WO2014020531A1 - Dérivés d'imidazo[1,2-b]pyridazin-6-amine utilisables en tant qu'inhibiteurs de la kinase jak2 - Google Patents

Dérivés d'imidazo[1,2-b]pyridazin-6-amine utilisables en tant qu'inhibiteurs de la kinase jak2 Download PDF

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WO2014020531A1
WO2014020531A1 PCT/IB2013/056241 IB2013056241W WO2014020531A1 WO 2014020531 A1 WO2014020531 A1 WO 2014020531A1 IB 2013056241 W IB2013056241 W IB 2013056241W WO 2014020531 A1 WO2014020531 A1 WO 2014020531A1
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methyl
pyridazin
imidazo
amine
chloro
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PCT/IB2013/056241
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English (en)
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Karolina DZWONEK
Michal MROCZKIEWICZ
Filip Stefaniak
Maciej Wieczorek
Krzysztof DUBIEL
Monika Lamparska-Przybysz
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Celon Pharma S.A.
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Publication of WO2014020531A1 publication Critical patent/WO2014020531A1/fr

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention related to novel heterocyclic compounds, imidazo[1 ,2-j ]- pyridazin-6-amine derivatives showing the activity of kinase JAK-2 inhibitors, pharmaceutical compositions containing these compounds, and their use as medicaments.
  • the compounds can be useful, in particular, in the treatment of myeloproliferative and neoplastic diseases.
  • Tyrosine kinases JAK1 , JAK2, JAK3, and TYK2 from the JAK kinases family are involved in intracellular signal transduction in the signaling pathway JAK-STAT, and play significant role in the activation of STAT proteins and initiation of transcription.
  • Activation of JAK kinases is believed to be one of the factors of the proliferation of neoplastic cells.
  • the activity of transcriptional factor STAT in a cell depends on its phosphorylation level. High STAT phosphorylation levels lead to pathological myeloproloferative disorders and leukemias.
  • Phosphorylation level is dependent on activity of JAK2 kinase - inhibition of JAK2 kinase causes lowering of the phosphorylation level and transcriptional activity of STAT.
  • Kinase JAK2 is also activated in the range of solid tumors and leukemias. Therefore, kinase JAK2 inhibitors block specific signaling pathway which can lead to excessive proliferation of cells and neoplasm development, and can find use in the treatment of myeloproliferative and neoplastic diseases.
  • WO2008/030579 discloses very broad group of imidazo[1 ,2-b]pyridazine derivatives as modulators of interleukine-1 receptors associated kinase (IRAK), potentially useful in the treatment of inflammatory diseases, cell proliferation disorders and immunological diseases mediated by IRAK.
  • IRAK interleukine-1 receptors associated kinase
  • WO2010/074947 discloses single compound 3-(4-chloro-2-fluorobenzyl)-2-methyl- N-(5-methyl-1 H-pyrazol-3-yl)-8-(morpholinomethyl)imidazo[1 ,2-b]pyridazin-6- amine as a selective JAK-2 inhibitor, of potential utility in the treatment myeloproliferative and neoplastic diseases.
  • This compound known now under code designation LY-2784544, shows selectivity with respect to JAK2 receptor against JAK3 and is currently in the second phase of clinical trials. There is no suggestion as to the possibility and the type of modification of the structure of the disclosed compound required to obtain further JAK-2 inhibitors. There is still a need for new highly effective compounds showing the ability to inhibit JAK2 kinase with high potency and/or high selectivity and potential use in the treatment of myeloproliferative and neoplastic diseases.
  • the invention relates to new compounds represented by the general formula (I)
  • R 1 represents H or C1 -C4 alkyl
  • R 2 represents phenyl substituted with one or two substituents selected from the group consisting of halogen atom and -OC1 -C4 alkyl;
  • R 3 represents phenyl or 5- to 10-membered monocyclic or bicyclic heteroaryl with 1 to 4 ring heteroatoms selected from the group consisting of N, S, and 0, which is unsubstituted or substituted with a substituent selected from halogen atom, Ci -C4-alkyl, and -C(0)0-Ci -C4-alkyl; and
  • X represents -CH 2 - group or -C(O)- group
  • the object of the invention is the compound of formula (I) as defined above for use as a medicament.
