Classifications

• • 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
  • C07D471/12—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 three hetero rings
  • C07D471/14—Ortho-condensed systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • 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
  • C07D471/22—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 systems contains four or more hetero rings

Definitions

• the present invention relates to (5,6-dihydro)pyrimido[4,5-e]indolizines, to pharmaceutical compositions comprising these compounds and their use in therapy.
• the present invention relates to the use of (5,6-dihydro)pyrimido[4,5-e]indolizines in the treatment of cancer.
• the present invention relates to chemical compounds, which modulate the activity of protein kinases, in particular inhibit the activity of the protein kinase TTK (EC 2.7.12.1).
• TTK commonly referred to as Mps1
• Mps1 is a component of the mitotic checkpoint, a machinery that ensures the fidelity of sister chromatid segregation over two daughter cells during cell division. Defects in or inhibition of the mitotic checkpoint leads to aneuploidy.
• aneuploidy is a common genetic alteration in solid human tumors (Lengauer, C.
• TTK expression is increased in breast cancer with chromosomal instability (Yuan, B. et al., Clin. Cancer Res.
• TTK kinase inhibitors are useful for the treatment of a variety of cancers and may be applied as single agents, or in combination with other chemotherapeutic agents.
• WO 2012/101032 A1 (Nerviano Medical Sciences SRL) relates to tricyclic pyrrolo derivatives which modulate the activity of protein kinases and are therefore useful in treating diseases caused by dysregulated protein kinase activity.
• Mps-BAY2b was shown to inhibit HeLa-Matu cervical tumor cell growth in a mouse xenograft model and increased the efficacy of paclitaxel in this model (Jemaà, M. et al.), thus confirming the chemotherapy sensitizing effect of TTK inhibition previously shown with RNA interference techniques (Janssen, A. et al.).
• pyrazolo-quinazolines are disclosed as inhibitors of TTK by Nerviano Medical Sciences S.R.L.
• a compound described in this parent, NMS-P715 inhibited TTK kinase activity with a half-maximal inhibitory concentration (IC 50 ) of 8 to 182 nM, depending on whether a pre-incubation step was included in the assay or not (Colombo, R., et al., Cancer Res. 70: 10255; 2010).
• IC 50 half-maximal inhibitory concentration
• NMS-P715 also inhibited the proliferation of cancer cell lines from different tumor origin with IC 50 s of 1 ⁇ M and higher, and inhibited tumor growth in A375 and A2780 mouse xenograft models (Columbo, R. et al.).
• WO2010/111406A2 compounds are disclosed as inhibitors of TTK by Myriad Pharmaceuticals, Inc.
• a representative compound, MPI 4079605 inhibited TTK with an IC 50 of 1.8 nM (Tardif, K. D., et al., Mol. Cancer Res. 10:2267; 2011).
• Treatment of cancer cell lines for 72 hours revealed many cell lines with little sensitivity (Tardif K. D., et al.).
• MPI 4079605 is structurally similar to reversine and MPS1-IN-1, two other published inhibitors of TTK (Kwiatkowski, N., et al., Nat. Chem. Biol. 6: 359; 2010; Santiguida, S., et al., J. Cell. Biol. 190: 73; 2010).
• TTK inhibitors with potent kinase inhibitory and anti-proliferative activity.
• the present invention provides (5,6-dihydro)pyrimido[4,5-e]indolizine derivatives.
• the present invention provides (5,6-dihydro)pyrimido[4,5-e]indolizine derivatives according to formula I or pharmaceutically acceptable salts thereof.
• the present invention provides compounds which inhibit TTK activity, their use for treatment of hyper-proliferative disorders, in particular cancers that are caused by, or associated with chromosomal instability or aneuploidy, as a sole agent or in combination with other active ingredients, as well as pharmaceutical compositions comprising such compounds and pharmaceutical carriers.
• the object of the present invention is to provide (5,6-dihydro)pyrimido[4,5-e]indolizines, to pharmaceutical compositions comprising these compounds and their use in therapy.
• the present invention relates to the use of (5,6-dihydro)pyrimido[4,5-e]indolizines in the treatment of cancer.
• the present invention provides (5,6-dihydro)pyrimido[4,5-e]indolizines according to Formula I or pharmaceutically acceptable salts thereof.
• R 2 is selected from the group consisting of:
• R 2 is selected from a group consisting of:
• R 21 and R 25 are R 2 can be H.
• the attachment point is at the last group.
• substituted means that one or more hydrogens on the designated atom/atoms is/are replaced with a selection from the indicated group, provided that the designated atom's normal valency under the existing circumstances is not exceeded, and that the substitution results in a stable compound. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.
• “Stable compound” or “stable structure” is defined as a compound or structure that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.
