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WO2008038011A1 - DÉrivÉs de la pyrimidine servant d'inhibiteurs de l'aurora A et de l'aurora B - Google Patents

DÉrivÉs de la pyrimidine servant d'inhibiteurs de l'aurora A et de l'aurora B Download PDF

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Publication number
WO2008038011A1
WO2008038011A1 PCT/GB2007/003688 GB2007003688W WO2008038011A1 WO 2008038011 A1 WO2008038011 A1 WO 2008038011A1 GB 2007003688 W GB2007003688 W GB 2007003688W WO 2008038011 A1 WO2008038011 A1 WO 2008038011A1
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Prior art keywords
alkyl
compound
piperidin
hydroxy
nitrogen atom
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PCT/GB2007/003688
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English (en)
Inventor
Lee David Walmsley
Martin James Drysdale
Christopher Graham
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Vernalis (R & D) Limited
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Publication of WO2008038011A1 publication Critical patent/WO2008038011A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • 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
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings

Definitions

  • This invention relates to substituted pyrimidine compounds having Aurora A and Aurora B inhibitory activity, to the use of such compounds in medicine, in relation to the treatment of disorders which are responsive to inhibition of Aurora A and Aurora B such as cancer, and to pharmaceutical compositions containing such compounds.
  • Aurora family of serine/threonine protein kinases are critical for proper regulation of mitosis in many organisms. They play a key role in diverse cell cycle events such as entry to mitosis, centrosome function, mitotic spindle formation, chromosome segregation and cytokinesis. Overexpression of Aurora kinases occur in a wide range of human tumours and have been implicated in human tumourigenesis. Mammals express three Aurora Kinase paralogues and at least two (Aurora A and Aurora B) are commonly overexpressed in human tumours. 1
  • the present invention relates to a class of substituted pyrimidine compounds useful as inhibitors of Aurora A and Aurora B, for example, for the treatment of cancer.
  • a core amino pyrimidine ring substituted on the heterocyclic ring with an oxymethyl piperidine is a principal characterising feature of the compounds with which the invention is concerned.
  • Re is hydrogen, C1-C3 alkyl, or fluoro(CrC 3 )alkyl
  • R 7 is Ci-C 3 alkyl, hydroxy-(Ci-C 6 )alkyl, hydroxy-(Ci-C 6 )alkyl substituted on the alkyl portion by phenyl, C 1 -C 3 alkoxy ⁇ (Ci-C 3 )alkyl, halo(Ci-C 4 )alkyl, or C 3 - C 6 cycloalkyl;
  • R 6 and R 7 taken together with the nitrogen atom to which they are attached form an optionally substituted monocyclic 5- or 6-membered heterocyclic ring;
  • Rs is selected from hydrogen, C 1 -C 3 alkyl, fluoro(Ci-C 3 )alkyl, or a radical of formula -AIk-N(Rg)-R 10 ;
  • R 9 and R10 are independently selected from hydrogen, C 1 -C 3 alkyl, or
  • Rg and R10 taken together with the nitrogen atom to which they are attached form an optionally substituted monocyclic 5- or 6-membered heterocyclic ring;
  • -AIk- is a divalent (Ci-C 4 )alkylene radical
  • X is halogen, cyano or triflouromethyl
  • Z is O or S
  • Y is selected from -NH-, -N(Ci-C 3 alkyl)-, -(CH 2 )-, or is absent;
  • Ar is aryl or heteroaryl, optionally substituted with one or more halogen atoms, C1-C3 alkyl radicals or trifluoromethyl radicals.
  • the active compounds of formula (I) are inhibitors of Aurora Kinases, both A and B paralogues, and are useful for the treatment, prevention and suppression of diseases mediated by Aurora Kinases.
  • the invention is concerned with the use of these compounds to selectively inhibit Aurora Kinases and, as such, in the treatment of cancer.
  • carboxy refers to a group of formula -COOH.
  • (C a -C b )alkyl wherein a and b are integers refers to a straight or branched chain alkyl radical having from a to b carbon atoms.
  • a 1 and b is 6, for example, the term includes methyl, ethyl, n- propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl and n-hexyl.
