL) was added dropwise 4 M HC1 in 1,4-dioxane (1 mL) and the mixture stirred at RT for 90 min. The reaction mixture was then diluted with diethyl ether (5 mL) and slurried at RT for 15 min, before removal of the supernatant solvent by pipette.
This was repeated a total of three times before removal of residual solvent under reduced pressure and freeze- drying to give the title compound (40 mg, quantitative yield) as a yellow solid. 1H NMR (500 MHz, methanol-d4) : δ 9.09 (s, 1H), 7.70 (br d, 2H) , 7.55 (dd, 1H) , 7.39-7.46 (m, 2H), 7.31 (dd, 1H) , 7.08 (d, 2H) , 3.37-3.45 (m, 8H) , 2.21 (s, 3H) . LCMS (Method A): RT = 0.87 min, m/z
450 [M+H]+.
Example 28: 6- ( 2-Chlorophenyl ) -2- (( 4- (piperazin-1- yl ) phenyl ) amino ) -5H-pyrano [ 2 , 3-d] pyrimidin-5-one .
Figure imgf000096_0001
Step 1: 2- ( 2-Chlorophenyl ) acetaldehyde : A solution of methyl 2- ( 2-chlorophenyl ) acetate (3.0 g, 16 mmol) in anhydrous dichloromethane (30 mL) was cooled to -78 °C followed by the dropwise addition of DIBAL-H (1 M in toluene, 16.3 mL, 16 mmol) over a period of 30 min. The reaction mixture was stirred at -78 °C under a nitrogen atmosphere for a further 30 min, then quenched by the addition of methanol (15 mL) and poured into saturated sodium potassium tartrate (aq) solution (100 mL) . The resulting mixture was stirred vigorously for 1 h and extracted into dichloromethane (3 x 50 mL) . The combined organic phases were dried over Na2SC>4, filtered and concentrated to dryness under reduced pressure to give the title compound (2.65 g, quantitative yield) as a pale yellow oil. XH NMR (500 MHz, CDC13): δ 9.76 (t, 1H) , 7.41-7.46 (m, 1H) , 7.22-7.29 (m, 3H) , 3.85 (d, 2H) .
Step 2: 2-Chloro-6- ( 2-chlorophenyl ) -5H-pyrano [ 2 , 3- d] pyrimidin-5-one : A solution of 2- (2- chlorophenyl ) acetaldehyde (730 mg, 4.7 mmol) in anhydrous THF (6 mL) was cooled to 0 °C followed by the dropwise addition of tert-butylmagnesium chloride (2 M in diethyl ether, 4.7 mL, 9.5 mmol) and stirring at RT for 10 min. The resulting suspension was re-cooled to 0 °C before the dropwise addition of a solution of 2,4- dichloropyrimidine-5-carbonyl chloride (1.00 g, 4.7 mmol) in anhydrous THF (3 mL) . The reaction mixture was stirred at RT for 60 min, then quenched by the addition of water (20 mL), neutralised with a few drops of citric acid (aq) solution and extracted into ethyl acetate (3 x 20 mL) . The combined organic phases were dried over Na2S04, filtered and concentrated to dryness under reduced pressure. The residue was purified by flash
chromatography (10-50%, EtOAc in cyclohexane) to give the title compound (190 mg, 14%) as a brown solid. 1ti NMR
(500 MHz, CDC13) : δ 9.45 (s, 1H) , 8.01 (s, 1H) , 7.51 (dd, 1H) , 7.40 (td, 1H), 7.35 (td, 1H) , 7.30 (dd, 1H) . LCMS (Method A): RT = 1.19 min, m/z = 293, 295 [M+H]+. Step 3: tert-Butyl 4- ( 4- (( 6- ( 2-chlorophenyl ) -5-oxo-5H- pyrano [ 2 , 3-d] pyrimidin-2-yl ) amino ) phenyl ) piperaz ine-1- carboxylate: A solution of 2-chloro-6- ( 2-chlorophenyl) - 5H-pyrano [ 2 , 3-d] pyrimidin-5-one (42 mg, 0.14 mmol), tert- butyl 4- ( 4-aminophenyl ) piperazine-l-carboxylate (40 mg, 0.14 mmol) and DIPEA (50 pL, 0.29 mmol) in anhydrous DMF (1 mL) was heated to 100 °C under a nitrogen atmosphere for 60 min. The reaction mixture was allowed to cool to RT, diluted with water (5 mL) and extracted into ethyl acetate (3 x 5 mL) . The combined organic phases were washed with 1:1 water/brine (3 x 5 mL) , dried over Na2S04, filtered, and concentrated to dryness under reduced pressure. The residue was purified by flash
chromatography (10-100%, MeOH in DCM) to give the title compound (50 mg, 65%) as a brown solid. 1H NMR (500 MHz, CDCI3) : δ 9.24 (s, 1H) , 7.94 (br s, 1H) , 7.80 (s, 1H) ,
7.54 (br d, 2H) , 7.49 (dd, 1H) , 7.30-7.38 (m, 3H) , 6.96 (d, 2H), 3.59 (t, 4H), 3.13 (t, 4H) , 1.49 (s, 9H) . LCMS (Method A): RT = 1.52 min, m/z = 534, 536 [M+H]+. Step 4: 6- ( 2-Chlorophenyl ) -2- ( ( 4- (piperazin-1- yl ) phenyl ) amino ) -5H-pyrano [ 2 , 3-d] pyrimidin-5-one , 2HC1 : To a solution of tert-butyl 4- ( 4- ( ( 6- ( 2-chlorophenyl) -5- oxo-5H-pyrano [ 2 , 3-d] pyrimidin-2- yl ) amino ) phenyl ) piperaz ine-l-carboxylate (50 mg, 0.094 mmol) in dichloromethane (1 mL) was added dropwise 4 M HC1 in 1,4-dioxane (1 mL) and the mixture stirred at RT for 2 h. The reaction mixture was then diluted with diethyl ether (5 mL) and slurried at RT for 15 min, before removal of the supernatant solvent by pipette.
This was repeated a total of three times before removal of residual solvent under reduced pressure and freeze- drying to give the title compound (32 mg, 67%) as a brown solid. XH NMR (500 MHz, methanol-d4) : δ 9.09 (s, 1H) , 7.70 (br d, 2H) , 7.55 (dd, 1H) , 7.39-7.46 (m, 2H) , 7.31 (dd, 1H), 7.08 (d, 2H) , 3.37-3.45 (m, 8H) , 2.21 (s, 3H) . LCMS (Method A): RT = 0.87 min, m/z = 434, 436 [M+H]+.
Example 29: 6- ( 2-Chlorophenyl ) -8-cyclopropyl-2- ( ( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one
Figure imgf000098_0001
Step 1: 1- ( 4-Chloro-2- (methylthio ) pyrimidin-5-yl ) -2 -( 2- chlorophenyl ) ethanone : To a solution of 2- (2- chlorophenyl ) acetic acid (2 g, 11.7 mmol) in THF (30 mL) was added tert-butylmagnesium chloride (11.7 mL, 23.5 mmol) . The brown suspension formed was stirred at RT for 30 min and ethyl 4-chloro-2- (methylthio ) pyrimidine-5- carboxylate (2.46 g, 10.6 mmol) in 10 mL THF added. The resulting yellow suspension was stirred for 16 h.
Aqueous HC1 (25%, 20 mL) and water (100 mL) were added and extracted with EtOAc. The combined organic layers were dried over Na2S04 and concentrated to give the title compound (2.2 g, 60%) as an oil that was used in the next step without further purification. LCMS (Method A) : RT = 1.48 min, m/z = 314 [M+H]+.
Step 2: 2- ( 2-Chlorophenyl ) -1- ( 4- ( cyclopropylamino ) -2- (methylthio ) pyrimidin-5-yl ) ethanone :
Following the procedure for ethyl 4- ( ( 3-ethoxy-3- oxopropyl) (methyl ) amino ) -2- (methylthio ) pyrimidine-5- carboxylate , 1- ( 4-chloro-2- (methylthio ) pyrimidin-5-yl ) -2- ( 2-chlorophenyl ) ethanone (2.2 g, 7.02 mmol) and
cyclopropanamine (0.401 g, 7.02 mmol) were reacted to afford the title compound (602 mg, 26%) as a clear oil that was used in the next step without further
purification. LCMS (Method A): RT = 1.57 min, m/z = 334 [M+H] +.
Step 3: 6- ( 2-Chlorophenyl ) -8-cyclopropyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one :
To a solution of 2- ( 2-chlorophenyl ) -1- ( 4-
( cyclopropylamino ) -2- (methylthio ) pyrimidin-5-yl ) ethanone (50 mg, 0.150 mmol) in toluene (1 mL) was added 1,1- dimethoxy-N, N-dimethylmethanamine (0.022 mL, 0.165 mmol) . The resulting mixture was heated for 16 h at 110 °C, cooled to RT, suspended in ethyl acetate, washed with saturated aqueous NaHCC>3 (100 ml), dried over magnesium sulfate, filtered, and concentrated in vacuo. The resultant residue was subjected to flash chromatography (0-100 %, EtOAc in cyclohexane) to afford the title compound (25 mg, 49%). LCMS (Method A): RT = 1.29 min, m/z = 344 [M+H]+. Step 4: 6- ( 2-Chlorophenyl ) -8-cyclopropyl-2- ( ( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one :
Following the procedure for 8-methyl-2- ( ( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) -6 -phenylpyrido [2,3- d] pyrimidin-5 ( 8H) -one, 6- ( 2-chlorophenyl ) -8-cyclopropyl- 2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one ( 25mg, 0.073 mmol) was reacted to give crude material. The resultant residue was subjected to preparative HPLC to afford the title compound (8.1 mg, 22%). Ή NMR (500
MHz, methanol-d4) : δ 9.20 (s, 1H) , 7.95 (s, 1H) , 7.84 (br s, 2H), 7.52 (m, 1H) , 7.39 (m, 3H) , 7.04 (d, 2H) , 3.60 (m, 1H), 3.21 (dd, 4H) , 2.66 (dd, 4H) , 2.37 (s, 3H) , 1.29 (dd, 2H) , 1.11 (dd, 2H) . LCMS (Method A): RT = 0.88 min, m/z = 487 [M+H]+.