• the invention relates to a compound according Formula I wherein R 13 is R 132 O, R 135 C(O), (2-7C)heterocycloalkyl or (1-5C)heteroaryl each heterocycloalkyl or heteroaryl optionally being substituted with (1-2C)alkyl, (1-6C)alkylcarbonyl, (1-6C)alkylsulfonyl, (1-5C)alkoxycarbonyl, (1-6C)alkylaminocarbonyl, (3-6C)cycloalkylcarbonyl or (2-7C)heterocycloalkylcarbonyl, each alkylcarbonyl, alkylsulfonyl, alkoxycarbonyl, alkylaminocarbonyl, cycloalkylcarbonyl or heterocycloalkylcarbonyl optionally being substituted with (1-2C)alkyl, fluoro, (1-2C)alkoxy; R 132 is selected from a group consisting of (1-6C)alkyl,
• the invention relates to a compound according to Formula I wherein R 13 is R 132 O, R 135 C(O); or R 13 is piperidinyl, piperazinyl, morpholinyl, pyrazolyl or isoxazolyl (each optionally being substituted with (1-2C)alkyl, (1-6C)alkylcarbonyl, (1-6C)alkylsulfonyl, (1-5C)alkoxycarbonyl, (1-6C)alkylaminocarbonyl, (3-6C)cycloalkylcarbonyl or (2-7C)heterocycloalkylcarbonyl each alkylcarbonyl, alkylsulfonyl, alkoxycarbonyl, alkylaminocarbonyl, cycloalkylcarbonyl or heterocycloalkylcarbonyl optionally being substituted with (1-2C)alkyl, fluoro or (1-2C)alkoxy.
• R 132 is selected from a group consisting of (1-6C)alkyl, piperidinyl, pyrrolidinyl or azetidinyl, each optionally being substituted with one or more groups selected from (1-2C)alkyl, (1-2C)alkoxy or di[(1-2C)alkyl]amino and R 135 is selected from a group consisting of piperidinyl, thiomorpholinyl, morpholinyl, homo-piperazinyl, (1-6C)alkylamino, (3-6C)cycloalkylamino or piperidinylamino, azetidinylamino, tetrahydropyranylamino or 3-oxabicyclo[3.1.0]hexan-6-amino, each optionally being substituted with one or more groups selected from (1-2C)alkyl, fluoro, hydroxyl or (1-2C)alkoxy, di[(1-2C)alkyl]amino, (2-7C)he
• the present invention relates to a compound according to Formula I wherein R 1 is selected from a group consisting of:
• the present invention relates to a compound according Formula I wherein R 12 and R 15 each are H and R 14 is H, fluoro, chloro or (1-2C)alkyl.
• the invention also relates to compounds according to Formula I wherein R 11 is H, (1-2C)alkyl or (1-2C)alkoxy, all alkyl and alkoxy groups optionally being substituted with one or more fluoro.
• the invention relates to a compound according to Formula I wherein R 2 is selected from group consisting of:
• the invention relates to a compound according to Formula I in which R 2 is:
• the invention relates to a compound according to Formula I wherein R 23 is H or (1-6C)alkyl and R 22 and R 24 each are H.
• the invention relates to a compound according to Formula wherein R 21 is selected from group consisting of H, halogen, (1-6C)alkyl or cyano.
• the invention relates to a compound according to Formula I wherein R 23 is H or (1-2C)alkyl and R 22 and R 24 each are H and R 21 and R 25 are independently selected from a group consisting of halogen, (1-3C)alkyl, methoxy, hydroxymethyl or cyano.
• the invention in another embodiment relates to a compound according to Formula I wherein R 26 is H, (1-6C)alkyl, oxetanyl, azetidinyl or (1-2C)alkoxy[(2-4C)alkoxy] n (1-6C)alkyl, wherein n represents an integer of 1 or 2, all alkyl, oxetanyl and azetidinyl groups optionally being substituted with one or more groups selected from (1-2C)alkyl, (1-2C)alkoxy, hydroxyl, di[(1-2C)alkyl]amino or oxetanyl.
• the invention also relates to those compounds wherein all specific definitions R 1 , R 2 , R 11-15 , R 21-26 and R 131-137 and all substituent groups in the various aspects of the inventions defined here above occur in any combination within the definition of the compound of the Formula I.
• the compounds of the invention have an inhibitory potency on TTK with an IC 50 of 10 ⁇ M or lower.
• the invention relates to compounds of Formula I which have an inhibitory potency on TTK with an IC 50 of less than 100 nM.
• the invention relates to compounds of Formula I which have an inhibitory potency on TTK with an IC 50 of less than 10 nM.
• IC 50 means the concentration of the test compound that is required for 50% inhibition of its maximum effect in vitro.
• IMAP Immobilized Metal Assay for Phosphochemicals
• FP fluorescence polarization
• IMAP uses fluorescein-labeled peptide substrates that, upon phosphorylation by a protein kinase, bind to so-called IMAP nanoparticles, which are derivatized with trivalent metal complexes. Binding causes a change in the rate of the molecular tumbling of the peptide, and results in an increase in the FP value observed for the fluorescein label attached to the substrate peptide (Gaudet, E. A. et al. J. Biomol. Screen 8: 164; 2003).