  • the object of the invention is also a pharmaceutical composition, comprising as an active ingredient a compound of the general formula (I) as defined above in combination with pharmaceutically acceptable excipients.
  • the compounds of formula (I) as defined above can find use in the treatment of myeloproliferative and neoplastic diseases. Accordingly, the object of the invention is the compound of formula (I ) as defined above for use in a method of treatment of myeloproliferative disorders and neoplastic diseases.
  • the object of the invention is also a use of the compound of formula (I ) as defined above for the preparation of a medicament for the treatment of myeloproliferative disorders and neoplastic diseases.
  • the object of the invention is also a method of treatment of myeloproliferative disorders and neoplastic diseases in a mammal subject, including humans, which comprises administration to the subject in need thereof a therapeutically effective amount of a compound of the general formula (I ) or a pharmaceutical composition as defined above.
  • One embodiment of the invention is the compound of the above formula (I ), wherein X represents -CH 2 - group (i.e. methylene group).
  • Another embodiment of the invention is the compound of the above formula (I ), wherein X represents -C(O)- group (i.e. carbonyl group).
  • Another embodiment of the invention is the compound of the above formula (I ), wherein R 1 represents H .
  • Another specific embodiment of the invention is the compound of the above formula (I ), wherein R 1 represents CH 3 .
  • R 2 represents phenyl substituted with halogen atom, especially fluorine atom, and -0Ci -C 4 alkyl, especially -OCH 3 .
  • Another specific embodiment of the invention is the compound of the above formula (I ), wherein R 3 represents phenyl.
  • R 3 represents 6-membered heteroaryl, which is unsubstituted or substituted with a substituent as specified above, in particular halogen atom, especially fluorine or chlorine atom, or Ci-C4-alkyl, especially methyl, or -C(0)0-C r C 4 -alkyl, especially -C(0)0-CH 2 -CH 3 .
  • 6-membered heteroaryl can be a pyridinyl, such as 2-pyridinyl, 3- pyridinyl or 4-pyridinyl, unsubstituted or substituted with a substituent such as specified above, in particular halogen atom, especially fluorine atom, or CrC 4 - alkyl, especially methyl, or -C(0)0-Ci-C4-alkyl, especially -C(0)0-CH 2 -CH 3 .
  • a substituent such as specified above, in particular halogen atom, especially fluorine atom, or CrC 4 - alkyl, especially methyl, or -C(0)0-Ci-C4-alkyl, especially -C(0)0-CH 2 -CH 3 .
  • 6-Membered heteroaryl can also be a heteroaryl containing 2 nitrogen atoms, in particular pyridazinyl or pyrimidinyl, unsubstituted or substituted with a substituent such as specified above, in particular halogen atom, especially chlorine atom.
  • R 3 represents 5-membered heteroaryl containing 2 nitrogen atoms, such as pyrazolyl or imidazolyl, 5-membered heteroaryl containing 2 nitrogen atoms and one sulphur atom, such as thiadiazolyl, or 5-membered heteroaryl containing one nitrogen atom and one sulphur atom, such as thiazolyl, which are unsubstituted or substituted with a substituent such as specified above, in particular halogen atom, especially fluorine atom, C1 -C4- alkyl, especially methyl, or C(0)0-CrC 4 -alkyl, especially -C(0)0-CH 3 .
  • R 3 represents 5-membered heteroaryl containing 2 nitrogen atoms, such as pyrazolyl or imidazolyl, 5-membered heteroaryl containing 2 nitrogen atoms and one sulphur atom, such as thiadiazolyl, or 5-membered heteroaryl containing one nitrogen atom and one s
  • the term encompasses -CH 3 (methyl), -CH 2 CH 3 (ethyl), -CH 2 CH 2 CH 3 (n-propyl), -CH(CH 3 ) 2 (isopropyl), -CH 2 CH 2 CH 2 CH 3 (n-butyl), -CH 2 CH(CH 3 ) 2 (isobutyl), -CH(CH 3 )CH 2 CH 3 (sec-butyl) and -C(CH 3 ) 3 (tert-butyl) groups.
  • the term encompasses fluorine (F), chlorine (CI), bromine (Br) and iodine (I ) atoms.