Example 30: 6- ( 4-Hydroxyphenyl ) -8-methyl-2- (( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin-
5 ( 8H) -one
Figure imgf000100_0001
Step 1: 6- ( 4-Hydroxyphenyl ) -8-methyl-2-
(methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one :
Palladium ( I I ) acetate (2.0 mg, 9.00 pmol) was added to a
10 mL vial containing 6-iodo-8-methyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one (60.0 mg, 0.180 mmol), ( 4-hydroxyphenyl ) boronic acid (37.3 mg, 0.270 mmol), tetrabutylammonium bromide (58.1 mg, 0.180 mmol) and potassium carbonate (62.2 mg, 0.450 mmol) in EtOH (1.0 mL) /Water (2.0 mL). The vessel was sealed and irradiated at 120 °C for 5 min (CEM Discover/Explorer24 ) . Analysis by LCMS showed some residual SM. The vessel was resealed and heated at 120 °C for 20 min (CEM Discover/Explorer24 ) . The solvents were removed in vacuo and the remaining residue was purified by flash
chromatography (0-100%, EtOAc in cyclohexane) to give the title compound (10.5 mg, 20%) as a pale yellow solid. LCMS (Method A): RT = 0.92 min, m/z = 300 [M+H]+.
Step 2: 4- ( 8-Methyl-2- (methylthio ) -5-oxo-5 , 8- dihydropyrido [ 2 , 3-d] pyrimidin-6-yl ) phenyl acetate: Acetic anhydride (0.014 mL, 0.147 mmol) was added to a stirred solution of 6- ( 4-hydroxyphenyl ) -8-methyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one (8.8 mg, 0.029 mmol) and triethylamine (0.5 mL, 3.59 mmol) in DCM (1.0 mL) at RT under nitrogen. After 1 h, the solvents were removed in vacuo and the remaining residue was purified by flash chromatography (0-100%, EtOAc in cyclohexane) to give the title compound (9.8 mg, 98%) as a white solid. LCMS (Method A): RT = 1.09 min, m/z = 342 [M+H] +.
Step 3: 6- ( 4-Hydroxyphenyl ) -8-methyl-2- ( ( 4- ( 4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one: mCPBA (<77% pure) (7.4 mg, 0.033 mmol) in DCM (0.5 mL) was added to a stirred solution of 4- ( 8-methyl- 2- (methylthio ) -5-oxo-5, 8-dihydropyrido [ 2 , 3-d] pyrimidin-6 - yl)phenyl acetate (9.8 mg, 0.029 mmol) in toluene (1.0 mL) at RT under nitrogen. After 15 min, DIPEA (0.015 mL, 0.086 mmol) and 4- ( 4-methylpiperazin-l-yl ) aniline (6.0 mg, 0.032 mmol) were added, sequentially, and the
temperature was increased to 60 °C. After 18 h, the reaction mixture was cooled to RT and loaded directly onto a KP-NH column and purified by flash chromatography (0-100%, EtOAc in cyclohexane; then 10% MeOH in EtOAc) to give the title compound (5.4 mg, 43%) as a yellow solid. H NMR (500 MHz, CDC13): δ 10.0 (br s, 1H) , 9.40 (br s, 1H) , 9.08 (s, 1H), 8.06 (s, 1H) , 7.69 (s, 2H) , 7.49 (d, 2H) , 6.94 (d, 2H), 6.78 (d, 2H) , 3.77 (s, 3H) , 3.10 (t, 4H) , 2.46 (t, 4H) , 2.22 (s, 3H) . LCMS (Method A): RT = 0.66 min, m/z = 443 [M+H]+.
Example 31: 6- ( 3-Hydroxyphenyl ) -8-methyl-2- ( ( 4- (piperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one
Figure imgf000102_0001
Step 1: 6- ( 3-Hydroxyphenyl ) -8-methyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one :
Bis ( triphenylphosphine ) palladium ( I I ) chloride (7.9 mg, 0.011 mmol) was added to a 10 mL vial containing 6-iodo- 8-methy1-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one (75 mg, 0.225 mmol), ( 3-acetoxyphenyl ) boronic acid (60.8 mg, 0.338 mmol) [commercially available] in 2 M sodium carbonate (aq) (0.5 mL)/THF (1.0 mL). The vessel was sealed and the reaction mixture was heated to 40 °C
(conventional) with stirring. After 3 h, LCMS showed the O-acetylated product and significant SM and therefore, the temperature was increased to 75 °C. After a further 17 h, the reaction mixture was partitioned between EtOAc and saturated sodium bicarbonate (aq) solution,
separated, dried (Phase Separator), the solvents were removed in vacuo and the remaining residue was purified by flash chromatography (0-100%, EtOAc in cyclohexane) to give the title compound (14.8 mg, 22%) as a pale yellow solid. LCMS (Method A): RT = 0.93 min, m/z = 300 [M+H]+. Step 2: 3- ( 8-Methyl-2- (methylthio ) -5-oxo-5 , 8- dihydropyrido [ 2 , 3-d] pyrimidin-6-yl ) phenyl acetate: Acetic anhydride (0.023 mL, 0.247 mmol) was added to a stirred solution of 6- ( 3-hydroxyphenyl ) -8-methyl-2- (methylthio ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one (14.8 mg,
0.049 mmol) in triethylamine (0.5 mL, 3.59 mmol)/DCM (2.0 mL) at RT under nitrogen. After 30 min, the solvents were removed in vacuo and the remaining residue was purified by flash chromatography (0-100%, EtOAc in cyclohexane) to give the title compound (11.6 mg, 69%) as a white solid. LCMS (Method A): RT = 1.12 min, m/z = 342 [M+H] +.
Step 3: tert-Butyl 4- ( 4- (( 6- ( 3-hydroxyphenyl ) -8-methyl-5- oxo-5 , 8-dihydropyrido [ 2 , 3-d] pyrimidin-2- yl ) amino ) phenyl ) piperaz ine-l-carboxylate : mCPBA (<77% pure) (8.8 mg, 0.039 mmol) in DCM (0.5 ml) was added to a stirred solution of 3- ( 8-methyl-2- (methylthio ) -5-oxo-5 , 8- dihydropyrido [ 2 , 3-d] pyrimidin-6-yl ) phenyl acetate (11.6 mg, 0.034 mmol) in toluene (1.0 mL) at RT under nitrogen. After 15 min, DIPEA (0.018 mL, 0.102 mmol) and tert-butyl 4- ( 4-aminophenyl ) piperazine-l-carboxylate (10.4 mg, 0.037 mmol) were added, successively, and the temperature was increased to 60 °C. After 16 h, the reaction mixture was cooled to RT and was loaded directly onto a KP-NH column and purified by flash chromatography (0-100%, EtOAc in cyclohexane; then 10% MeOH in EtOAc) to give the title compound (9.7 mg, 54%) as a yellow solid. LCMS (Method A): RT = 1.25 min, m/z = 529 [M+H]+.
Step 4: 6- ( 3-Hydroxyphenyl ) -8-methyl-2- ( ( 4- (piperaz in-1- yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one : TFA (1.0 mL, 13.0 mmol) was added to a stirred solution of tert-butyl 4- ( 4- ( ( 6 - ( 3-hydroxyphenyl ) -8-methyl-5-oxo-5 , 8- dihydropyrido [ 2 , 3-d] pyrimidin-2- yl ) amino ) phenyl ) piperaz ine-l-carboxylate (9.7 mg, 0.018 mmol) in DCM (1.0 mL) at RT under nitrogen. After 1 h, the solvents were removed in vacuo and the remaining residue was partitioned between DCM and saturated sodium bicarbonate (aq) solution, separated, extracted (2 x DCM), dried (Phase Separator), and the solvents were removed in vacuo to give the title compound (2.9 mg, 33%) as a pale yellow solid. LCMS (Method A): RT = 0.66 min, m/z = 429 [M+H]+.
Example 32: 3- ( 2-chlorophenyl ) -1- ( 4-methoxyphenyl ) -7- (( 4- ( 4-methylpiperaz in-1-yl ) phenyl ) amino ) pyrimido [4,5-
Figure imgf000104_0001
Step 1: 1- ( 4-chloro-2- (methylthio ) pyrimidin-5-yl ) -2 -( 2- chlorophenyl ) -2- ( 2- ( 4-methoxyphenyl ) hydrazono ) ethanone : To a solution of 4-methoxyaniline (0.170 g, 1.382 mmol) in cone. HC1 (0.433 ml, 5.20 mmol) with 1.5 ml of water at 0 °C was added sodium nitrite (0.095 g, 1.382 mmol) in 0.5 ml of water. To this was added a suspension of l-(4- chloro-2- (methylthio ) pyrimidin-5-yl ) -2- ( 2- chlorophenyl ) ethanone (0.433 g, 1.382 mmol) in ethanol (4 ml) and water (1ml) with sodium acetate (0.624 g, 7.60 mmol) . The resulting suspension was warmed to room temperature and left for 16 hours. The reaction mixture was suspended in ethyl acetate (100 ml) and washed with water (100 ml), dried over magnesium sulphate, filtered and concentrated in vacuo to give a yellow residue which was purified by flash chromatography (0-100%, EtOAc in cyclohexane) to afford the title compound (402 mg, 65%) . LCMS (Method A): RT = 1.52 min, m/z = 447 [M+H]+.