• TTK inhibitors can be measured, in proliferation assays with tumor cell lines.
• the activity of the compounds on tumor cells can also be determined in colony formation assays, and in the context of an animal model, in mice grafted with human or mouse cell lines or tumor tissue.
• the compounds of Formula I can form salts which are also within the scope of this invention.
• Reference to a compound of Formula I herein is understood to include reference to salts thereof, unless otherwise indicated.
• the term “salt(s)”, as employed herein, denotes acidic salts formed with inorganic and/or organic acids, as well as basic salts formed with inorganic and/or organic bases.
• zwitterions inner salts
• Pharmaceutically acceptable salts are preferred.
• Salts of the compounds of the Formula I may be formed, for example, by reacting a compound of formula I with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization.
• Exemplary acid addition salts include acetates, ascorbates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, fumarates, hydrochylorides, hydrobromides, hydroiodides, lactates, maleates, methanesulfonates, naphthanelesulfonates, nitrates, oxalates, phosphates, propionates, salicylates, succinates, sulfates, tartrates, thiocyanates, toluenesulfonates (also known as tosylates) and the like.
• Exemplary basic salts include ammonium salts, alkali metal salts such as sodium, lithium, and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases (for example, organic amines) such as dicyclohexylamines, tert-butyl amines, and salts with amino acids such as arginine, lysine and the like.
• alkali metal salts such as sodium, lithium, and potassium salts
• alkaline earth metal salts such as calcium and magnesium salts
• salts with organic bases for example, organic amines
• organic amines such as dicyclohexylamines, tert-butyl amines
• salts with amino acids such as arginine, lysine and the like.
• Basic nitrogen-containing groups may be quarternized with agents such as lower alkyl halides (e.g., methyl, ethyl, and butyl chlorides, bromides and iodides), dialkyl sulfates (e.g., dimethyl, diethyl, and dibutyl sulfates), long chain halides (e.g., decyl, lauryl, and stearyl chlorides, bromides and iodides), aralkyl halides (e.g., benzyl and phenethyl bromides), and others.
• lower alkyl halides e.g., methyl, ethyl, and butyl chlorides, bromides and iodides
• dialkyl sulfates e.g., dimethyl, diethyl, and dibutyl sulfates
• long chain halides e.g., decyl, lauryl, and
• the compounds of Formula I may have the ability to crystallize in more than one form, a characteristic known as polymorphism, and it is understood that such polymorphic forms (“polymorphs”) are within the scope of Formula I.
• Polymorphism generally can occur as a response to changes in temperature or pressure or both and can also result from variations in the crystallization process.
• Polymorphs can be distinguished by various physical characteristics known in the art such as x-ray diffraction patterns, solubility and melting point.
• the compounds of Formula I may contain asymmetric or chiral centers, and, therefore, exist in different stereoisomeric forms. It is intended that all stereoisomeric forms of the compounds of Formula I as well as mixtures thereof, including racemic mixtures, form part of the present invention.
• the present invention embraces all geometric and positional isomers. For example, if a compound of Formula I incorporates a double bond or a fused ring, both the cis- and trans-forms, as well as mixtures, are embraced within the scope of the invention.
• Diastereomeric mixtures can be separated into their individual diastereomers on the basis of their physical chemical differences by methods well known to those skilled in the art, such as, for example, by chromatography and/or fractional crystallization.
• Enantiomers can be separated by converting the enantiomeric mixture into a diastereomeric mixture by reaction with an appropriate optically active compound (e.g. chiral auxiliary such as a chiral alcohol or Mosher's acid chloride), separating the diastereomers and converting (e.g. hydrolyzing) the individual diastereomers to the corresponding pure enantiomers.
• an appropriate optically active compound e.g. chiral auxiliary such as a chiral alcohol or Mosher's acid chloride
• some of the compounds of Formula I may be atropisomers (e.g. substituted biaryls) and are considered as part of this invention.
• Enantiomers can also be separated by use of chiral a HPLC column.
• All stereoisomers (for example, geometric isomers, optical isomers and the like) of the present compounds including those of the salts, solvates, esters and prodrugs of the compounds as well as the salts, solvates and esters of the prodrugs), such as those which may exist due to asymmetric carbons on various substituents, including enantiomeric forms (which may exist even in the absence of asymmetric carbons), rotameric forms, atropisomeres, and diastereomeric forms, are contemplated within the scope of this invention.
• the chiral centers of the present invention can have the S or R configuration as defined by the IUPAC 1974 Recommendations.
• salt is intended to equally apply to the salt, solvate, ester and prodrug of enantiomers, stereoisomers, rotamers, tautomers, positional isomers, racemates or prodrugs of the compounds according to the invention.
• the compounds having Formula (I) or the pharmaceutically accepted salts may form hydrates or solvates. It is known to those of skill in the art that charged compounds from hydrated species when lyophilized with water, or form solvated species when concentrated in a solution with an appropriate organic solvent.