  • 5-membered monocyclic heteroaryls including heteroaryls containing 1 nitrogen atom, such as pyrrolyl, 2 nitrogen atoms, such as pyrazolyl and imidazolyl, 3 nitrogen atoms, such as triazolyl, such as 1 ,2,4- triazolyl, and 4 nitrogen atoms, such as 1 H-tetrazolyl.
  • heteroaryls containing 1 nitrogen atom and 1 sulphur atom such as thiazolyl and isothiazolyl
  • heteroaryls containing 2 nitrogen atoms and 1 sulphur atom such as thiadiazolyl.
  • 6-membered monocyclic heteroaryls including heteroaryls containing 1 nitrogen atom, such as pyridinyl, and heteroaryls containing 2 nitrogen atom (diazines), such as pyridazinyl, pyrimidinyl and pirazynyl.
  • heteroaryls containing 1 nitrogen atom such as pyridinyl
  • diazines such as pyridazinyl, pyrimidinyl and pirazynyl.
  • heteroaryls containing 1 nitrogen atom such as indolyl, isoindolyl and indolizynyl
  • heteroaryls containing 2 nitrogen atoms such as benzimidazolyl and indazolyl
  • heteroaryls containing 4 nitrogen atoms such as purinyl
  • 10-membered bicyclic heteroaryls including heteroaryls containing 1 nitrogen atom, such as quinolinyl and isoquinolinyl, and heteroaryls containing 2 nitrogen atoms, such as quinazolinyl, quinoxalinyl and cinnolinyl.
  • Acid addition salts of the compounds of formula (I) of the invention include salts with inorganic or organic acids. Preferred are salts that are pharmaceutically acceptable. Inorganic and organic acids that can form pharmaceutically acceptable salts with compounds having basic nitrogen atom are well known in the art. Salts with inorganic acids especially comprise those of hydrochloric, hydrobromic, sulfuric, and phosphoric acids. Salts with organic acids especially comprise those of methanesulfonic, ethanesulfonic, toluenesulfonic, benzenesulfonic, naphthalenedisulfonic, formic, acetic, propionic, lactic, tartaric, malic, citric, fumaric, maleic, and benzoic acids.
  • the compounds of formula (I) of the invention can be prepared by reaction of a compound of formula (II), wherein R 1 , R 2 and X have the meanings as given above for formula (I), with an amine compound of formula (III), wherein R 3 has the meaning as given above for formula (I), in accordance with Scheme 1.
  • the reaction between compound (II) and amine compound (III) can be carried out as Buchwald-Hartwig coupling reaction, in a solvent, in the presence of a palladium catalyst, a phosphine ligand and an inorganic or organic base.
  • Solvents used in the reaction can be aprotic solvents such as benzene, toluene, xylenes, tetrahydrofurane, dioxane, dimethoxyethane, diethoxyethane, or protic solvents such as butanol, water or a mixture of these solvents.
  • Amine R 3 -NH 2 is used in the amount of 1 to 3 molar equivalents per 1 equivalent of the compound of formula (II).
  • Palladium catalyst can be tn ' s(dibenzylideneacetone)dipalladium(0) (a preferred one), bis(dibenzylideneacetone)palladium(0), palladium(ll) acetate or 1 ,1'-bis(diphenylphosphino)ferrocene.
  • Palladium catalyst is used in the amount of 0.05 to 0.10 molar equivalents per 1 equivalent of the compound of formula (II).
  • Phosphine ligands used in the reaction can be Xantphos - 4,5- bis(diphenylphosphino)-9,9-dimethylxanthene or BINAP - 2,2 -bis(diphenyl- phosphino)-1 , 1 '-binaphtyl.
  • Phosphine ligand is used in the amount of 0.10 to 0.20 molar equivalents per 1 equivalent of the compound of formula (II).
  • lithium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, cesium carbonate, or potassium tert- butanolate in the amount of 1.05 to 1.5 molar equivalents per 1 equivalent of the compound of formula (II) can be used.
  • the reaction is carried out in strictly oxygen-free conditions in the atmosphere of inert gas such as argon or nitrogen.
  • the reaction is carried out at the solvent reflux temperature.