Step 2: 3- ( 2-chlorophenyl ) -1- ( 4-methoxyphenyl ) -7- (methylthio) pyrimido [ 4, 5-c] pyridazin-4 ( IH) -one : To a solution of 1- ( 4-chloro-2- (methylthio ) pyrimidin-5-y1 ) -2- ( 2-chlorophenyl ) -2- ( 2- ( 4-methoxyphenyl ) hydrazono ) ethanone (0.4 g, 0.894 mmol) in acetonitrile (8 ml) was added potassium carbonate (0.148 g, 1.073 mmol) . The resulting mixture was heated for 1 hour at 90 °C, cooled to room temperature, suspended in ethyl acetate and washed with brine (100 ml), dried over magnesium sulphate, filtered and concentrated in vacuo. The resultant residue was purified by flash chromatography (0-100%, EtOAc in cyclohexane) to afford the title compound (205 mg, 54%) . LCMS (Method A) : RT = 1.48 min, m/z = 411 [M+H]+.
Step 3: 3- ( 2-chlorophenyl ) -1- ( 4-methoxyphenyl ) -7- (( 4- ( 4- methylpiperaz in-l-yl ) phenyl ) amino ) pyrimido [4,5- c] pyridazin-4 ( IH) -one : Following the procedure for 8- methyl-2- ( ( 4- ( 4-methylpiperaz in-l-yl ) phenyl ) amino ) -6 - phenylpyrido [ 2 , 3-d] pyrimidin-5 ( 8H) -one, 3- (2- chlorophenyl ) -1- ( 4-methoxyphenyl ) -7-
(methylthio) pyrimido [ 4, 5-c] pyridazin-4 ( IH) -one (200 mg, 0.49 mmol) was reacted to give crude material which was purified by flash chromatography (KP-NH column, 0-100% EtOAc in cyclohexane) to afford the title compound as a yellow solid (26 mg, 9%) . Ή NMR (500 MHz, DMSO-d6) : δ 10.40 (bs, IH), 9.18 (s, IH) , 7.41-7.61 (m, 8H) , 7.15 (d, 2H) , 6.73 (d, 2H), 3.86 (s, 3H) , 3.06-3.08 (m, 4H) , 2.45- 2.47 (m, 4H) , 2.23 (s, 3H) . LCMS (Method A): RT = 1.03 min, m/z = 554 [M+H]+.
Example 33: 3- ( 2-chlorophenyl ) -l-methyl-7- ( ( 4- (piperazin- 1-y1 ) phenyl ) amino ) pyrimido [ 4 , 5-c ] pyridaz in-4 ( 1H ) -one
Figure imgf000106_0001
Step 1: 1- ( 4-chloro-2- (methylthio) pyrimidin-5-yl ) -2- (2- chlorophenyl ) ethanone : To a solution of 2- (2- chlorophenyl ) acetic acid (10 g, 58.6 mmol) in THF (120 mL) under nitrogen, was added tert-butylmagnesium
chloride 2M in ether (73.3 mL, 147 mmol) at 0°C. The resulting solution was warmed to room temperature and a white precipitate crashed out of solution. Ethyl 4- chloro-2- (methylthio ) pyrimidine-5-carboxylate (12.3 g, 52.8 mmol) in THF (50 mL) was added giving a clear yellow solution and then after 10 minutes a yellow precipitate crashed out. The reaction mixture was left stirring at room temperature for 16 hours. 2M HC1 (200 mL) was added and the product extracted with EtOAc. The combined organic layers were dried over Na2S04, and then
concentrated in vacuo. The residue was purified by flash chromatography on silica (0-100% DCM in cyclohexane) to afford the title compound as a yellow solid (13.2 g, 72%). Ή NMR (500 MHz, DMSO-d6): δ 9.15 (s, 1H) , 7.49-7.45 (m, 1H), 7.44-7.38 (m, 1H) , 7.39-7.33 (m, 2H) , 4.55 (s, 2H) , 2.61 (s, 3H) . LCMS (Method A): RT = 1.45 min, m/z = 313-315 [M+H]+. Step 2: di-tert-butyl 3- ( 2-chlorophenyl ) -7- (methylthio ) - 4-oxo-3 , 4-dihydropyrimido [ 4 , 5-c ] pyridaz ine-1 , 2- dicarboxylate : A solution of 1- ( 4-chloro-2- (methylthio ) pyrimidin-5-yl ) -2- ( 2-chlorophenyl ) ethanone (1.0 g, 3.19mmol) in anhydrous THF (20 mL) was cooled to -78°C. Lithium bis ( trimethylsilyl ) amide 1 M in THF
(3.83mL, 3.83mmol) was added drop-wise at -78°C. The reaction mixture was stirred for 30 minutes at -78°C. Then ( E ) -di- tert-butyl diazene-1 , 2-dicarboxylate (0.888 g, 3.83mmol) in THF (40 mL) was added drop-wise, stirred at -78°C for 2h and the solution allowed to warm to room temperature over 16 hours. The reaction was quenched with NH4C1 saturated solution and the product extracted twice with EtOAc. The combined organic layers were dried over Na2SC>4 and then concentrated in vacuo. The residue was purified by flash chromatography on silica (0-100% DCM in cyclohexane) to afford the title compound as a yellow solid (700 mg, 43%). Ή NMR (500 MHz, DMSO-d6): δ 9.05 (s, 1H), 7.65(d, 1H) , 7.47(t, 1H) , 7.28(t, 1H) , 7.07 (d, 1H), 6.32 (bs, 1H) , 2.66 (s, 3H) , 1.51 (s, 9H) , 1.23 (s, 9H) . LCMS (Method A): RT = 1.73 min, m/z = 506-508 [M+H] +.
Step 3: 3- ( 2-chlorophenyl ) -7- (methylthio ) pyrimido [ 4 , 5- c] pyridazin-4-ol : To a solution of di-tert-butyl 3-(2- chlorophenyl ) -7- (methylthio) -4-oxo-3, 4- dihydropyrimido [ 4 , 5-c ] pyridaz ine-1 , 2-dicarboxylate
(4.0 g, 7.89 mmol) in dichloromethane ( 20 mL) was added trifluoroacetic acid (20 mL, 260 mmol) at 0°C. The resulting solution was stirred for 2h at room
temperature. The reaction mixture was concentrated in vacuo. The residue was dissolved in acetonitrile (20 mL) and 4, 5-dichloro-3 , 6-dioxocyclohexa-l , 4-diene-l, 2- dicarbonitrile (1.79 g, 7.89 mmol) was added in a microwave sealed tube. The reaction mixture was heated by microwave irradiation at 100°C for 10 minutes. The reaction mixture was concentrated in vacuo. The residue was triturated with water and the precipitate was filtrated and washed with water and ethyl acetate to afford the title compound as a brown powder (1 g, 42%) . Ή NMR (500 MHz, DMSO-d6): δ 14.10 (s, 1H) , 9.22 (s, 1H) , 7.57 (d, 1H), 7.53-7.43 (m, 3H) , 2.65 (s, 3H) . LCMS (Method A): RT = 1.10 min, m/z = 305-307 [M+H]+.
Step 4: 3- ( 2-chlorophenyl ) -l-methyl-7-
(methylthio) pyrimido [ 4, 5-c] pyridazin-4 ( 1H) -one : To a solution of 3- ( 2-chlorophenyl ) -7-
(methylthio) pyrimido [ 4, 5-c] pyridazin-4-ol (100 mg,
0.33 mmol) in DMF (2 mL) was added methyl iodide (0.041 mL, 0.656 mmol) and cesium carbonate (180 mg, 0.55 mmol) and the resulting suspension was heated at 70°C for 1 hour. 1M HC1 was added and the product extracted with EtOAc. The combined organic layers were dried over Na2S04 and then concentrated in vacuo. The residue was purified by flash chromatography on silica (0-20% EtOAc in
cyclohexane) to afford the title compound as a yellow- beige solid (60 mg, 40%). Ή NMR (500 MHz, CDC13): δ 9.32 (s, 1H), 7.50 (d, 1H), 7.43-7.35 (m, 3H) , 4.11 (s, 3H) , 2.67 (s, 3H) . LCMS (Method A): RT = 1.31 min, m/z = 319- 321 [M+H]+.