• the compounds of this invention include the hydrates or solvates of the compounds listed.
• the atoms may exhibit their natural isotopic abundances, or one or more of the atoms may be artificially enriched in a particular isotope having the same atomic number, but an atomic mass number different from the atomic mass or mass number predominantly found in nature.
• the present invention is meant to include all suitable isotopic variations of the compounds of Formula I.
• different isotopic forms of hydrogen (H) include protium ( 1 H) and deuterium ( 2 H).
• Protium is the predominant hydrogen isotope found in nature.
• Isotopically labelled compounds of Formula I can generally be prepared by following procedures analogous to those disclosed in the Schemes and/or in the Examples hereinbelow, by substituting an appropriate isotopically labeled reagent for a non-isotopically labeled reagent.
• the compounds of the present invention can be used for therapy.
• the compounds of the present invention can be used for the treatment of TTK-mediated diseases or conditions.
• the compounds of Formula I or their salts, and pharmaceutical compositions thereof can be used to treat diseases and conditions caused by or associated with overexpression or over-activity of the TTK protein, and/or abnormal expression, activity, or regulation of any regulators of TTK activity, or other regulators of the mitotic checkpoint, such as MAD1, MAD2, BUB1, BUBR1, BUB3 and others (Kops, G. J. P. L., et al.; Nature Rev. Cancer 5: 773; 2005).
• the compounds according to Formula I or pharmaceutically acceptable salts thereof can be used to treat diseases and conditions caused by or associated with overexpression or over-activity of the TTK protein.
• compounds according to Formula I or a pharmaceutically acceptable salt thereof can be used to treat hyperproliferative disorders.
• the invention thus relates to a method of regulating, modulating, or inhibiting TTK for the prevention and/or treatment of hyperproliferative disorders.
• the compounds of the present invention can be used for the treatment of diseases or conditions caused by abnormal cell proliferation, and/or diseases associated with chromosomal instability, chromosomal rearrangements, and/or aneuploidy.
• the compounds of the present invention can be used for the treatment of cancer, in particular for the treatment or prophylaxis of diseases caused by, or associated with uncontrolled cell growth, cell proliferation and/or cell survival.
• the compounds of the present invention can be used for the treatment of solid tumors, haematological tumors, and/or metastases thereof, e.g., mammary and gynaecological tumours, head and neck tumors, brain tumors and brain metastases, tumors of the thorax including non-small cell and small cell lung tumors, gastrointestinal tumors, endocrine tumors, urological tumors including renal, bladder and prostate tumors, skin tumor, and sarcomas, leukaemias and myelodysplastic syndrome, malignant lymphomas, and/or metastases thereof.
• solid tumors, haematological tumors, and/or metastases thereof e.g., mammary and gynaecological tumours, head and neck tumors, brain tumors and brain metastases, tumors of the thorax including non-small cell and small cell lung tumors, gastrointestinal tumors, endocrine tumors, urological tumors including renal, bladder and prostate tumors
• a further aspect of the invention resides in the use of a compound of Formula I or a pharmaceutically salt thereof for the manufacture of a medicament to be used to treat diseases and conditions caused by or associated with overexpression or over-activity of the TTK protein and for the treatment of disorders in which hyperproliferative cells play a prominent role.
• compositions in which at least one compound of Formula I or a pharmaceutically salt thereof is administered in combination with at least one other active agent.
• the other active agent can be a chemotherapeutic agent, an antibody, or an active polypeptide.
• the another aspect the invention concerns a compound of Formula I in combination with one or more other drug(s).
• the invention further provides a pharmaceutical composition, which comprises a compound of Formula I and salts thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients.
• the carrier(s), diluent(s) or excipient(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
• the active agent may be compressed into solid dosage units, such as pills, tablets, or be processed into capsules or suppositories.
• the active agent can be applied as a fluid composition, e.g. as an injection preparation, in the form of a solution, suspension, emulsion, or as a spray, e.g. a nasal spray.
• compositions of the present invention may be presented in unit dose forms containing a predetermined amount of active ingredient per unit dose.
• a unit may contain, for example 5 ⁇ g to 1 g, preferably 1 mg to 700 mg, more preferably 5 mg to 100 mg of a compound of the Formula I, depending on the condition being treated, the route of administration and the age, weight and condition of the patient.
• Such unit doses may therefore be adiministered more than once a day.
• Preferred unit dosage compositions are those containing a daily dose or sub-dose (for administration more than once a day), as here in above recited, or an appropriate fraction thereof, of an active ingredient.
• such pharmaceutical compositions may be prepared by any of the methods well known in the pharmacy art.
• compositions of the present invention may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, topical, inhaled, nasal, ocular, sublingual, subcutaneous, local or parenteral (including intravenous and intramuscular) route, and the like, all in unit dosage forms for administration.
• Such compositions may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s).
• Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, and the like.
• the compound of the present invention can also be administered as a protein-drug conjugate.
• the compound can be covalently bound, optionally with a linker molecule to a peptide or protein, such as a binding protein for example an antibody.