  • R 3 in formula (III) is a heteroaryl group containing nitrogen atom substituted with hydrogen (-NH-) as a member of the ring, which may compete in the coupling reaction leading to undesired side product, it is recommended to protect such nitrogen atom in order to maximise yield. Therefore, in such a case coupling reaction is carried out using a compound of formula (III) with R 3 as a heteroaryl group containing protected nitrogen atom. After completion of the coupling reaction protecting group is removed to obtain corresponding unprotected compound of formula (I).
  • R 3 group is derived from pyrrol, pyrazol, imidazol, triazol and tetrazol, and is selected from 1H-pyrolyl, 1 H-pyrazolyl, 1 H-imidazolyl, 1H- 1 ,2,3-triazolyl, 4tf-1 ,2,4-triazolyl, 1 H-1 ,2,4-triazolyl, 1 H-tetrazolyl, and 2H- tetrazolyl.
  • Typical nitrogen atom protecting groups chosen from teri-butyl, tert- butoxycarbonyl, benzyl, para-methoxybenzyl or dimethoxybenzyl groups can be used.
  • Protecting groups are introduced in accordance with methods of protecting nitrogen functional groups commonly known in the literature, that is by introducing tert-butoxycarbonyl group (Boc) in the reaction with tert- butoxycarbonyl anhydride, introducing benzyl group (Bz) in the reaction with halide such as benzyl chloride or bromide, introducing para-methoxybenzyl group (Pmb) in the reaction with para-methoxybenzyl halide such as para- methoxybenzyl chloride or bromide, introducing dimethoxybenzyl group (Dmb) in the reaction with dimethoxybenzyl halide such as dimethoxybenzyl chloride or bromide.
  • Boc tert-butoxycarbonyl group
  • Bz benzyl group
  • Pmb para-methoxybenzyl group
  • Dmb dimethoxybenzyl group
  • Removal of the -NH- group protecting group from substituent R 3 in the compound of formula (I ) is also carried out in a known, conventional manner. Conditions of deprotection step depend on the type of protecting group.
  • f erf- Butyl group can be removed in the reaction with trifluoroacetic acid with the addition of water.
  • a solvent such as dichloromethane or chloroform. It is advantageous to use the mixture of trifluoroacetic acid and water at the volume ratio from 1 : 4 to 1 : 5 using from 15 to 40 molar equivalents of trifluoroacetic acid per 1 equivalent of deprotected compound.
  • the reaction is carried out in the temperature range 0 to 120° C.
  • the reaction is carried out in the temperature range 80 to 100° C.
  • tert- Butoxycarbonyl group can be removed in the reaction with an acid, such as acetic, trifluoroacetic, hydrochloric or sulfuric acid with or without the addition of water.
  • an acid such as acetic, trifluoroacetic, hydrochloric or sulfuric acid with or without the addition of water.
  • Benzyl, para-methoxybenzyl, and dimethoxybenzyl groups can be removed in the reaction with trifluoroacetic acid with or without the addition of water.
  • Amines R 3 -NH 2 of formula (III) are commercially available.
  • amines (III) can be prepared from commercially available corresponding nitro derivatives by reduction of nitro group to amine group using reductive conditions, such as Pd/C catalyzed hydrogenation, tin(ll ) chloride SnCl 2 in aqueous medium, or iron in aqueous medium.
  • Advantageous R' group is methyl
  • advantageous R" group is methyl. It is advantageous to use the excess of the compound of formula (V) with respect to the compound of formula (IV) in the range from 1.2 to 3 molar equivalents.
  • the reaction is carried out in an aprotic solvent such as benzene, toluene, xylenes, alkyl (C 2 -C 4 ) ether, tetrahydrofuran, 1 ,4-dioxane.
  • Preferred solvent is toluene.
  • the reaction is carried out in the temperature range 60 - 120°C, preferably 80 - 90 °C.
  • compound of formula (VI) is cyclized with 2- chloroethanone of formula (VII) to form a compound of formula (VIII).
  • Cyclization is carried out in a solvent with the addition of a bromide salt.
  • the solvent can be dimethylformamide, N-methylpyrrolidone, acetonitrile, acetone, preferably dimethylformamide.