  • divalent (C a -C b )alkylene radical wherein a and b are integers refers to a saturated hydrocarbon chain having from a to b carbon atoms and two unsatisfied valences.
  • cycloalkyl refers to a saturated carbocyclic radical having from 3-8 carbon atoms and includes, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.
  • aryl refers to a mono-, bi- or tri-cyclic carbocyclic aromatic radical. Illustrative of such radicals are phenyl, biphenyl and napthyl.
  • Carbocyclic refers to a cyclic radical whose ring atoms are all carbon, and includes monocyclic aryl, cycloalkyl and cycloalkenyl radicals.
  • heteroaryl refers to a mono-, bi- or tri-cyclic aromatic radical containing one or more heteroatoms selected from S, N and O.
  • Illustrative of such radicals are thienyl, benzthienyl, furyl, benzfuryl, pyrrolyl, imidazolyl, benzimidazolyl, thiazolyl, benzthiazolyl, isothiazolyl, benzisothiazolyl, pyrazolyl, oxazolyl, benzoxazolyl, isoxazolyl, benzisoxazolyl, isothiazolyl, triazolyl, benztriazolyl, thiadiazolyl, oxadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, indolyl and indazolyl.
  • heterocyclyl or “heterocyclic” includes “heteroaryl” as defined above, and in particular means a mono-, bi- or tricyclic non-aromatic radical containing one or more heteroatoms selected from S, N and O, and to groups consisting of a monocyclic non-aromatic radical containing one or more such heteroatoms which is covalently linked to another such radical or to a monocyclic carbocyclic radical.
  • radicals are pyrrolyl, furanyl, thienyl, piperidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, pyrazolyl, pyridinyl, pyrrolidinyl, pyrimidinyl, morpholinyl, piperazinyl, indolyl, morpholinyl, benzfuranyl, pyranyl, isoxazolyl, benzimidazolyl, methylenedioxyphenyl, ethylenedioxyphenyl, maleimido and succinimido groups.
  • substituted as applied to any moiety herein means substituted with up to four compatible substituents, each of which independently may be, for example, (Ci-C 6 )alkyl, (Ci-C 6 )alkoxy, hydroxy, hydroxy(C- ⁇ -C 6 )alkyl, mercapto, mercapto(Ci-C 6 )alkyl, (Ci-C 6 )alkylthio, halo (including fluoro, bromo and chloro), trifluoromethyl, trifluoromethoxy, nitro, nitrile (-CN), oxo, phenyl, - COOH, -COOR A , -COR A , -SO 2 R A , -CONH 2 , -SO 2 NH 2 , -CONHR A , -SO 2 NHR A , -CONR A R B , -SO 2 NR A R B
  • an “optional substituent” may be one of the foregoing substituent groups.
  • substituents Ci-C 6 )alkyl, halo, trifluoromethyl, trifluoromethoxy, trifluoromethylsulfonyl, and phenyl are those most commonly regarded as lipophilic.
  • substituents listed which contain alkyl groups may be lipophilic depending on the particular alkyl groups present.
  • salt includes base addition, acid addition and quaternary salts.
  • Compounds of the invention which are acidic can form salts, including pharmaceutically or veterinarily acceptable salts, with bases such as alkali metal hydroxides, e.g. sodium and potassium hydroxides; alkaline earth metal hydroxides e.g. calcium, barium and magnesium hydroxides; with organic bases e.g. N-methyl-D-glucamine, choline tris(hydroxymethyl)amino- methane, L-arginine, L-lysine, N-ethyl piperidine, dibenzylamine and the like.
  • bases such as alkali metal hydroxides, e.g. sodium and potassium hydroxides; alkaline earth metal hydroxides e.g. calcium, barium and magnesium hydroxides; with organic bases e.g. N-methyl-D-glucamine, choline tris(hydroxymethyl)amino- methane, L-arginine, L-lysine, N-
  • Those compounds (I) which are basic can form salts, including pharmaceutically or veterinarily acceptable salts with inorganic acids, e.g. with hydrohalic acids such as hydrochloric or hydrobromic acids, sulphuric acid, nitric acid or phosphoric acid and the like, and with organic acids e.g. with acetic, tartaric, succinic, fumaric, maleic, malic, salicylic, citric, methanesulphonic, p-toluenesulphonic, be4nzoic, benzenesunfonic, glutamic, lactic, and mandelic acids and the like.