• the conjugate can be delivered to the target tissue. Methods to prepare such conjugates are well known to those skilled in the art.
• the compound of the prewsent invention when administered in combination with other therapeutic agents normally administered by the inhaled, intravenous, oral or intranasal route, that the resultant pharmaceutical composition may be administered by the same routes.
• a therapeutically effective amount of a compound of the present invention will depend upon a number of factors including, for example, the age and weight of the animal, the precise condition requiring treatment and its severity, the particular compound having Formula I, the nature of the formulation, and the route of administration, and will ultimately be at the discretion of the attendant physician or veterinarian.
• an effective amount of a compound of Formula I for the treatment of diseases or conditions associated with inappropriate TTK activity will generally be in the range of 5 ⁇ g to 100 mg/kg body weight of recipient (mammal) per day and more usually in the range of 5 ⁇ g to 10 mg/kg body weight per day.
• This amount may be given in a single dose per day or more usually in a number (such as two, three, four, five or six) of sub-doses per day such that the total daily dose is the same.
• An effective amount of a salt or solvate, thereof, may be determined as a proportion of the effective amount of the compound of Formula I per se.
• a dosage for humans preferably contains 0.001-25 mg of a compound of Formula I or pharmaceutically salts thereof per kg body weight.
• the desired dose may be presented as one dose or as multiple sub-doses administered at appropriate intervals throughout the day, or, in case of female recipients, as doses to be administered at appropriate daily intervals throughout the menstrual cycle.
• the dosage, as well as the regiment of administration, may differ between a female and a male recipient.
• the present invention also relates to a pharmaceutical composition
• a pharmaceutical composition comprising a compound of Formula I or pharmaceutically salt thereof in a mixture with pharmaceutically acceptable auxiliaries and optionally other therapeutic agents.
• the auxiliaries must be “acceptable” in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipients thereof.
• the invention further includes a pharmaceutical composition comprising at least one compound of Formula I or pharmaceutically salts thereof in combination with at least one other therapeutically active agent.
• a compound of Formula I may be combined with one or more anticancer agents.
• anticancer agents can be found in Cancer Principles and Practice of Oncology by V. T. Devita and S. Hellman (editors), 6 th edition (Feb. 15, 2001), Lippincott Williams & Wilkins Publishers. A person of ordinary skill in the art would be able to discern which combinations of agents would be useful based on the particular characteristics of the drugs and the cancer involved.
• the (5,6-dihydro)pyrimido[4,5-e]indolizine derivatives of the present invention can be prepared by methods well known in the art of organic chemistry. See, for example, J. March, ‘Advanced Organic Chemistry’ 4 th Edition, John Wiley and Sons. During synthetic sequences it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules concerned. This is achieved by means of conventional protecting groups, such as those described in T. W. Greene and P. G. M. Wutts ‘Protective Groups in Organic Synthesis’ 3 rd Edition, John Wiley and Sons, 1999. The protective groups are optionally removed at a convenient subsequent stage using methods well known in the art.
• the products of the reactions are optionally isolated and purified, if desired, using conventional techniques, including but not limited to, filtration, distillation, crystallization, chromatography and the like. Such materials are optionally characterized using conventional means, including physical constants and spectral data.
• 5-Bromo-2-chloro-pyrimidin-4-amine (II) can be prepared from commercial available 5-bromo-2,4-dichloro-pyrimidine using 25% aqueous ammonia at ambient temperature. The resulting product can then be reacted with sodium methanolate in methanol at elevate temperatures to obtain 5-bromo-2-methoxy-pyrimidin-4-amine (III).
• Compound IV can, subsequently, be prepared from compound III using ethylacrylate in the presence of a suitable palladium catalyst system, for example palladium(II) acetate, an organic base like triethylamine or inorganic base like potassium carbonate, cesium carbonate or potassium phosphate in a suitable solvent system like combinations of dioxane and water or dimethylformamide.
• a suitable palladium catalyst system for example palladium(II) acetate, an organic base like triethylamine or inorganic base like potassium carbonate, cesium carbonate or potassium phosphate in a suitable solvent system like combinations of dioxane and water or dimethylformamide.
• Reduction of the double bond and subsequent cyclisation can be accomplished by hydrogenation in the presence of a suitable catalyst system and solvent, for example palladium on charcoal in methanol to provide lactam V.
• Ethylester VI can be prepared from lactam V using [1-ethoxycarbonyl)cyclopropyl]tris(phenyl)phosphonium tetrafluoroborate in THF and a suitable base such as sodium hydride. Oxidation of VI can be accomplished using oxidizing reagents such as manganese-oxide or lead(IV) acetate to provide derivative VII. Compound VII can be converted to derivative IX, after appropriate deprotection with trimethylsilyliodide and subsequent conversion using phosphorus(V) oxychloride under heating conditions.