  • the bromide salt can be lithum bromide, sodium bromide, or potassium bromide, preferably sodium bromide.
  • the amount of the bromide salt is in the range 0.9 to 1 .0 molar equivalents per 1 equivalent of the compound of formula (VI).
  • the amount of the compound of formula (VII) is in the range 0.9 to 1 .1 molar equivalents per 1 equivalent of the compound of formula (VI ).
  • the reaction is carried out under the inert gas atmosphere such as nitrogen or argon, in the temperature range 0 to 120°C. Preferred temperature is in the range 25 to 40° C.
  • the solvent can be an alcohol, such as methanol, ethanol, propanol, isopropanol, or dichloromethane or chloroform. Preferred solvent is ethanol.
  • Vanadyl acetylacetonate can be used as the catalyst, in the amount 0.05 to 0.25 molar equivalents per 1 equivalent of the compound of formula (VIII ).
  • the amount of the catalyst is 0.20 molar equivalents per 1 equivalent of the compound of formula (VIII ).
  • the reaction is carried out under inert gas atmosphere, such as nitrogen or argon, in the temperature range 0 to 100° C. Preferred temperature is in the range 35 to 45°C.
  • Reduction of carbonyl group in the compound of formula (II) to the group -CH 2 - is carried out using triethylsilane in trifluoroacetic acid as a solvent.
  • Triethylsilane is used in the amount of 5 to 10 molar equivalents per 1 equivalent of the compound of formula (II).
  • trifluoroacetic acid is used in the excess with respect to the compound of formula (II) of 10 to 12 molar equivalents per 1 equivalent of the compound of formula (II ).
  • the reaction is carried out under inert gas atmosphere, such as nitrogen or argon.
  • the reaction is carried out at the solvent reflux temperature.
  • reduction of carbonyl group in the compound of formula (II ) to -CH 2 - group can be carried out in two steps.
  • carbonyl group in the compound of formula (II ) is reduced to hydroxyl group using sodium borohydride (preferably) or lithium borohydride as a reducing agent, in methanol (preferably) or ethanol as a solvent.
  • hydroxyl group is reduced to methylene group with triethylsilane as the reducing agent in trifluoroacetic acid as a solvent.
  • the compounds of formula (I) can be administered in the treatment in the form of a pharmaceutical composition or preparation containing them.
  • the object of the invention is therefore also a pharmaceutical composition
  • a pharmaceutical composition comprising as an active ingredient a compound or compounds of formula (I ) as defined above in the mixture with pharmaceutically acceptable excipients.
  • the invention relates also to a method for treating myeloproliferative and neoplastic diseases in a mammal subject, including humans, which comprises administration to the subject in need thereof of a therapeutically effective amount of the compound of the above formula (I) or a pharmaceutical composition comprising said compound of the above formula (I) as an active ingredient.
  • the compounds of formula (I) of the invention can be administered as a chemical compound, but usually will be used in the form of pharmaceutical compositions comprising the compound of the invention or its pharmaceutically acceptable salt such as defined above as the active ingredient, in combination with pharmaceutically acceptable carriers and excipients.
  • the compositions of the invention will be administered by any route, preferably by oral route or parenteral route and will have the form of a preparation destined for use in medicine, depending on the intended route of administration.
  • compositions for oral administration can have a form of solid or liquid preparations.
  • Solid preparations can have the form of, for example, tablets or capsules produced in a conventional manner from pharmaceutically acceptable inactive excipients such as binders (for example, pregelatinised corn starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (for example lactose, saccharose or calcium hydrogenphosphate), lubricants (for example magnesium stearate, talc or silica), wetting agents (for example sodium laurylsulfate). Tablets can be coated with simple coatings, delayed/controlled- release coatings or enteral coatings well known in the art.
  • Liquid preparations for oral administration can be in the form of, for example, solutions, syrups or suspensions, or can have the form of a dry solid product for reconstitution with water or other suitable vehicles before use.
  • Such liquid preparations can be prepared using conventional means from pharmaceutically acceptable excipients, such as suspending agents (for example sorbitol syrup, cellulose derivatives or hydrogenated edible oils), emulsifiers (for example lecithine or acacia gum), nonaqueous vehicles (for example mandelic oil, oil esters, ethyl alcohol or fractionated vegetable oils), and preservatives (for example methyl or propyl p-hydroxybenzoate or sorbic acid).