  • hydrohalic acids such as hydrochloric or hydrobromic acids, sulphuric acid, nitric acid or phosphoric acid and the like
  • organic acids e.g. with acetic, tartaric, succinic, fumaric, maleic, malic, salicylic, citric, methanesulphonic, p-toluene
  • lipophilic as used herein in relation to a substituent means that it has a positive substituent hydrophobicity constant (D).
  • D hydrophobicity constant
  • 'solvate' is used herein to describe a molecular complex comprising the compound of the invention and a stoichiometric amount of one or more pharmaceutically acceptable solvent molecules, for example, ethanol.
  • solvent molecules for example, ethanol.
  • 'hydrate' is employed when said solvent is water.
  • Compounds with which the invention is concerned which may exist in one or more stereoisomeric form, because of the presence of asymmetric atoms or rotational restrictions, can exist as a number of stereoisomers with R or S stereochemistry at each chiral centre or as atropisomeres with R or S stereochemistry at each chiral axis.
  • the invention includes all such enantiomers and diastereoisomers and mixtures thereof.
  • So-called 'pro-drugs' of the compounds of formula (I) are also within the scope of the invention.
  • certain derivatives of compounds of formula (I) which may have little or no pharmacological activity themselves can, when administered into or onto the body, be converted into compounds of formula (I) having the desired activity, for example, by hydrolytic cleavage.
  • Such derivatives are referred to as 'prodrugs'.
  • Further information on the use of prodrugs may be found in Pro-dru ⁇ s as Novel Delivery Systems. Vol. 14, ACS Symposium Series (T. Higuchi and W. Stella) and Bioreversible Carriers in Drug Design. Pergamon Press, 1987 (ed. E. B. Roche, American Pharmaceutical Association).
  • Prodrugs in accordance with the invention can, for example, be produced by replacing appropriate functionalities present in the compounds of formula (I) with certain moieties known to those skilled in the art as 'pro-moieties' as described, for example, in Design of Prodrugs by H. Bundgaard (Elsevier, 1985).
  • metabolites of compounds of formula (I), that is, compounds formed in vivo upon administration of the drug are also included within the scope of the invention.
  • Some examples of metabolites include
  • R 1 is selected from - AIk- N(Re)-R 7 , or -O-Alk-N(R 6 )-R 7 .
  • AIk is a divalent (Ci-C 4 )alkylene radical
  • R 6 is hydrogen or C 1 -C 3 alkyl
  • R 7 is Ci-C 3 alkyl, hydroxy-(Ci- Ce)alkyl, hydroxy-(C-i-C 6 )alkyl substituted on the alkyl portion by phenyl, Ci-C 3 alkoxy-(Ci-C 3 )alkyl, halo C 1 -C 4 alkyl, or C 3 -C 6 cycloalkyl; or alternatively R 6 and R 7 taken together with the nitrogen atom to which they are attached form an optionally substituted monocyclic 5- or 6-membered heterocyclic ring.
  • AIk is methylene, -(CH 2 ) 2 - or -(CH 2 ) 3 -, R 6 is hydrogen, methyl or ethyl, and R 7 is methyl, ethyl, 2-hydroxyethyl, 2-methoxyethyl, 2,2,2- trifluoroethyl, cyclopentyl, -CH(IPr)-CH 2 -OH Or-CH(Ph)-CH 2 -OH.
  • R 6 and R 7 taken together with the nitrogen atom to which they are attached form an optionally substituted monocyclic 6-membered heterocyclic ring, with morpholinyl, piperidinyl or piperazinyl optionally substituted by hydroxy methyl, fluoro, hydroxy, methyl, 2-hydroxyethyl, or trifluoromethyl presently preferred.