Step 5: tert-butyl 4- ( 4- (( 3- ( 2-chlorophenyl ) -l-methyl-4- oxo-1 , 4-dihydropyrimido [ 4 , 5-c ] pyridaz in-7- yl ) amino ) phenyl ) piperaz ine-l-carboxylate : 3- (2- chlorophenyl ) -1-methyl-7- (methylthio ) pyrimido [4,5- c] pyridazin-4 ( 1H) -one (100 mg, 0.314 mmol) was dissolved in anhydrous dichloromethane . 3-Chloroperbenzoic acid 70% (93mg, 0.378 mmol) was added portionwise at 0°C. After stirring at room temperature for 30 minutes, all the starting material was converted into the
corresponding sulfoxide. The reaction mixture was
concentrated in vacuo, and the residue was dissolved in anhydrous toluene (5 mL) . Diisopropylethylamine (0.165 mL, 0.944mmol) and tert-butyl 4- (4- aminophenyl ) piperazine-l-carboxylate (lOOmg, 0.378 mmol) were added, and the reaction mixture was stirred at 60°C for 18 hours. After concentration in vacuo, the residue was purified by flash chromatography on silica (0-20%
EtOAc in cyclohexane) to afford the title compound as a yellow solid (35 mg, 20%). LCMS (Method A): RT = 1.57 min, m/z = 549 [M+H]+. Step 6: 3- ( 2-chlorophenyl ) -l-methyl-7- ( ( 4- (piperazin-1- yl ) phenyl ) amino ) pyrimido [ 4 , 5-c ] pyridaz in-4 ( IH ) -one :
To a solution of tert-butyl 4- ( 4- (( 3- ( 2-chlorophenyl ) -1- methyl-4-oxo-l , 4-dihydropyrimido [ 4 , 5-c ] pyridaz in-7- yl ) amino ) phenyl ) piperaz ine-l-carboxylate (35 mg, 0.064 mmol) in dichloromethane (3 mL) and methanol (1 mL) was added 4M HC1 in dioxane (3 mL) . The resulting solution was stirred for 16 hours at room temperature until the starting material had been consumed (LC analysis) . The suspension was filtered and washed with diethyl ether to yield the title compound as a yellow solid (13 mg, 42%) . Ή NMR (500 MHz, MeOD) : δ 9.21 (s, IH) , 7.80 (s, 2H) , 7.53 (d, IH), 7.42-7.49 (m, 3H) , 7.15 (d, 2H) , 4.08 (s, 3H) , 3.45-3.47 (m, 4H) , 3.42-3.44 (m, 4H) . LCMS (Method A): RT = 0.92 min, m/z = 449 [M+H]+.
Example 34: 3- ( 2-chlorophenyl ) -l-methyl-7- (( 3-
( (methylamino ) methyl ) -4- morpholinophenyl ) amino ) pyrimido [ 4 , 5-c ] pyridaz in-4 ( IH) -one
Figure imgf000110_0001
Step 1: tert-butyl 5- ( ( 3- ( 2-chlorophenyl ) -l-methyl-4-oxo- 1 , 4-dihydropyrimido [ 4 , 5-c ] pyridaz in- 7-yl ) amino ) -2- morpholinobenzyl (methyl ) carbamate : Following the
procedure of tert-butyl 4- ( 4- (( 3- ( 2-chlorophenyl ) -1- methyl-4-oxo-l , 4-dihydropyrimido [ 4 , 5-c ] pyridaz in- 7- yl ) amino ) phenyl ) piperaz ine-l-carboxylate , 3- ( 2- chlorophenyl ) -1 -methyl- 7- (methylthio ) pyrimido [4,5- c] pyridazin-4 ( 1H) -one (50 mg, 0.15 mmol) was reacted with tert-butyl 5-amino-2-morpholinobenzyl (methyl ) carbamate (55mg, 0.17 mmol) to give a crude mixture which was purified by flash chromatography on silica (0-20% EtOAc in cyclohexane) to afford the title compound as a yellow solid (60 mg, 65%) . Ή NMR (400 MHz, DMSO-d6) : δ 10.62 (bs, 1H), 9.15 (s, 1H), 7.57 (d, 2H) , 7.53-7.44 (m, 4H) , 7.22 (d, 1H), 4.51 (s, 3H) , 3.98 (s, 3H) , 3.74 (t, 4H) , 2.86-2.78 (m, 6H) , 1.42 (s, 9H) . LCMS (Method A) : RT = 1.57 min, m/z= 592-594 [M+H]+. Step 2: 3- ( 2-chlorophenyl ) -l-methyl-7- (( 3- ( (methylamino ) methyl ) -4- morpholinophenyl ) amino ) pyrimido [ 4 , 5-c ] pyridaz in-4 ( 1H) - one: tert-butyl 5- (( 3- ( 2-chlorophenyl ) -l-methyl-4-oxo- 1 , 4-dihydropyrimido [ 4 , 5-c ] pyridaz in- 7-yl ) amino ) -2- morpholinobenzyl (methyl ) carbamate (25 mg, 0.042 mmol) was dissolved in dichloromethane (1 mL) . Then TFA (1 mL, 12.98 mmol) was added at 0°C. The resulting solution was stirred for 16 hours at room temperature until the starting material had been consumed (LC analysis) . The reaction mixture was poured on an SCX-2 cartridge pre- treated with DCM/MeOH (9/1) . The cartridge was flushed 3 times with DCM/MeOH (9/1) . Then the target compound is eluted by flushing 3 times with DCM/NH3 (2M) in MeOH
(9/1), and concentrated in vacuo to afford the title compound as a yellow powder (10 mg, 48%) . 1 H NMR (500MHz, DMSO-d6) : δ 10.54 (bs, 1H) , 9.15(s, 1H) , 8.07(bs, 1H) , 7.68 (bs, 1H), 7.57 (d, 1H) , 7.53-7.45(m, 3H) , 7.16(d, 1H) , 4.01(s, 3H), 3.78 (s, 2H) , 3.75 (t, 4H) , 2.88- 2.77 (m, 4H) , 2.38 (s, 3H) . LCMS (Method A) : RT = 0.86 min, m/z = 492-494 [M+H]+.
Example 35: 3- ( 2-chlorophenyl ) -7- (( 3- (hydroxymethyl ) -4- (piperaz in-l-yl ) phenyl ) amino ) -pyrimido [ 4 , 5-c ] pyridaz in-
4 (1H) -one
Figure imgf000111_0001
Step 1: tert-butyl 4- ( 4- (( 3- ( 2-chlorophenyl ) -4-oxo-l , 4- dihydropyrimido [ 4 , 5-c ] pyridaz in-7-yl ) amino ) -2- ( hydroxymethyl ) phenyl ) piperazine-l-carboxylate : Following the procedure of tert-butyl 4- ( 4- (( 3- ( 2-chlorophenyl ) -1- methyl- 4-oxo-l , 4-dihydropyrimido [ 4 , 5-c ] pyridaz in-7- yl ) amino ) phenyl ) piperaz ine-l-carboxylate , 3- ( 2- chlorophenyl ) -7- (methylthio ) pyrimido [ 4 , 5-c ] pyridaz in- 4(lH)-one (50 mg, 0.16 mmol) was reacted with tert-butyl 4- ( 4-amino-2- (hydroxymethyl ) phenyl ) piperazine-1- carboxylate (50mg, 0.16 mmol) to give a crude mixture which was purified by flash chromatography on silica (0- 20% EtOAc in cyclohexane) to afford the title compound as a yellow solid (30 mg, 32%) . XH NMR (400MHz, DMSO-d6) :
δ 9.12 (s, 1H), 7.73 (m, 1H) , 7.57 (d, 1H) , 7.52-7.44 (4H, m) , 7.07 (d, 1H) , 5.07 (t, 1H) , 4.60(d, 2H) , 3.47 (t, 4H) , 2.81 (t, 4H) , 1.25 (s, 9H) . LCMS (Method A): RT = 1.31min, m/z = 564-566 [M+H]+.
Step 2: 3- ( 2-chlorophenyl ) -7- (( 3- (hydroxymethyl ) -4- (piperaz in-l-yl ) phenyl ) amino ) -pyrimido [ 4 , 5-c ] pyridazin- 4(lH)-one: tert-butyl 4- ( 4- (( 3- ( 2-chlorophenyl ) -4-oxo- 1 , 4-dihydropyrimido [ 4 , 5-c ] pyridaz in-7-yl ) amino ) -2- (hydroxymethyl ) phenyl ) piperazine-l-carboxylate (30 mg, 0.053 mmol) was dissolved in dichloromethane ( 1 mL) . Then TFA (1 mL, 13 mmol) was added at 0°C. The resulting solution was stirred for 16 hours at room temperature until the starting material had been consumed (LC
analysis) . The reaction mixture was poured on an SCX-2 cartridge pre-treated with DCM/MeOH (9/1) . The cartridge was flushed 3 times with DCM/MeOH (9/1) . Then the target compound was collected by flushing 3 times with DCM/NH3 (2M) in MeOH (9/1), and concentrated in vacuo to afford the product as a yellow powder (12 mg, 48%) . XH NMR (500
MHz, DMSO-d6) : δ 10.30 (bs, 1H) , 9.12 (s, 1H) , 7.82 (bs, 1H), 7.71 (s, 1H), 7.56 (d, 1H) , 7.52-7.42 (m, 4H) , 7.04 (d, 1H), 5.07 (t, 1H), 4.58 (d, 2H) , 2.86 (t, 4H) , 2.78
(t, 4H) . LCMS (Method A): RT = 0.70 min, m/z = 464-466
[M+H] + . Example 36: 3- ( 2-chlorophenyl ) -7- (( 3- (hydroxymethyl ) -4- (piperaz in-l-yl ) phenyl ) amino ) -1-methylpyrimido [4,5- c] pyridaz in-4 ( 1H) -one
Figure imgf000112_0001
Step 1: tert-butyl 4- ( 4- (( 3- ( 2-chlorophenyl ) -l-methyl-4- oxo-1 , 4-dihydropyrimido [ 4 , 5-c ] pyridaz in-7-yl ) amino ) -2- (hydroxymethyl ) phenyl ) piperazine-l-carboxylate : Following the procedure of tert-butyl 4- ( 4- (( 3- ( 2-chlorophenyl ) -1- methyl-4-oxo-l , 4-dihydropyrimido [ 4 , 5-c ] pyridaz in-7- yl ) amino ) phenyl ) piperaz ine-l-carboxylate , 3- ( 2- chlorophenyl ) -1-methyl-7- (methylthio ) pyrimido [4,5- c] pyridazin-4 ( 1H) -one (50 mg, 0.15 mmol) was reacted with tert-butyl 4- ( 4-amino-2- (hydroxymethyl ) phenyl ) piperazine- 1-carboxylate to give a crude mixture which was purified by flash chromatography on silica (0-20% EtOAc in
cyclohexane) to afford the title compound as a yellow solid (25 mg, 15%). LCMS (Method A): RT = 1.45 min, m/z =
578-580 [M+H]+.