  • suspending agents for example sorbitol syrup, cellulose derivatives or hydrogenated edible oils
  • emulsifiers for example lecithine or acacia gum
  • nonaqueous vehicles for example mandelic oil, oil esters, ethyl alcohol or fractionated vegetable oils
  • preservatives for example
  • Preparations for oral administration can be formulated so as to obtain controlled release of the active compound using methods known for a person skilled in the art.
  • compositions for parenteral administration can, for example, have the form of unit dosage form, such as ampoules, or multidosage containers, with the addition of a preservative.
  • Compositions can have the form such as suspensions, solutions or emulsions in oily or aqueous vehicles, and can include excipients such as suspending agents, stabilizers, and/or dispersing agents.
  • the active ingredient can have the form of a powder for reconstitution before use in a suitable carrier, for example sterile, pyrogen-free water.
  • the method of treatment with the use of the compounds of the present invention will comprise administration of a therapeutically effective amount of the compound of the invention, preferably in the form of a pharmaceutical composition, to the subject in need of such treatment.
  • Proposed dosage of the compounds of the invention is from 0.1 to about 1000 mg per day, in a single dose or in divided doses. It will be apparent for a person skilled in the art that the selection of a dosage required for obtaining desirable biological effect will depend on many factors, for example specific compound, the application, the manner of administration, the age and condition of a patient and that exact dosage will be ultimately found by a responsible physician.
  • tert- Butylhydrazine hydrochloride (15.2 g, 122 mmol) was added to the aqueous solution of sodium hydroxide (60 mL, 2M, 122 mmol) and stirred until dissolution of a solid.
  • 3-aminobut-2-enenitrile (10 g, 122 mmol) was added.
  • the reaction mixture was stirred while heating at 90°C for 18 hours, then cooled to room temperature and extracted with dichloromethane (3 ⁇ 50 mL). Organic layers were combined, washed with brine, dried (Na 2 S0 4 ) and concentrated under reduced pressure to obtain title product as a white, amorphous solid with the yield of 92% (17.2 g, 1 12 mmol).
  • Reaction mixture was degassed, purged with argon stream for 30 minutes, and then stirred at 100°C for 44 hours.
  • Reaction mixture was cooled to room temperature, diluted with ethyl acetate (10 mL), filtered through celite layer and washed with ethyl acetate. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography (silica gel, eluent: heptane/AcOEt 20:80, AcOEt 100% to AcOEt/methanol 98:2, v/v). After evaporation and drying title product was obtained in the form of amorphous white solid with the yield of 96% (253 mg, 0.53 mmol).
  • reaction mixture was degassed, purged with argon stream for 30 minutes, and then stirred while heating at 100° C for 44 hours.
  • the reaction mixture was cooled to room temperature, diluted with ethyl acetate (10 ml_), filtered through a Celite bed and washed with ethyl acetate. Filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography (silica gel, eluent: heptane/AcOEt 20:80, AcOEt 100% to AcOEt/methanol 98:2, v/v).
  • Title product was obtained in the form of an amorphous, white solid with the yield of 89% (204 mg, 0.437 mmol).
  • Example 1 3-(4-Chloro-2-fluorobenzyl)-2-methyl-N-(1 -methyl-1 H-pyrazol-4-yl)- 8-(morpholinomethyl)imidazo[1 ,2-b]pyridazin-6-amine
  • the mixture was suspended in degassed toluene (5 ml_).
  • the reaction mixture was degassed, purged with argon stream for 30 minutes, and then stirred while heating at 100° C for 20 hours.
  • the reaction mixture was cooled to room temperature, filtered through a Celite of and washed with ethyl acetate. Filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography (silica gel, eluent: AcOEt 100% to AcOEt/ methanol 97:3, v/v). Fractions were concentrated to obtain yellow oil, which was dissolved in ethyl acetate and added with heptane until precipitation of crystals. Title product was obtained in the form of creamy crystals with the yield of 41 % (93 mg, 0.1 98 mmol).