• R 1 NH 2 substituted with ethyl 2-chloro-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxylate with R 1 NH 2 can be carried out under acidic conditions using trifluoroacetic acid or concentrated hydrogen chloride solution and an appropriate solvent like n-butanol or isopropanol under microwave radiation.
• R 1 NH 2 can be introduced in the presence of a suitable palladium catalyst system, for example palladium(II)acetate or tetrakis(triphenylphosphine)palladium(0) in the presence of an inorganic base such as potassium carbonate, cesium carbonate or potassium phosphate in a suitable solvent system like dioxane and water to generate derivative X.
• a suitable palladium catalyst system for example palladium(II)acetate or tetrakis(triphenylphosphine)palladium(0) in the presence of an inorganic base such as potassium carbonate, cesium
• Oxidation of VII can be performed using oxidizing reagents like manganese-oxide, oxygen or DDQ to provide derivative XI.
• Compound XI can be converted to chloride XIII, after appropriate deprotection with trimethylsilyliodide and subsequent conversion using phosphorus(V) oxychloride under heating conditions.
• Substitution of ethyl 2-chloro-pyrimido[4,5-e]indolizine-7-carboxylate with R 1 NH 2 can be carried out under acidic conditions using trifluoroacetic acid or concentrated hydrogen chloride solution and an appropriate solvent like n-butanol or isopropanol under microwave radiation.
• R 1 NH 2 can be introduced in the presence of a suitable palladium catalyst system, for example palladium(II) acetate or tetrakis(triphenylphosphine)palladium(0) in the presence of an inorganic base such as potassium carbonate, cesium carbonate or potassium phosphate in a suitable solvent system like dioxane and water to generate derivative XIV.
• a suitable palladium catalyst system for example palladium(II) acetate or tetrakis(triphenylphosphine)palladium(0)
• an inorganic base such as potassium carbonate, cesium carbonate or potassium phosphate
• conversion of derivative XIV to compounds with Formula I can be accomplished either first by saponification of the ester functionality of compound XIV and subsequent condensation to the amide, using methods well known in the art, or through aminolysis of the ester functionality using a strong base such as lithium bis(trimethylsilyl)amide.
• Method LCMS (B) Method LCMS (B) NTRC_C18.M Method name Column Waters XTerra C18-MS, 50 ⁇ 4.6 mm ID, 2.5 ⁇ m Flow 0.5 ml/min. Temperature 40° C.
• N,N-dimethylaniline (182 mg, 191 uL, 1.50 mmol) was added to a solution of ethyl 2-hydroxy-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxylate (3.89 g, 15.0 mmol) in acetonitrile (100 mL).
• a solution of phosphorus(V) oxychloride (11.5 g, 7.00 mL, 75.0 mmol) in acetonitrile (15 mL) was added dropwise to the reaction mixture.
• the yellow suspension was heated for 4 hours to 65° C. during which the suspension turned into a clear solution. After cooling, the mixture was slowly poured in a stirred mixture of 25% aq.
• tert-Butyl N-tert-butoxycarbonyl-N-[2-chloro-4-methylpiperazin-1-yl)phenyl]carbamate (260 mg; 0.61 mmol) was dissolved in DCM (4 mL). TFA (4 mL) was added and the reaction mixture was stirred for 1 hour at room temperature. The mixture was concentrated and the residue was dissolved in DCM (10 mL) and poured into a 5% sodium bicarbonate solution (10 mL). The water layer was extracted with DCM (2 ⁇ 10 mL). The combined organic layers were filtered over a PE-filter and concentrated in vacuo to give a brown oil (105 mg, 76%) that was used without further purification.
• Diphenylphosphoryl azide (2.51 mL, 11.6 mmol) was added to a solution of 3-ethyl-5-methyl-isoxazole-4-carboxylic acid (1.5 g, 9.67 mmol), triethylamine (2.7 mL, 19.3 mmol), and tert-butylalcohol (0.92 mL, 9.67 mmol) in toluene (50 mL) and stirred for 4 h at 100° C. The solvent was removed by evaporation and the residue taken up in EtOAc (50 mL). The organic layer washed with water and brine, dried over sodium sulfate, filtered and concentrated in vacuo.
• Ethyl 2-chloro-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxylate (Intermediate 1, 200 mg, 0.72 mmol), 4-amino-N-(2-hydroxy-2-methyl-propyl)-3-methoxy-benzamide (Intermediate A, 172 mg, 0.72 mmol) and cesium carbonate (937 mg; 2.89 mmol) were suspended in dioxane (20 mL). Nitrogen was bubbled through the mixture at 30° C.
• LiHDMS (1M in THF/ethylbenzene, 0.61 mL, 0.61 mmol) was added to a cold (0° C.) solution of 2,6-dimethylaniline (38.5 ⁇ L, 0.31 mmol) in THF (1 mL).
• ethyl 2-[4-[(2-hydroxy-2-methyl-propyl)carbamoyl]-2-methoxy-anilino]-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxylate 50 mg, 0.10 mmol
• THF 3 mL
• This compound was prepared from its corresponding acid chloride, using the same sequence of reactions, as described for Intermediate 2, using commercially available 4-(4-methylpiperazino)aniline as starting material.