Step 2: 3- ( 2-chlorophenyl ) -7- (( 3- (hydroxymethyl ) -4- (piperaz in-l-yl ) phenyl ) amino ) -1-methylpyrimido [4,5- c] pyridazin-4 ( 1H) -one : tert-butyl 4-(4-((3-(2- chlorophenyl ) -l-methyl-4-oxo-l , 4-dihydropyrimido [4, 5- c ] pyridaz in-7-yl ) amino ) -2- ( hydroxymethyl ) phenyl )
piperazine-l-carboxylate (25 mg, 0.042 mmol) was
dissolved in dichloromethane (lmL) . Then TFA (1 mL,
12.98 mmol) was added at 0°C. The resulting solution was stirred for 16 hours at room temperature until the starting material had been consumed by LC analysis. The reaction mixture was poured on an SCX-2 cartridge pre- treated with DCM/MeOH (9/1) . The cartridge was flushed 3 times with DCM/MeOH (9/1) . Then the target compound was collected by flushing 3 times with DCM/NH3 (2M) in MeOH (9/1), and concentrated in vacuo to afford the product as a yellow powder (5 mg, 30%). XH NMR (400 MHz, DMSO-d6): δ 10.57 (bs, 1H), 9.15 (s, 1H) , 8.16 (bs, 1H) , 7.67 (m,
1H) , 7.57 (d, 1H), 7.53-7.43 (m, 4H) , 7.10 (d, 1H) , 5.14 (t, 1H), 4.59 (d, 2H), 4.10 (s, 3H) , 3.00 (t, 4H) , 2.86 (t, 4H) . LCMS (Method A): LCMS RT = 0.77 min, m/z = 478-
480 [M+H]+. Example 37: 3- ( 2-chlorophenyl ) -l-methyl-7- ( ( 3-methyl-4- (piperaz in-1-yl ) phenyl ) amino ) pyrimido [ 4 , 5-c ] pyridaz in- 4 ( 1H) -one
Figure imgf000114_0001
Step 1: tert-butyl 4- ( 4- (( 3- ( 2-chlorophenyl ) -l-methyl-4- oxo-1, 4-dihydropyrimido [ 4 , 5-c ] pyridaz in-7-yl ) amino ) -2- methylphenyl ) piperazine-l-carboxylate : Following the procedure of tert-butyl 4- ( 4- (( 3- ( 2-chlorophenyl ) -1- methyl-4-oxo-l , 4-dihydropyrimido [ 4 , 5-c ] pyridaz in-7- yl ) amino ) phenyl ) piperaz ine-l-carboxylate , 3- ( 2- chlorophenyl ) -l-methyl-7- (methylthio ) pyrimido [4,5- c] pyridazin-4 ( 1H) -one (100 mg, 0.314 mmol) was reacted with tert-butyl 4- ( 4-amino-2-methylphenyl ) piperazine-l- carboxylate to give a crude mixture which was purified by flash chromatography on silica (0-50% EtOAc in
cyclohexane, KP-NH column) to afford the title compound as an orange solid (63 mg, 36%) . LCMS (Method A) : RT = 1.74 min, m/z = 562 [M+H]+.
Step 2: 3- ( 2-chlorophenyl ) -l-methyl-7- (( 3-methyl-4-
(piperaz in-1-yl ) phenyl ) amino ) pyrimido [ 4 , 5-c ] pyridaz in-
4(lH)-one: Following the procedure of 3- ( 2-chlorophenyl ) - l-methyl-7- ( ( 3- ( (methylamino ) methyl ) -4-morpholinophenyl ) amino) pyrimido [ 4, 5-c] pyridazin-4 ( 1H) -one, tert-butyl 4- ( 4- ( ( 3- ( 2-chlorophenyl ) -1-methyl-4-oxo-l , 4- dihydropyrimido [ 4 , 5-c ] pyridaz in-7-yl ) amino ) -2- methylphenyl ) piperazine-l-carboxylate (63 mg, 0.112 mmol) was reacted with TFA to afford the title compound as an orange solid (48 mg, 94%). XH NMR (400 MHz, DMSO-d6): δ 10.50 (bs, IH), 9.14 (s, IH) , 7.70 (bs, 2H) , 7.57 (d, IH) , 7.53-7.45 (m, 4H) , 7.03 (d, IH) , 3.99 (s, 3H) , 2.87- 2.85 (m, 4H) , 2.77-2.75 (m, 4H) , 2.28 (s, 3H) . LCMS
(Method A): LCMS RT = 0.88 min, m/z = 462 [M+H]+.
Example 38: 3- ( 2-chlorophenyl ) -7- ( ( 3-methoxy-4- (piperaz in-l-yl ) phenyl ) amino ) -1-methylpyrimido [4,5- c] pyridaz in-4 ( IH) -one
Figure imgf000115_0001
Step 1: tert-butyl 4- ( 4- (( 3- ( 2-chlorophenyl ) -l-methyl-4- oxo-1, 4-dihydropyrimido [ 4 , 5-c ] pyridaz in-7-yl ) amino ) -2- methoxyphenyl ) piperazine-l-carboxylate : Following the procedure of tert-butyl 4- ( 4- (( 3- ( 2-chlorophenyl ) -1- methyl-4-oxo-l , 4-dihydropyrimido [ 4 , 5-c ] pyridaz in-7- yl ) amino ) phenyl ) piperaz ine-l-carboxylate , 3- ( 2- chlorophenyl ) -1-methyl-7- (methylthio ) pyrimido [4,5- c] pyridazin-4 ( IH) -one (100 mg, 0.314 mmol) was reacted with tert-butyl 4- ( 4-amino-2-methoxyphenyl ) piperazine-l- carboxylate to give a crude mixture which was purified by flash chromatography on silica (0-50% EtOAc in
cyclohexane, KP-NH column) to afford the title compound as an orange solid (49 mg, 27%) . LCMS (Method A) : RT = 1.58 min, m/z = 578 [M+H]+.
Step 2: 3- ( 2-chlorophenyl ) -7- (( 3-methoxy-4- (piperaz in-1 yl ) phenyl ) amino ) -1-methylpyrimido [ 4 , 5-c ] pyridaz in-4 ( IH ) one: Following the procedure of 3- ( 2-chlorophenyl ) -1- methyl-7- ( ( 3- ( (methylamino ) methyl ) -4- morpholinophenyl ) amino ) pyrimido [ 4 , 5-c ] pyridaz in-4 ( IH) - one, tert-butyl 4- ( 4- ( ( 3- ( 2-chlorophenyl ) -l-methyl-4-oxo- 1 , 4-dihydropyrimido [ 4 , 5-c ] pyridaz in-7-yl ) amino ) -2- methoxyphenyl ) piperazine-l-carboxylate (49 mg, 0.085 mmol) was reacted with TFA to afford the title compound as an orange solid (22 mg, 54%) . XH NMR (400 MHz, DMSO- d6) : δ 10.53 (bs, 1H) , 9.15 (s, 1H) , 7.72 (bs, 1H) , 7.57 (d, 1H), 7.53-7.45 (m, 4H) , 7.29 (d, 1H) , 6.89 (d, 1H) , 4.02 (s, 3H), 3.83 (s, 3H) , 2.87-2.85 (m, 8H) . LCMS
(Method A): LCMS RT = 0.84 min, m/z = 478 [M+H]+.
Example 39: 3- ( 2-chlorophenyl ) -7- (( 3- (hydroxymethyl ) -4- ( 4-isopropylpiperaz in-l-yl ) phenyl ) amino ) -1- methylpyrimido [ -c] pyridaz in-4 ( 1H) -one
Figure imgf000116_0001
Following the procedure of tert-butyl 4-(4-((3-(2- chlorophenyl ) -l-methyl-4-oxo-l , 4-dihydropyrimido [4, 5- c ] pyridaz in-7-yl ) amino ) phenyl ) piperaz ine-l-carboxylate , 3- ( 2-chlorophenyl ) -1-methyl-7- (methylthio ) pyrimido [4,5- c] pyridazin-4 ( 1H) -one (100 mg, 0.314 mmol) was reacted with ( 5-amino-2- ( 4-isopropylpiperaz in-l- yl ) phenyl ) methanol to give a crude mixture which was purified by flash chromatography on silica (0-100% EtOAc in cyclohexane, KP-NH column) to afford the title
compound as an yellow solid (59 mg, 36%). 1ti NMR (400 MHz, DMSO-d6) : δ 10.56 (bs, 1H) , 9.14 (s, 1H) , 8.17 (bs, 1H) , 7.64 (bs, 1H), 7.56 (d, 1H) , 7.50-7.44 (m, 3H) , 7.07 (d, 1H) , 5.12 (t, 1H), 4.58 (d, 2H) , 4.00 (s, 3H) , 2.82 (bs, 4H) , 2.70-2.67 (m, 1H) , 2.58 (bs, 4H) , 1.01 (d, 6H) . LCMS (Method A): LCMS RT = 0.83 min, m/z = 520 [M+H]+.