  • Example 12 3-(4-Chloro-2-fluorobenzyl)-2-methyl-/V-(1 -methyl- 1 H-imidazol-4- yl)-8-(morpholinomethyl)imidazo[1 ,2-b]pyridazin-6-amine
  • Example 1 3 Ethyl 3-(3-(4-chloro-2-fluorobenzyl)-2-methyl-8-(morpholino- methyl)imidazo[1 , 2-b]pyridazin-6-ylamino)- 1 H-pyrazole-4-carboxylate
  • Example 14 3-(4-Chloro-2-fluorobenzyl)-2-methyl-8-(morpholinomethyl)-N-(1 H- 1 ,2,4-triazol-3-yl)imidazo[1 ,2-b]pyridazin-6-amine
  • Example 17 Methyl 2-(3-(4-chloro-2-fluorobenzyl)-2-methyl-8-(morpholino- methyl)imidazo[1 ,2-t»]pyridazin-6-ylamino)thiazole-5-carboxylate
  • Example 28 3-(2,4-Difluorobenzyl)-2-methyl-N-(1 -methyl- 1 H-imidazol-4-yl)-8- (morpholinomethyl)imidazo[1 ,2-b]pyridazin-6-amine
  • reaction buffer 50 mM Tris pH 7.5, 10 mM MgCl 2 , 0.25 mM EGTA, 0.1 mM Na 3 V0 4 , 0.01 % Triton X-100, 2.5 mM DTT.
  • Recombinant JAK2 kinase (Carna Biosciences) was diluted to final concentration of 0.1 ng/ ⁇ in the dilution buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 0.05% Triton X-100, 1 mM DTT).
  • Enzymatic reaction was initiated by adding 15 ⁇ _ of solution consisting of: 5x concentrated reaction buffer (50 mM Tris pH 7.5, 10 mM MgCl 2 , 0.25 mM EGTA, 0.1 mM Na 3 V0 4 , 0.01 % Triton X-100, 2.5 mM DTT), water, 50 ⁇ ATP, and 16.67 ⁇ IGF-1 Rtide peptide (MUlpore).
  • 5x concentrated reaction buffer 50 mM Tris pH 7.5, 10 mM MgCl 2 , 0.25 mM EGTA, 0.1 mM Na 3 V0 4 , 0.01 % Triton X-100, 2.5 mM DTT
  • MUlpore 16.67 ⁇ IGF-1 Rtide peptide
  • IC50 values were computed by fitting each point to the curve in non-linear regression model in Graph Pad software (ver. 5.03). Each compound was analyzed at least 6 times (in 6 wells) on two 96-well plates with at least three wells of each control.
  • the level of phosphorylation of STAT3 transcription factor in a cell depends on the activity of JAK2 kinase - inhibition of JAK2 kinase by an inhibitor (tested compound) causes decrease of STAT3 phosphorylation, what is observed in Western blot assay as a decline of a band immunodetected with anti-pSTAT3 antibody.
  • the inhibition of JAK2 kinase should not interfere with total amount of STAT3 protein - based on the immunodetection of STAT3.
  • the loading control in this assay includes the immunodetection of beta-tubulin - a protein which is present at stable level in the cells.
  • HEL-92.1.7 erythroleukemia cell line model harboring JAK2 kinase mutation [V617F], entailing constant kinase activation in these cells.
  • This cell line is the accepted model to study biological activity of JAK2 kinase inhibitors (H. Quentmeier et al., Leukemia 2006, 20, 471 -476).
  • the cells were seeded into 6-well plates with the density of 0.5x10 6 /ml_ in the medium without the inhibitor. After 24 hours, cells were treated with the compound in the final concentration of 500 nM, for 2 hours. Then the cells were lysed with RIPA buffer (Sigma-Aldrich) containing proteases inhibitors (Halt Protease Inhibitor Cocktail, Thermo) and phosphatase inhibitors (PhosSTOP, Roche). The protein concentration in cell lysates were measured with BCA assay (Pierce) according to manufacturer's instruction. Cell lysates were separated with SDS-PAGE through 2 hours at 100 V in Mini Protean III system (BioRad).
  • Electrophoretically-fractionated proteins were subsequently electrotransferred onto the nitrocellulose membrane through 1 hour at 100 V in Mini Protean III system.