  • AIk is methylene or -(CH 2 ) 2 -
  • R 6 is hydrogen, methyl or ethyl
  • R 7 is 2-hydroxyethyl, 2-methoxyethyl, - CH(JPr)-CH 2 -OH Or-CH(Ph)-CH 2 -OH
  • AIk is methylene or -(CH 2 ) 2 -, and R 6 and R 7 taken together with the nitrogen atom to which they are attached form 1 -fluoro-piperidin-3-yl, 1 -hydroxy-piperidin-3-yl, 1 -hydroxymethyl-piperidin-3- yl, 1-hydroxymethyl-piperidin-4-yl, 1 -methyl-piperidin-3-yl, 1-(2-hydroxyethyl)- piperidin-3-yl, or 1-trifluoromethyl-piperidin-3-yl.
  • R 8 is selected from hydrogen, Ci-C 3 alkyl or a radical of formula -AIk-N(Rg)-RiO, wherein AIk is a divalent (C 1 -C 4 )alkylene radical and R 9 and R 1 O are independently selected from hydrogen or Ci-C 3 alkyl; or Rg and R 10 taken together with the nitrogen atom to which they are attached form an optionally substituted monocyclic 5- or 6-membered heterocyclic ring.
  • R 8 is a radical of formula -AIk- N(Rg)-R 10 , wherein AIk is methylene or -(CH 2 ) 2 -, and Rg and Ri 0 taken together with the nitrogen atom to which they are attached form optionally substituted piperidinyl or pyrrolidinyl.
  • Particulary preferred compounds are those wherein AIk is methylene or -(CH 2 ) 2 -, and R 9 and R 10 taken together with the nitrogen atom to which they are attached form piperidin-1-yl, 1- methyl-piperidin-2-yl, 1-hydroxy-piperidin-4-yl, or 1 -ethyl-pyrrolidin-2-yl.
  • Ri is selected from hydroxy(Ci-C 3 )alkyl. It is presently preferred that Ri is hydroxymethyl or 2- hydroxyethyl.
  • Ri is selected from carboxy(Ci-C 3 )alkyl. It is presently preferred that Ri is carboxymethyl or 2- carboxyethyl.
  • R2, R3, R 4 and R 5 are independently selected as for R 1 .
  • R 1 , R 2 , R 3 , R 4 and R 5 generally at least three are hydrogen. Preferred cases are wherein Ri, R 2 and R 5 are hydrogen; R-i, R 2 , R 4 and R5 are hydrogen; or R 1 , R 2 , R 3 and R 5 are hydrogen. Particularly preferred cases are wherein R 1 , R 2 , R 4 and R 5 are hydrogen; or Ri, R 2 , R3 and R 5 are hydrogen.
  • Group X X is halogen, cyano or triflouromethyl. Preferred structures are those wherein X is halogen, with fluoro particularly preferred.
  • Z is oxygen or sulphur and Y is selected from -NH-, -N(Ci-C 3 alkyl)-, -(CH 2 )-, or is absent.
  • Z is oxygen and Y is selected from -NH- , -(CH 2 )-, or is absent.
  • preferred compounds Z is oxygen and Y is nitrogen.
  • Ar is aryl or heteroaryl, optionally substituted with one or more halogen atoms, C 1 -C 3 alkyl radicals or trifluoromethyl radicals. It is presently preferred that aryl is phenyl and heteroaryl is pyridyl or N-oxido-pyridyl. Particularly preferred structures are those wherein Ar is 3-fluorophenyl, 3,4-difluorophenyl, 3,5- difluorophenyl, 2,4 difluorophenyl, 3-chlorophenyl or 3-methylphenyl.
  • R 3 is hydrogen or C 1 -C 3 alkyl
  • R 4 is C- 1 -C 3 alkyl, hydroxy-(Ci-C 6 )alkyl, hydroxy-(Ci-C- 6 )alkyl substituted on the alkyl portion by phenyl, C 1 -C 3 alkoxy-(CrC 3 )alkyl, halo C 1 -C 4 alkyl, or C 3 -C 6 cycloalkyl;
  • R 3 and R 4 taken together with the nitrogen atom to which they are attached form an optionally substituted monocyclic 6-membered heterocyclic ring;
  • R 5 and R 6 taken together with the nitrogen atom to which they are attached form an optionally substituted monocyclic 5- or 6-membered heterocyclic ring;
  • Ar 1 is -1 ,3-phenylene or -1 ,4-phenylene
  • Y is -NH-, -CH 2 -, or is absent
  • Ar 2 is halo- or C 1 -C 3 alkyl- substituted phenyl.