• the acid chloride was subsequently reacted with 6-ethyl-o-toluidine according to procedures described in Example 2. Purification was performed using preparative HPLC to afford the title compound (18 mg, 58%). Data: LCMS (B) R t : 10.456 min; m/z 522.3 (M+H) + .
• This compound was prepared from its corresponding acid chloride, using the same sequence of reactions as described for Intermediate 2, using commercially available 4-bromo-2-methoxyaniline as starting material.
• the acid chloride was subsequently reacted with 2,6-dimethylaniline according to procedures described in Example 2. Purification was performed using preparative HPLC to afford the title compound.
• N,N-Dimethylaniline (47 mg, 50 ⁇ L, 1.50 mmol) was added to a solution of ethyl 2-hydroxypyrimido[4,5-e]indolizine-7-carboxylate (1.0 g, 3.89 mmol) in acetonitrile (30 mL).
• a solution of phosphorus(V) oxychloride (2.99 g, 1.81 mL, 19.5 mmol) in acetonitrile (4 mL) was added dropwise to the reaction mixture.
• the brown/red suspension was heated to 65° C. for 4 hours. After cooling, the mixture was slowly poored in a stirred mixture of 25% aq.
• This compound was prepared from its corresponding ethyl ester, using the same sequence of reactions as described for Example 1, starting from Intermediate 3 and Intermediate H as starting material.
• the ethyl ester was subsequently reacted with 2,6-dimethylaniline according to procedures described in Example 1.
• purification was performed using preparative HPLC to afford the title compound (1.4 mg, 5%).
• This compound was prepared from its corresponding ethyl ester, using the same sequence of reactions as described for Example 1, starting from Intermediate 1 and Intermediate H as starting material.
• the ethyl ester was subsequently reacted with 2,6-diethylaniline according to procedures described in Example 1.
• purification was performed using preparative HPLC to afford the title compound (20.7 mg, 72%).
• This compound was prepared from its corresponding bromide, using the same sequence of reactions, as described for Example 38-a, using 4-bromo-2-ethylaniline as starting material. The bromide was subsequently reacted with N-methylpiperazine according to procedures described in Example 38-b. Purification was performed using preparative HPLC to afford the title compound (6.5 mg, 15%). Data: LCMS (B) R t : 12.136 min; m/z 564.4 (M+H) + .
• This compound was prepared from its corresponding acid chloride, using the same sequence of reactions, as described for Example 60, using N,N,N′-trimethyl-1,3-propanediamine as starting material. The acid chloride was subsequently reacted with Intermediate Ae according to procedures described in Example 2. Purification was performed using preparative HPLC to afford the title compound (13.8 mg, 20%). Data: LCMS (B) R t : 10.220 min; m/z 658.4 (M+H) + .
• This compound was prepared from its corresponding acid chloride, using the same sequence of reactions, as described for Intermediate 2, using commercially available 4-bromo-2-methoxyaniline as starting material.
• the acid chloride was subsequently reacted with 2,6-dimethylaniline according to procedures described in Example 2 to afford the title compound (1.35 g, 84%).
• This compound was prepared from its corresponding carboxylic acid (Example 103-c) and tert-butyl (3S,4R)-4-amino-3-fluoro-piperidine-1-carboxylate, using standard HATU-coupling procedures as described in Example 9. After deprotection of the Boc-group, purification was performed using preparative HPLC to afford the title compound (2.2 mg, 5%). Data: LCMS (B) R t : 11.671 min; m/z 584.3 (M+H) + .
• This compound was prepared from its corresponding carboxylic acid (Example 103-c) and 1-N-Boc-cis-1,4-cyclohexyldiamine hydrochloride, using standard HATU-coupling procedures as described in Example 9. After deprotection of the Boc-group, purification was performed using preparative HPLC to afford the title compound (4.6 mg, 15%). Data: LCMS (B) R t : 11.581 min; m/z 580.3 (M+H) + .
• IMAP® assay (Molecular Devices) was used. Compounds were serially diluted in dimethylsulfoxide (DMSO) and subsequently in 4% DMSO in IMAP reaction buffer, which consists of 10 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , 0.01% Tween-200, 0.1% NaN 3 and 1 mM freshly prepared dithiotreitol (DTT). Compound solution was mixed with an equal volume of full-length TTK enzyme (Life Technologies, Cat. no. PV 3792) in IMAP reaction buffer.
• DMSO dimethylsulfoxide
• IMAP reaction buffer which consists of 10 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , 0.01% Tween-200, 0.1% NaN 3 and 1 mM freshly prepared dithiotreitol (DTT).