  • Western blot analysis of selected proteins was performed according to antibodies manufacturers' instructions. In this analysis, the following primary antibodies were used: anti-pSTAT3, anti-STAT3 (Cell Signaling Technology) and anti-i efa-tubulin (Millipore).
  • the secondary horseradish peroxidase-conjugated antibodies were used (Sigma-Aldrich). Immobilized proteins were visualized with LumiLight substrate (Roche) and subsequently exposed to Light Film BioMax (Kodak) which was developed.
  • Fig. 1 presents the results of the analysis of STAT phosphorylation in HEL cells treated with selected compounds of invention in concentration of 500 nM for 2 hours: A) pSTAT3 and B) STAT3. Abbreviation used: C - control; 21 - compound from example 21 ; 22 compound from example 22.
  • the pharmacokinetic features including bioavailability of compounds of the invention were evaluated in a rat model as follows.
  • Fig. 2 shows the example of pharmacokinetic analysis result for the compound of the invention (Example 12) in reference to JAK2 kinase inhibitor LY-2784544 known from WO2010/074947.
  • the compound of invention showed greater AUC than reference inhibitor (373.5 and 268.0, respectively).
  • the Cmax for compound from Example 12 was higher (81 .43 ng/mL) than for LY-2784544 (37.43 ng/mL).

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Abstract

La présente invention concerne un composé représenté par la formule générale (I), dans laquelle R1 représente H ou un groupe alkyle en C1 à C4 ; R2 représente un groupe phényle substitué par un ou deux substituants choisis dans le groupe constitué d'un atome d'halogène et d'un groupe O-alkyle en C1 à C4 ; R3 représente un groupe phényle ou un groupe hétéroaryle monocyclique ou bicyclique à 5 à 10 chaînons comportant de 1 à 4 hétéroatomes cycliques choisis dans le groupe constitué de N, S et O, et pouvant être non substitué ou substitué par un substituant choisi parmi un atome d'halogène, un groupe alkyle en C1 à C4 et un groupe -C(O)O-alkyle en C1 à C4 ; et X représente un groupe -CH2- ou carbonyl-C(O) ; et leurs sels d'addition acides ; à l'exception de la 3-(4-chloro-2-fluorobenzyl)-2-méthyl-N-(5-méthyl-1H-pyrazol-3-yl)-8- (morpholinométhyl)-imidazo[1,2-b]pyridazine-6-amine et de ses sels. Lesdits composés sont des inhibiteurs de la kinase JAK2 et peuvent être utilisés comme médicaments, notamment pour traiter les maladies prolifératives et le cancer.
PCT/IB2013/056241 2012-08-01 2013-07-30 Dérivés d'imidazo[1,2-b]pyridazin-6-amine utilisables en tant qu'inhibiteurs de la kinase jak2 WO2014020531A1 (fr)

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PL400213A PL400213A1 (pl) 2012-08-01 2012-08-01 Pochodne imidazo[1,2-b]pirydazyno-6-aminy jako inhibitory kinazy JAK-2
PLP.400213 2012-08-01

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008030579A2 (fr) 2006-09-07 2008-03-13 Biogen Idec Ma Inc. Modulateurs de la kinase associée au récepteur de l'interleukine-1
WO2010074947A1 (fr) 2008-12-16 2010-07-01 Eli Lilly And Company Composé amino pyrazole

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008030579A2 (fr) 2006-09-07 2008-03-13 Biogen Idec Ma Inc. Modulateurs de la kinase associée au récepteur de l'interleukine-1
WO2010074947A1 (fr) 2008-12-16 2010-07-01 Eli Lilly And Company Composé amino pyrazole

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DAVID MITCHELL ET AL: "Development and a Practical Synthesis of the JAK2 Inhibitor LY2784544", ORGANIC PROCESS RESEARCH & DEVELOPMENT, vol. 16, no. 1, 20 January 2012 (2012-01-20), pages 70 - 81, XP055084316, ISSN: 1083-6160, DOI: 10.1021/op200229j *
H. QUENTMEIER ET AL., LEUKEMIA, vol. 20, 2006, pages 471 - 476
ORG. PROCESS. RES. REV., vol. 16, 2012, pages 70 - 81

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