Example 40: (R) -3- ( 2-chlorophenyl ) -1-methyl-7- ( ( 4- ( 3- methylpiperaz in-l-yl ) phenyl ) amino ) pyrimido [4,5- c] pyridaz in-4 ( 1H) -one
Figure imgf000117_0001
Step 1: (R) -tert-butyl 4- ( 4- (( 3- ( 2-chlorophenyl ) -1- methyl-4-oxo-l , 4-dihydropyrimido [ 4 , 5-c ] pyridaz in-7- yl ) amino ) phenyl ) -2-methylpiperaz ine-l-carboxylate :
Following the procedure of tert-butyl 4-(4-((3-(2- chlorophenyl ) -l-methyl-4-oxo-l , 4-dihydropyrimido [4, 5- c ] pyridaz in-7-yl ) amino ) phenyl ) piperaz ine-l-carboxylate , 3- ( 2-chlorophenyl ) -1-methyl-7- (methylthio ) pyrimido [4,5- c] pyridazin-4 ( 1H) -one (100 mg, 0.314 mmol) was reacted with ( R) -tert-butyl 4- ( 4-aminophenyl ) -2-methylpiperazine- 1-carboxylate to give a crude mixture which was purified by flash chromatography on silica (0-50% EtOAc in
cyclohexane, KP-NH column) to afford the title compound as an orange solid (82 mg, 47%) . LCMS (Method A) : RT =
1.67 min, m/z = 562 [M+H]+.
Step 2: (R) -3- ( 2-chlorophenyl ) -l-methyl-7- (( 4- ( 3- methylpiperaz in-l-yl ) phenyl ) amino ) pyrimido [4,5- c] pyridazin-4 ( 1H) -one : (R) -tert-butyl 4-(4-((3-(2- chlorophenyl ) -l-methyl-4-oxo-l , 4-dihydropyrimido [4, 5- c] pyridaz in-7-yl ) amino ) phenyl ) -2-methylpiperaz ine-1 - carboxylate (82 mg, 0.146 mmol) was reacted with HC1 (3 mL, 4 M in dioxane) in DCM (3mL) for 4h at RT . The solvents were removed in vacuo and the crude taken up in MeOH and transferred onto a SCX-2 column. The column washed three times with MeOH and then 3 times with NH3 in MeOH (2 M) . The solvents were removed in vacuo to afford the title compound as an orange solid (58 mg, 86%) .
1E NMR (400 MHz, DMSO-d6): δ 10.45 (bs, 1H) , 9.11 (s, 1H) , 7.72 (bd, 2H), 7.56 (d, 1H) , 7.50-7.44 (m, 3H) , 6.96 (d, 2H), 3.96 (s, 3H), 3.49 (t, 2H) , 2.95 (d, 1H) , 2.84-2.78 (m, 2H) , 2.17 (t, 1H) , 1.02 (d, 3H) . LCMS (Method A):
LCMS RT = 0.85 min, m/z = 462 [M+H]+.
Method 1: Measurement of Wee-1 kinase activity
In the measurement of Wee-1 activity, a commercial peptide Poly(Lys Tyr(4:l)) hydrobromide was purchased from Sigma Aldrich and used as the substrate. Activated Wee-1 kinase was purchased from Invitrogen (PV3817) and an ADP-Glo luminescent kit was purchased from Promega.
All reactions took place in 60 pL volumes in reaction buffer containing 40 mM Tris-HCl and 20 mM magnesium chloride, supplemented with 0.1 mg/mL bovine serum albumin and 2 mM DTT. Compounds were serially diluted in buffer and 5 pL of each concentration pipetted into a white 384 well plate (Sigma Aldrich M6186) . A 5 pL aliquot of the Wee-1 enzyme was added to each well and the plate centrifuged for 1 min to ensure mixing of the enzyme and inhibitor.
The plate was incubated at room temperature for 30 minutes before the addition of 2 pg/mL of substrate and 30 pM ATP in a 5 pL aliquot. The plate was centrifuged for one minute and incubated for 1 h at RT . 15 \1~L of ADP-Glo stop reagent was added to each well to quench the reaction and deplete unconverted ATP. The plate was incubated for a further 40 min in the dark at RT .
30 pL of ADP-Glo kinase detection reagent was added to each well, converting ADP to ATP, catalysing the
generation of luciferin by luciferase. The plate was shaken for 1 min, and incubated in the dark for an additional hour.
Luminescence from each well was detected using the Biotek Synergy4 HD plate reader and the percentage inhibition of kinase activity calculated for each inhibitor tested. Positive (kinase only) and negative (no kinase) controls were added to each plate to ensure specific interaction of kinase and inhibitor. The IC50 concentration for each inhibitor was calculated by plotting the percentage kinase inhibition against concentration of inhibitor and the curve generated by non-linear regression fitting.
Method 2: Determining the effect of compounds on the phosphorylation of cdc2 at Tyrl5.
The colorectal cancer cell lines HT-29 and HCT-116 were purchased from the ATCC and routinely maintained in
McCoy's Medium (Invitrogen) supplemented with 10% Foetal Calf Serum.
The cells were trypsinised from their growing vessel and counted, 100 \1~L of cell suspension containing 6000 cells was pipetted into black 96 well Co-star plates and incubated overnight to allow adherence to the surface at a temperature of 37°C and an atmosphere of 5% CO2. Test compounds were formulated in DMSO and diluted in foetal calf serum supplemented medium. Incubating medium was removed by aspiration and diluted drug supplemented medium added to each well.
The plate was returned to the incubator for an additional eight hours at 37°C and an atmosphere of 5% CO2. Post incubation, the drug supplemented medium was aspirated from each well and the cells were washed once in ice-cold phosphate buffered saline (PBS) . 100 pL of cell lysis buffer (Cell Signalling Technologies #9803) containing 20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton- X100, 2.5 mM sodium pyrophosphate, 1 mM glycerophosphate, 1 mM Na3VC>4 and 1 pg/mL leupeptin was added to each well of the 96 well plate and incubated at 4 °C for 30 min. The samples on the plate were snap frozen at -80 °C until required. Immediately before the continuation of the assay, the sample plate was thawed and centrifuged at 4 °C for 10 min and the supernatant transferred to
secondary tubes or 96 well plate.
Cell supernatant was mixed in a ratio of 1:1 with sample dilutent buffer and vortexed for one minute. 100 pL of diluted sample was pipetted into pre-coated plates containing a rabbit polyclonal antibody for phospho-cdc2 (Tyrl5) (Cell Signalling Technologies PathScan kit
#7176) . The plate was sealed and incubated overnight at 4 °C.
The plate seal was removed and the well contents
aspirated, followed by 3 x 5 min washes with 200 pL of diluted wash buffer. Between each wash the plate was tapped firmly onto blotting paper to ensure the removal of all kit solution. 100 pL of kit detection antibody was added to each well and the plate re-sealed and incubated at 37°C for 1 h. Post incubation the plate was washed and processed in a similar manner to that previously
described.
100 pL of horseradish peroxidise-linked secondary
antibody was added to each test well, the plate sealed and incubated for thirty minutes at 37°C. Post
incubation, the plate was washed as previously stated, followed by the addition of 100 pL of 3, 3', 5, 5'
tetramethylbenz idine (TMB reagent) . The plate was sealed and incubated at RT for 30 min. 100 pL of stop solution was added to each well and the underside of the plate wiped with a lint-free tissue, prior to spectrophotometric determination. Absorbance from each well was read at 450 nm within 30 min of the addition of the stop solution.
The percentage of phospho-cdc2 was calculated compared to DMSO control and plotted versus the concentration of inhibitor using GraphPad Prism. Data was fitted using non-linear regression analysis and IC5o values generated.
Method 3: Determining the anti-proliferative properties of Wee-1 inhibitors in the HT-29 cell line Cell Titre Glo (Promega) is a highly sensitive
homogeneous reagent used to determine the viability of cells. The reagent uses a stable form of luciferase to measure ATP as an output of viability. The luminescent values generated in the assay are directly proportional to the number of viable cells in your assay.
The following materials were used: white clear bottomed 96 well assay plates (Costar) ; Cell titre Glo reagents; HT-29 (ATCC) cells were grown in McCoy's medium
(Invitrogen) supplemented with 10% Foetal calf serum ( Invitrogen ) . HT-29 cells were washed, detached and re-suspended in their respective fresh media. The cells were pelleted by centrifugation (Eppendorf 5414) and the spent supernatant was discarded. The cells were re-suspended by vortex mixing, counted and seeded into clear bottom white 96 well plates at a density of 5000 cells per well. The cells were incubated overnight at 37 °C (95% O2 / 5% CO2) , and next day treated with increasing concentrations of test compound formulated in fresh medium. The plates were returned to the incubator for 72 h.
Cell Titre Glo (Promega) was prepared by mixing the supplied reagents as per manufacturer's instructions and left to stand at RT . The cell plates were removed from the incubator and 80 pL of the Cell Titre Glo solution added to each well. The plate was shaken for five minutes to ensure homogenous mixing of reagents and cells, then left to stand for 10 min at RT .
The cell viability post compound treatment was determined by the luminescent intensity emitted from the drug treated wells in the plate. In brief, the assay plate was placed in the Biotex Synergy 4 Hybrid plate reader and the luminescence read in each well. The compound treated wells were compared to vehicle treated wells and the % inhibition of cell viability calculated.
The data was analysed using GraphPad Prism, with IC5o values generated using non-linear regression of the data set .
Method 4: Determination of CLint estimates using human liver microsomes.
Test compounds (final concentration =1μΜ; final DMSO concentration = 0.1%) were incubated in 0.1M phosphate buffer pH 7.4 with human liver microsomes ( 0.5mg of protein/mL) at 37°C. Reactions were started by addition of NADPH in 0.1M phosphate buffer pH 7.4 (final
concentration ImM) . 40pL aliquots were removed at 2, 5, 10, 15, 20, 30, 40 and 50minutes. Reactions were quenched in 80pL of ice-cold methanol. Samples were subsequently frozen overnight then centrifuged at 3500rpm for
20minutes at 4°C. The supernatants were removed and transferred into analytical plates and analysed by
LC/MS/MS .