  • a method of treatment of a disorder mediated by Aurora Kinases comprising administration to a subject in need of such treatment an effective dose of the compound of formula (I), or a pharmaceutically acceptable salt or prodrug thereof.
  • the present invention is particularly directed to a hyperproliferative disease such as cancer, wherein the cancer is colorectal, breast, lung, prostate, bladder, renal or pancreatic cancer, or leukaemia or lymphoma.
  • the present invention may be employed in respect of a human or animal subject, more preferably a mammal, more preferably a human subject.
  • treatment includes prophylactic treatment.
  • the compound of formula (I) may be used in combination with one or more additional drugs useful in the treatment of the disorders mentioned above, the components being in the same formulation or in separate formulations for administration simultaneously or sequentially.
  • a suitable dose for orally administrabie formulations will usually be in the range of 0.1 to 3000 mg, once, twice or three times per day, or the equivalent daily amount administered by infusion or other routes.
  • optimum dose levels and frequency of dosing will be determined by clinical trials as is conventional in the art.
  • the compounds with which the invention is concerned may be prepared for administration by any route consistent with their pharmacokinetic properties.
  • the orally administrabie compositions may be in the form of tablets, capsules, powders, granules, lozenges, liquid or gel preparations, such as oral, topical, or sterile parenteral solutions or suspensions.
  • Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricant, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants for example potato starch, or acceptable wetting agents such as sodium lauryl sulphate.
  • the tablets may be coated according to methods well known in normal pharmaceutical practice.
  • Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use.
  • Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, glucose syrup, gelatin hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, fractionated coconut oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and if desired conventional flavouring or colouring agents.
  • suspending agents for example sorbitol, syrup, methyl cellulose, glucose syrup, gelatin hydrogenated edible fats
  • emulsifying agents for example lecithin, sorbitan monooleate, or acacia
  • non-aqueous vehicles which may include edible oils
  • almond oil fractionated coconut oil
  • oily esters such as glycerine, propylene
  • the drug may be made up into a cream, lotion or ointment.
  • Cream or ointment formulations which may be used for the drug are conventional formulations well known in the art, for example as described in standard textbooks of pharmaceutics such as the British Pharmacopoeia.
  • the active ingredient may also be administered parenterally in a sterile medium.
  • the drug can either be suspended or dissolved in the vehicle.
  • adjuvants such as a local anaesthetic, preservative and buffering agents can be dissolved in the vehicle.
  • example 15 was identical to example 1 LCMS (RT 1.89 minutes, [M+H] + 567)
  • example 3 The method used to prepare example 3 was identical to example 1 LCMS (RT 1.87 minutes, [M+H] + 567)
  • Examples 4 to 42 listed in the following table were prepared by methods analogous to Examples 1 , 2 and 3 above. All 42 compounds were tested for activity in kinase assays described below in the Assay section.
  • characterization and/or purification were performed using standard spectroscopic and chromatographic techniques, including liquid chromatography-mass spectroscopy (LC-MS) and high performance liquid chromatography (HPLC) 1 using the conditions described in methods A and B.
  • LC-MS liquid chromatography-mass spectroscopy
  • HPLC high performance liquid chromatography
  • Ionization was positive or negative ion electrospray Molecular weight scan range was 120-1000
  • Ionization was positive or negative ion electrospray
  • Aurora A Assays for the Aurora A Kinase activity were carried out by monitoring the phosphorylation of a synthetic peptide, Kemptide (LRRASLG).
  • the assay mixture containing the inhibitor, Aurora A enzyme, and peptide was mixed together in a microtiter plate in a final volume of 50 ⁇ l and incubated for 30min at 30°C.
  • the assay mixture contained 0.01 mM unlabeled ATP, 0.01 ⁇ Ci/ ⁇ l 33 P- ⁇ -ATP, 0.2mM peptide, 0.1 mg/ml BSA, 7.5mM magnesium acetate, 0.04M MOPS, pH 7, 1mM EDTA.