• DTT dithiotreitol
• the MOLT-4 cancer cell line was purchased from the American Type Culture Collection (ATCC, Manassas, Va., U.S.A.) and cultured in RPMI 1640 medium (LifeTechnologies, Bleiswijk, The Netherlands), supplemented with 10% bovine calf serum. Compounds were serially diluted in 3.16 fold steps in 100% DMSO, followed by further dilution in aqueous buffer. Cells in medium were seeded at 45 ⁇ L per well in the wells of a 384-well plate, and incubated for 24 hours in a humidified atmosphere of 5 % CO 2 at 37° C.
• ATPlite 1StepTM PerkinElmer, Groningen, The Netherlands
• Luminescence was recorded on an Envision multimode reader. The cell signal at the start of incubation was recorded separately in order to distinguish between cell population growth and cell death.
• maximum growth was determined by incubation of a duplicate without compound in the presence of 0.4% DMSO. Percentage growth was used as the main y-axis signal.
• IC 50 s were fitted by non-linear regression using IDBS XLfitTM5 using a 4-parameter logistic curve, yielding a maximum signal, minimum signal, hill-parameter and IC 50 .
• IC 50 values of all exemplified compounds were found to be smaller than 300 nM.
• Compounds of examples 3, 6, 7, 10, 11, 14-16, 20, 23-29, 31, 32, 34, 36, 38, 44, 52, 53, 77, 88, 90-92, 98, 102, 104, 128, 131, 132, 136, 140 and 142 showed an IC 50 value ⁇ 50 nM- ⁇ 150 nM and compounds of examples 1, 2, 4, 5, 9, 13, 17-19, 21, 22, 33, 35, 37, 39-43, 45-51, 56, 58-61, 62-76, 78-84, 86, 93-97, 99, 100, 103, 105, 107, 109-114, 116-124, 126, 127, 129, 130, 133-135, 137, 138, 141, 144 and 145 showed an IC 50 of ⁇ 50 nM.
• LANCE® Ultra TR-FRET assay Perkin Elmer
• Compounds were serially diluted in dimethylsulfoxide (DMSO) and subsequently in 4% DMSO in LANCE® kinase buffer, which consists of 50 mM Hepes, pH 7.5, 10 mM MgCl 2 , 1 mM EGTA, 0.01% Tween-20, and 2 mM dithiotreitol (DTT).
• DMSO dimethylsulfoxide
• LANCE® kinase buffer which consists of 50 mM Hepes, pH 7.5, 10 mM MgCl 2 , 1 mM EGTA, 0.01% Tween-20, and 2 mM dithiotreitol (DTT).
• 2.5 ⁇ l of compound solution was mixed with an equal volume of full-length Aurora A enzyme (Carna Biosciences, cat. no. 05-101) in LANCE® kinase buffer.
• Time-resolved fluorescence was measured on an Envision multilabel reader (Perkin Elmer, Waltham, Mass., USA).
• IC 50 were calculated using XLfitTM5 software (ID Business Solutions, Ltd., Surrey, U.K.).
• Compounds of examples 33, 138, 141, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159 and 160 showed an IC 50 value ⁇ 100 nM.
• LANCE® Ultra TR-FRET assay Perkin Elmer
• Compounds were serially diluted in dimethylsulfoxide (DMSO) and subsequently in 4% DMSO in LANCE® kinase buffer, which consists of 50 mM Hepes, pH 7.5, 10 mM MgCl 2 , 1 mM EGTA, 0.01% Tween-20, and 2 mM dithiotreitol (DTT).
• DMSO dimethylsulfoxide
• LANCE® kinase buffer which consists of 50 mM Hepes, pH 7.5, 10 mM MgCl 2 , 1 mM EGTA, 0.01% Tween-20, and 2 mM dithiotreitol (DTT).
• 2.5 ⁇ l of compound solution was mixed with an equal volume of full-length Aurora C enzyme (Carna Biosciences, cat. no. 05-103) in LANCE® kinase buffer.
• TRF-0203 was added and the incubation was continued for 1 hour in the dark at room temperature. Time-resolved fluorescence was measured on an Envision multilabel reader (Perkin Elmer, Waltham, Mass., USA). IC 50 were calculated using XLfitTM5 software (ID Business Solutions, Ltd., Surrey, U.K.).
• LANCE® Ultra TR-FRET assay Perkin Elmer
• DMSO dimethylsulfoxide
• LANCE® kinase buffer which consists of 50 mM Hepes, pH 7.5, 10 mM MgCl 2 , 1 mM EGTA, 0.01% Tween-20, and 2 mM dithiotreitol (DTT).
• DTT dithiotreitol
• TRF-0214 was added and the incubation was continued for 1 hour in the dark at room temperature. Time-resolved fluorescence was measured on an Envision multilabel reader (Perkin Elmer, Waltham, Mass. USA). IC 50 were calculated using XLfitTM5 software (ID Business Solutions, Ltd., Surrey, U.K.).
• AurA Selectivity means IC 50 AurA/IC 50 TTK (biochemical assays)
• AurC Selectivity means IC 50 AurC/IC 50 TTK (biochemical assays)
• c PLK1 Selectivity means IC 50 PLK1/IC 50 TTK (biochemical assays)

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