LC/MS/MS method:
All samples were analysed on a Waters Acquity I-Class coupled to a Waters Xevo TQD mass spectrometer. A Waters BEH C18 2.1x50mm 1.7pm column was used and mobile phases were water and methanol containing 0.1% formic acid as modifier. Analysis was by multiple reaction monitoring and conditions were optimised for each test compound.
Data analyses: From a plot of In peak area against time, the gradient of the line is determined. Subsequently, half-life and
intrinsic clearance are calculated using the equations below :
Eliminated rate constant (k) = (- gradient)
0.693
Half life (ti/2) (min) =
VxO.693
Intrinsic Clearance (CLint) (pL/min/mg) =
Figure imgf000124_0001
where V=Incubation volume (pL)/mg protein
The table below highlights the selectivity of
representative compounds of the invention against a panel of 50 kinases (assessed using the Invitrogen SelectScreen ® Kinase Profiling Service) . Unexpectedly, representative Wee-1 inhibitors from the present invention show greater selectivity than a representative compound described in
BMCL, 2005, pl931.
Compound 1 BMCL, 2005, pl931
Example Example
(Wee-1 2 3
IC50 150nM, Src IC50 6nM)
ABL1 - - +
ACVR1B (ALK4) - - +
AKT1 (PKB alpha) - - -
AMPK A1/B1/G1 - - -
AURKA (Aurora A) - - -
BTK - - +
CDKl/cyclin B - - -
CHEK1 (CHK1) - - -
CSNK1G2 (CK1 gamma 2) - - - CSNK2A1 (CK2 alpha 1) — — —
DYRK3 - - -
EGFR (ErbBl) + + +
EPHA2 - - +
ERBB2 (HER2) - -
FGFR1 - -
FLT3 - - +
FRAP1 (mTOR) - - -
GSK3B (GSK3 beta) - - -
IGF1R - - -
IKBKB (IKK beta) - - -
INSR - - -
IRAK4 - - -
JAK3 - - +
KDR (VEGFR2) - - +
KIT - - +
LCK - - +
MAP2K1 (MEK1) - - -
MAP4K4 (HGK) - - +
MAPK1 (ERK2) - - -
MAPK14 (p38 alpha) - - +
MAPK8 (JNK1) - - -
MAPKAPK2 - - -
MARK2 - - +
MET (cMet) - - -
NEK1 - - -
NTRK1 (TRKA) - - -
PAK4 - - -
PDGFRB (PDGFR beta) - - +
PHKG2 - - -
PIM1 - - -
PLK1 - - -
PRKACA (PKA) - - -
PRKCB1 (PKC beta I) - - -
RET - - +
ROCK1 - - -
RPS6KA3 (RSK2) - - - RPS6KB1 (p70S6K) — — —
SRC - - +
SYK - - -
TEK (Tie2) - - -
Inhibition <75% @ 300-350nM;
"+": Inhibition >75% at 300-350nM
Table 1
Figure imgf000126_0001
Compound 1: Bioorg & Med Chem Lett Vol 15, pp 1931-1935.

Claims

CLAIMS : compound of Formula
Figure imgf000127_0001
(I) or a pharmaceutically acceptable salt or iV-oxide
derivative thereof, wherein:
X is an oxygen atom or a nitrogen atom;
Y is a carbon atom or a nitrogen atom;
R1 is an optionally substituted aryl group;
R2 is a hydrogen atom, an optionally substituted alkyl group or an optionally substituted amino group when Y is a carbon atom and R2 is absent when Y is a nitrogen atom;
R3 is a hydrogen atom, an optionally substituted alkyl group or an optionally substituted aryl group when X is a nitrogen atom and R3 is absent when X is an oxygen atom;
R4 is an optionally substituted alkyl group or an optionally substituted aryl group.
2. The compound of claim 1 or a pharmaceutically acceptable salt or iV-oxide derivative thereof, wherein R4 is a group represented by the formula (a) :
Figure imgf000127_0002
(a)
Z is a nitrogen atom or an optionally substituted methine group;
R4a is a hydrogen atom, a halo group, an optionally substituted C1-C6 alkyl group, an optionally substituted Ci~C6 alkoxy group or is a nitrogen-containing
heterocyclic group optionally substituted with a
substituent selected from the group consisting of a halo group, an optionally substituted C1-C6 alkyl group, an oxo group and an optionally substituted amino group;
R4 is a hydrogen atom, a halo group, an optionally substituted C1-C6 alkyl group or an optionally substituted C1-C6 alkoxy group;
or, when R4a and R4 exist on adjacent ring atoms of the group of the formula (b) :
Figure imgf000128_0001
(b)
R4a and R4 and the ring atoms to which they are attached may form, as taken together, a C3-C7 alkyl group, in which one or two methylene groups constituting the C3- C7 alkyl group may be each independently replaced by an oxygen atom or a group of -N(Rle)-, and the C3-C7 alkyl group may be substituted with one or more substituents selected from the group consisting of a halo group and a C1-C6 alkyl group;
or R4a and R4 and the ring atoms to which they are attached may form, as taken together, a spiro ring or a bicyclo ring to be formed of a 5-membered to 7-membered aliphatic ring and any other 3-membered to 7-membered aliphatic ring, in which one or two or more methylene groups constituting the spiro ring or the bicyclo ring may be each independently replaced by an oxygen atom, a sulfur atom, a sulfinyl group, a sulfonyl group, an oxo group or a group of -N(Rlf)-, and the spiro ring or the bicyclo ring may be each independently substituted with a substituent selected from the group consisting of a halo group, a hydroxyl group or a Ci~C6 alkyl group; and
Rle and Rlf are each independently a hydrogen atom or a C1-C6 alkyl group optionally substituted with a
substituent selected from the group consisting of a halo group, a hydroxyl group, a cyano group, an oxo group, a C1-C6 alkyl group, a C1-C6 alkoxy group, an amino group, a substituted amino group and a nitrogen-containing
heterocyclic group.
3. The compound of claim 2 or a pharmaceutically acceptable salt or iV-oxide derivative thereof, wherein R4 is a group represented by the formula (c) :
Figure imgf000129_0001
(c)
wherein R4a is a hydrogen atom, a halo group, a C1-C6 alkyl group or an optionally substituted C1-C6 alkoxy group, or is a nitrogen-containing heterocyclic group optionally substituted with a substituent selected from the group consisting of a halo group, a C1-C6 alkyl group, an oxo group and a group of -Q1-N (Rla) Rl ;
R4 is a hydrogen atom, a halo group, an optionally substituted C1-C6 alkyl group or an optionally substituted C1-C6 alkoxy group; Rla and Rl each independently is a hydrogen atom, a C1-C6 alkyl group, or as taken together, they may form an optionally substituted C2-C6 alkyl group; and
Q1 is a single bond or a C1-C3 alkyl group.
4. The compound of claim 2 or claim 3, or a
pharmaceutically acceptable salt or N-oxide derivative thereof, wherein
R4a is a substituted C1-C6 alkoxy group or a nitrogen- containing heterocyclic group substituted with a
substituent selected from the group consisting of a halo group and a C1-C6 alkyl group; and
R4 is a hydrogen atom.
5. The compound of any of the preceding claims, or a pharmaceutically acceptable salt or N-oxide derivative thereof, wherein
R4 is a roup represented by the formula (d) :
Figure imgf000130_0001
(d) wherein R m is selected from the group consisting of a hydrogen atom and an optionally substituted C1-C6 alkyl group .
6. The compound of any of the preceding claims, or a pharmaceutically acceptable salt or iV-oxide derivative thereof, wherein R1 is a group represented by the formula
(e) :
Figure imgf000131_0001
(e)
wherein R a is a hydrogen atom, a halo group, a hydroxyl group, a cyano group, a C1-C6 alkyl group, a C1-C6 alkoxy group or a C1-C6 alkoxy-Ci-C6 alkyl group;
R3a and R3 are each independently selected from the group consisting of a hydrogen atom, a hydroxyl group, an amino group, an alkoxy group and a C1-C6 alkyl group; and
T is a nitrogen atom, or a methine group which may be substituted with a halo group, a hydroxyl group, a cyano group, a C1-C6 alkyl group, a C1-C6 alkoxy group or a C1-C6 alkoxy-Ci-C6 alkyl group.
7. The compound of any of the preceding claims, or a pharmaceutically acceptable salt or iV-oxide derivative thereof, wherein R1 is a group represented by the formula
(g) :
Figure imgf000131_0002
(g)
wherein R a is a hydrogen atom, a halo group, a hydroxyl group, a cyano group, a C1-C6 alkyl group, a alkoxy group or a C1-C6 alkoxy-Ci-C6 alkyl group; and T is a nitrogen atom, or a methine group which may be substituted with a halo group, a hydroxyl group, a cyano group, a C1-C6 alkyl group, a C1-C6 alkoxy group or a C1-C6 alkoxy-Ci-C6 alkyl group.
8 The compound of claim 6 or claim 7,
pharmaceutically acceptable salt or iV-oxide derivative thereof, wherein
R2a is a halo group, a C1-C6 alkyl group or a C1-C6 alkoxy group, and
T is a methine group or is a methine group
substituted with a halo group or a C1-C6 alkyl group.
9. The compound of any of claims 6 to 8, or a
pharmaceutically acceptable salt or iV-oxide derivative thereof, wherein
R2a is a halo group; and
T is a methine group substituted with a halo group. 10. The compound of any of the preceding claims, or a pharmaceutically acceptable salt or iV-oxide derivative thereof, wherein R1 is a 2 , 6 -dichlorophenyl group.