  • the reaction was stopped by adding 50 ⁇ l of 5OmM phosphoric acid.
  • Assays for Aurora Kinase activity and compound inhibition were carried out by monitoring the phosphorylation of a synthetic peptide, STK substrate-biotin in a HTRF-assay format.
  • the assay mixture contains the inhibitor, Aurora enzyme, peptide and ATP mixed together in a microtiter plate in a final volume of 10ml and incubated for 15 minutes at room temperature.
  • 10ul of development solution containing streptavidin-XL665 and STK antibody crypatate, in Hepes 25OmM (pH 7), BSA 0.1%, KF 0.8M and EDTA is added.
  • the plate was incubated at room temperature for 1 hour and read using time resolved fluorescence at excitation wavelength 360nm and emission at 620and 655nm, on an Anaylst plate reader.
  • Example 17 gave an IC 50 versus Aurora A kinase of 0.056 ⁇ M. All examples demonstrate greater than 50-fold selectivity for Aurora kinases over AGC kinases PDK1 , CDK2, PKA, AKT, Gsk3b and CHK1. In a broader kinase panel, Example 17 exhibited selectivity of greater than 1000-fold versus the kinases listed in the table below.
  • Fluorescence-activated cell-sorting is a trademarked employed by Becton-Dickinson to describe their method of flow cytometry (FCM).
  • FCM Fluorescence-activated cell-sorting
  • FCM flow cytometry
  • a flow cytometer operates by causing a fluid stream to pass single file through a beam of light usually generated by a laser.
  • the photons of light emitted by the cells, following their interaction with the laser beam, are separated into constituent wavelengths by a series of filters and mirrors. This separated light falls upon individual detectors that generate electrical impulses or analogue signals proportional to the amount of light striking the detectors.
  • Each analogue signal is converted to a digital signal which is accumulated in a frequency distribution or histogram (see figure below).
  • FCM is commonly used to quantify the volume and morphological complexity of cells, enzymatic activity and the quantification and measurement of DNA degradation.
  • the assay has been designed for DNA cell cycle analysis using Propidium Iodide staining of the DNA.
  • HCT-116 cells are grown and maintained by methods that will be familiar to those skilled in the art. For flow cytometry cells are counted and split into a 24-well plate at 30000 cells/well. The next day putative Aurora inhibitors are added at a range of appropriate concentrations (usually tripling dilution series are used). 48 hours later, floating cells are removed, then combined with the corresponding adherent cells that released by treatment with trypsin using methods familiar to those skilled in the art.
  • the combined cell population is pelleted by 5 minutes centrifugation at 200 x g, washed with phosphate buffered saline, then resuspended in a solution containing (50 ug/ml propidium iodide and 0.5 mg/ml RNAase A). 1 hour of incubation at 37 C is sufficient for the digestion of cellular RNAs enabling the amount of propidium iodide associated with cells to be proportional to their DNA content.
  • Samples are then read on a BD FACSArray with the amount of propidium iodine staining being measured for each cell; this yields a fluorescence histogram that plots the number of cells with a particular fluorescence against propidium iodide fluorescence.
  • Untreated cells are bimodally distributed on such a fluorescence histogram with a large peak representing cells with a 2n DNA content (G 1 cells) and a smaller peak composed of cells with approximately double the fluorescence of the first peak representing cells with a 4n DNA content (cells in G2 and mitosis); cells with an intermediate fluorescence represent cells in S phase.
  • Treatment with a pure Aurora A inhibitor is predicted to lead to a sharp increase in the number of cells with a 4n DNA content (though this was not observed for our compounds).
  • Treatment of cells with an Aurora B inhibitor leads to failed cell division, followed by an attempt to re-replicate the genome which leads to the appearance of an 8n peak.
  • the co- inhibition of kinases required for replication will prevent the appearance of the 8n peak and the co-inhibition of kinases required for survival under the culture conditions of the HCT116 cells will result in the appearance of apoptotic cells, which due to the activation of nucleases, will have a DNA content of ⁇ 2n.