11. The compound of any of claims 1 to 5, wherein Y is a nitrogen atom.
12. The compound of claim 11, wherein R1 is a group represented by the formula (h) :
Figure imgf000132_0001
R2a' (h)
wherein R2a is a hydrogen atom, a hydroxyl group, a cyano group, a C1-C6 alkyl group, a C1-C6 alkoxy group or a C1-C6 alkoxy-Ci-C6 alkyl group;
R3a and R3 are each independently selected from the group consisting of a hydrogen atom, a hydroxyl group, an amino group, an alkoxy group and a C1-C6 alkyl group; and T' is a nitrogen atom, or a methine group which may be substituted with a halo group, a hydroxyl group, a cyano group, a C1-C6 alkyl group, a C1-C6 alkoxy group or a C1-C6 alkoxy-Ci-C6 alkyl group.
13. The compound of claim 11 or claim 12, wherein R1 is a 2-chlorophenyl group.
14. The compound of any of claims 11 to claim 13, wherein R3 is a methyl group.
15. The compound of any of claims 11 to 14, wherein R4a is a nitrogen-containing heterocyclic group optionally substituted with a methyl group; and
R4 is a hydrogen atom, a methyl group, a methoxy group or a methyl group substituted by a hydroxy group. 16. The compound of claim 1, or a pharmaceutically acceptable salt or iV-oxide derivative thereof, wherein the compound is selected from the following:
( 1 ) 8-Methy1-2- ( ( 4- ( 4-methylpiperaz in-1-yl ) phenyl ) amino ) - 6 -phenylpyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one ;
(2) 6- (2, 6-Dichlorophenyl) -8-methyl-2- ( (4- (4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 (8H) -one;
( 3 ) 6- ( 2-Chlorophenyl ) -8-methyl-2- ( ( 4- ( 4-methylpiperaz in- 1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one ; (4) 6- (2-Methoxyphenyl) -8-methyl-2- ( (4- (4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one;
( 5 ) 8-Methy1-2- ( ( 4- ( 4-methylpiperaz in-1-yl ) phenyl ) amino ) - 6-(o-tolyl) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one ;
(6) 6- (2, 4-Dichlorophenyl) -8-methyl-2- ( (4- (4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one;
(7) 6- (2, 5-Dichlorophenyl) -8-methyl-2- ( (4- (4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one;
(8) 6- (2, 3-Dichlorophenyl) -8-methyl-2- ( (4- (4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one;
(9) 8-Methyl-2- (( 4- ( 4-methylpiperaz in-l-yl ) phenyl ) amino ) - 6 - ( thiophen-3-yl ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one ;
(10) 8-Methyl-6- (5-methyl-l, 3, 4-oxadiazol-2-yl ) -2- ( (4- (piperaz in-l-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one;
(11) 6- (3-Chloropyridin-2-yl) -8-methyl-2- ( (4- (4- methylpiperaz in-l-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one;
(12) 6 - ( 2 , 6 -Dichlorophenyl ) -8-methy1-2- ( ( 4- (piperaz in-l- yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one ;
(13) 8-Allyl-6- (2, 6-dichlorophenyl) -2- ( (4- (4- methylpiperaz in-l-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one;
(14) 6- (2, 6 -Dichlorophenyl) -8- ( 4-methoxybenzyl ) -2- ( (4- (4- methylpiperaz in-l-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one;
( 15 ) 6- ( 2 , 6 -Dichlorophenyl ) -2- ( ( 4- ( 4-methylpiperaz in-l- yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one ;
(16) 6- (2, 6-Dichlorophenyl) -8-ethyl-2- ( (4- (4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one;
(17) Ethyl 2- (6- (2, 6-dichlorophenyl) -2- ( (4- (4- methylpiperaz in-1-yl ) phenyl ) amino ) -5-oxopyrido [2,3- d] pyrimidin-8 ( 5H) -yl ) acetate ;
(18) 2-(6-(2,6 -Dichlorophenyl ) -2- ( ( 4- ( 4-methylpiperaz in-
1-yl ) phenyl ) amino ) -5-oxopyrido [ 2 , 3-d] pyrimidin-8 ( 5H ) - yl) acetic acid.2HCl;
(19) 6- (2, 6-Dichlorophenyl) -8- (3-hydroxypropyl) -2- ( (4- (4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one;
(20) 6 - ( 2 , 5-Dimethyl-lH-pyrrol-l-yl ) -8- ( 4-methoxybenzyl ) -
2- ( ( 4- ( 4-methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [2,3- d] pyrimidin-5 ( 8H) -one;
(21) 6 - ( 2 , 6 -Dichlorophenyl ) -2- ( ( 6 - (piperaz in-1- yl ) pyridin-3-yl ) amino ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one ;
(22) 6- (2, 6-Dichlorophenyl) -2- ( (4- (2-
( diethylamino ) ethoxy ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one;
( 23 ) 6- ( 2 , 6-Dichlorophenyl ) -2- ( (pyridin-4- ylmethyl ) amino ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one ;
(24) 6 - ( 2 , 6 -Dichlorophenyl ) -2- ( ( 1-methylpiperidin-4- yl ) amino ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one ;
(25) 7-Amino-6- (2-chlorophenyl) -8-methyl-2- ( (4- (4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin-
5 ( 8H) -one;
(26) 6- (2-Chlorophenyl) -7, 8-dimethyl-2- ( (4- (4- methylpiperaz in-1-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5(8H)-one;
(27) 6 - ( 2-Chlorophenyl ) -7-methy1-2- ( ( 4- (piperaz in-1 - yl ) phenyl ) amino ) -5H-pyrano [ 2 , 3-d] pyrimidin-5-one ;
(28) 6 - ( 2-Chlorophenyl ) -2- ( (4- (piperaz in-1- yl ) phenyl ) amino ) -5H-pyrano [ 2 , 3-d] pyrimidin-5-one .2HC1 ;
(29) 6- (2-Chlorophenyl) -8-cyclopropyl-2- ( (4- (4- methylpiperaz in-l-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one;
(30) 6- ( 4-Hydroxyphenyl) -8-methyl-2- ( (4- (4- methylpiperaz in-l-yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin- 5 ( 8H) -one; and
( 31 ) 6- ( 3-Hydroxyphenyl ) -8 -methy1-2- ( ( 4- ( piperaz in-l- yl ) phenyl ) amino ) pyrido [ 2 , 3-d] pyrimidin-5 ( 8H ) -one . 17. The compound of claim 1, or a pharmaceutically acceptable salt or iV-oxide derivative thereof, wherein the compound is selected from the following:
( 32 ) 3- ( 2-chlorophenyl ) -1- ( 4-methoxyphenyl ) -7- ( ( 4- ( 4- methylpiperaz in-l-yl ) phenyl ) amino ) pyrimido [4,5- c] pyridazin-4 ( IH) -one;
( 33 ) 3- ( 2-chlorophenyl ) -1 -methyl- 7- ( ( 4- ( piperaz in-l- yl ) phenyl ) amino ) pyrimido [ 4 , 5-c ] pyridaz in-4 ( IH ) -one ;
(34) 3- (2-chlorophenyl) -1 -methyl- 7- ( (3- ( (methylamino ) methyl ) -4- morpholinophenyl ) amino ) pyrimido [ 4 , 5-c ] pyridaz in-4 {IH) - one ;
( 35 ) 3- ( 2-chlorophenyl ) -7- ( (3- ( hydroxymethyl ) -4-
( piperaz in-l-yl ) phenyl ) amino ) -pyrimido [ 4 , 5-c ] pyridaz in-
4 {IH) -one;
(36) 3- ( 2-chlorophenyl ) -7- (( 3- (hydroxymethyl ) -4-
( piperaz in-l-yl ) phenyl ) amino ) - 1 -methylpyrimido [4,5- c] pyridazin-4 ( IH) -one;
(37) 3- (2-chlorophenyl) -l-methyl-7- ( (3-methyl-4-
( piperaz in-l-yl ) phenyl ) amino ) pyrimido [ 4 , 5-c ] pyridaz in- 4(lH)-one;
(38) 3- ( 2-chlorophenyl ) -7- ( ( 3-methoxy-4- (piperaz in- 1- yl ) phenyl ) amino ) -1 -methylpyrimido [ 4 , 5-c ] pyridaz in-4 ( IH) - one;
(39) 3- (2-chlorophenyl) -7- ( (3- ( hydroxymethyl ) -4- (4- isopropylpiperaz in-l-yl ) phenyl ) amino ) -1- methylpyrimido [4, 5-c] pyridazin-4 ( 1H) -one; and
(40) (R) -3- (2-chlorophenyl) -1-methyl-7- ( (4- (3- methylpiperaz in-l-yl ) phenyl ) amino ) pyrimido [4,5- c] pyridazin-4 ( 1H) -one .
18. The compound as defined in any of claims 1 to 17, or a pharmaceutically acceptable salt or iV-oxide derivative thereof, and at least one pharmaceutically acceptable excipient .
19. A pharmaceutical composition comprising a compound as defined in any of claims 1 to 17, or a
pharmaceutically acceptable salt or iV-oxide derivative thereof, and at least one pharmaceutically acceptable excipient .
20. The pharmaceutical composition as defined in claim 19 comprising one or more further pharmaceutically active agents .
21. The compound as defined in any of claims 1 to 17, or a pharmaceutically acceptable salt or iV-oxide derivative thereof, or the pharmaceutical composition as defined in claim 19 or 20 for use in therapy.
22. The compound of any of claims 1 to 17 for use as a medicament .
23. The compound of any of claims 1 to 17 for use in treating or preventing cancer.
24. Use of the compound as defined any of claims 1 to 17 for the manufacture of a medicament for treating or preventing cancer. 24. A method of treating or preventing cancer in a human or animal patient comprising administering to a patient in need thereof an effective amount of a compound
according to any one of claims 1 to 17 or a
pharmaceutical composition according to claim 19 or claim 20.
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