  • the range of doses that leads to the appearance of a peak of 8n cells, without the induction of a major sub 2n peak can be used as an indication of the dose range at which an inhibitor imposes an Aurora B blockade without confounding effects on other targets.
  • Example 17 shows 8n down to 37nM.
  • Example 26 shows 8n down to 33OnM.

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Abstract

L'invention se rapporte à des composés de formule (I) et à leur utilisation thérapeutique, en particulier pour le traitement d'une affection sensible à l'inhibition de la kinase Aurora A et/ou B, formule (I) dans laquelle R1, R2, R3, R4 et R5 sont choisis indépendamment les uns des autres parmi l'hydrogène et les groupes hydroxy, alkyle en C1-C3, fluoroalkyle (en C1-C3), hydroxyalkyle (en C1-C3), alkoxy (en C1-C3), fluoroalkoxy (en C1-C3), -N(R6)-R7, Alk-N(R6)-R7, (O-Alk-N(R6)-R7, -C(O)OH, carboxyalkyle en (C1-C3) ou -C(O)-NH-R8 ; R6, R7, R8 et -Alk- sont comme définis dans les présentes ; X est un halogène, un groupe cyano ou un groupe trifluorométhyle ; Z représente O ou S ; Y est choisi parmi -NH-, -N(alkyle en C1-C3)-, -(CH2)- ou est absent ; et Ar représente un groupe aryle ou hétéroaryle, éventuellement substitué avec au moins un atome d'halogène, au moins un radical alkyle en C1-C3 ou au moins un radical trifluorométhyle ; ou encore à un sel, un hydrate ou un solvate, acceptable sur le plan pharmaceutique, desdits composés.
PCT/GB2007/003688 2006-09-30 2007-09-28 DÉrivÉs de la pyrimidine servant d'inhibiteurs de l'aurora A et de l'aurora B WO2008038011A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011039344A1 (fr) 2009-10-02 2011-04-07 Boehringer Ingelheim International Gmbh Dérivés de pyrimidine utilisables en tant qu'inhibiteurs de la protéine qu'est la tyrosine kinase 2
WO2011147951A1 (fr) 2010-05-28 2011-12-01 Prosidion Limited Dérivés de cycloamino comme antagonistes du gpr119
US8785464B2 (en) 2008-11-24 2014-07-22 Boehringer Ingelheim International Gmbh Pyrimidine derivatives that inhibit FAK/PTK2
US8846689B2 (en) 2008-11-24 2014-09-30 Boehringer Ingelheim International Gmbh Substituted pyrimidines for the treatment of diseases such as cancer
US11578061B2 (en) 2021-03-23 2023-02-14 Halia Therapeutics, Inc. Inhibitors of LRRK2 kinase

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8785464B2 (en) 2008-11-24 2014-07-22 Boehringer Ingelheim International Gmbh Pyrimidine derivatives that inhibit FAK/PTK2
US8846689B2 (en) 2008-11-24 2014-09-30 Boehringer Ingelheim International Gmbh Substituted pyrimidines for the treatment of diseases such as cancer
US9676762B2 (en) 2008-11-24 2017-06-13 Boehringer Ingelheim International Gmbh Pyrimidine compounds containing seven-membered fused ring systems
WO2011039344A1 (fr) 2009-10-02 2011-04-07 Boehringer Ingelheim International Gmbh Dérivés de pyrimidine utilisables en tant qu'inhibiteurs de la protéine qu'est la tyrosine kinase 2
CN102695700A (zh) * 2009-10-02 2012-09-26 贝林格尔.英格海姆国际有限公司 用作蛋白酪氨酸激酶2抑制剂的嘧啶衍生物
US8466155B2 (en) 2009-10-02 2013-06-18 Boehringer Ingelheim International Gmbh Pyrimidines
WO2011147951A1 (fr) 2010-05-28 2011-12-01 Prosidion Limited Dérivés de cycloamino comme antagonistes du gpr119
US11578061B2 (en) 2021-03-23 2023-02-14 Halia Therapeutics, Inc. Inhibitors of LRRK2 kinase
US11866423B2 (en) 2021-03-23 2024-01-09 Halia Therapeutics, Inc. Inhibitors of LRRK2 kinase

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