WO2024256569A1 - Nouveaux agents de dégradation de cdk - Google Patents

Nouveaux agents de dégradation de cdk Download PDF

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WO2024256569A1
WO2024256569A1 PCT/EP2024/066424 EP2024066424W WO2024256569A1 WO 2024256569 A1 WO2024256569 A1 WO 2024256569A1 EP 2024066424 W EP2024066424 W EP 2024066424W WO 2024256569 A1 WO2024256569 A1 WO 2024256569A1
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Alexander Dömling
Markella KONSTANTINIDOU
Jan Jasper Molenaar
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Princess Maxima Center
Rijksuniversiteit Groningen
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Princess Maxima Center
Rijksuniversiteit Groningen
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    • 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

  • CDKs cyclin and cyclin dependent kinases
  • CDK9 Concomitant inhibition of several CDKs can have advantageous antiproliferative effects.
  • inhibition of CDK9 suppresses transcription, leading to disruption of the pTEFb complex, and by extension, RNA Polll.
  • CDK9 regulates cellular transcriptional elongation and mRNA maturation and has become an attractive therapeutic target for many cancers, especially those caused by dysregulation of transcription.
  • the object of the present invention to provide novel CDK degraders which may be used for the treatment of diseases that are associated with CDK activity, such as cancer and inflammation.
  • the following invention preferably describes triple targeting PROTAC degraders against CDK4/6/9 with superior anticancer efficacies.
  • the present invention provides compounds of formula (I): wherein n is 1 or 2;
  • X is a C1-6 alkylene group, a C2-6 alkenylene group, a C1-6 heteroalkylene group, an optionally substituted C3-5 cycloalkylene group, or an optionally substituted aralkylene group;
  • Y is a NH group or is selected from the following groups:
  • Z is hydrogen or a methyl group; or a salt thereof.
  • Z is hydrogen.
  • the present invention further provides compounds of formula (la): wherein
  • X is a Ci-6 alkylene group, a C2-6 alkenylene group, a C1-6 heteroalkylene group, an optionally substituted C3-5 cycloalkylene group, or an optionally substituted aralkylene group;
  • Y is a NH group or is selected from the following groups: or a salt thereof.
  • the present invention moreover provides compounds of formula (II):
  • X is a Ci-6 alkylene group, a C2-6 alkenylene group, a C1-6 heteroalkylene group, an optionally substituted C3-5 cycloalkylene group, or an optionally substituted aralkylene group;
  • Y is a NH group or is selected from the following groups: or a salt thereof.
  • Y is a NH group.
  • Y is selected from the following groups:
  • Y is the following group:
  • X is a C1-6 alkylene group or a C1-6 heteroalkylene group.
  • X is selected from the following groups:
  • the most preferred compounds of the present invention are the compounds disclosed in the examples, or a salt thereof.
  • alkenyl groups have one or two (especially preferably one) double bond(s), and alkynyl groups have one or two (especially preferably one) triple bond(s).
  • the expression C2-6 alkenyl refers to a straight-chain or branched hydrocarbon group that contains from 2 to 6 (e.g., 2, 3 or 4) carbon atoms and at least one double bond.
  • alkyl, alkenyl and alkynyl refer to groups in which one or more hydrogen atoms have been replaced by a halogen atom (preferably F or Cl) such as, for example, a 2,2,2-trichloroethyl or a trifluoromethyl group.
  • a halogen atom preferably F or Cl
  • heteroalkyl refers to an alkyl, alkenyl or alkynyl group in which one or more (preferably 1 to 8; especially preferably 1 , 2, 3 or 4) carbon atoms have been replaced by an oxygen, nitrogen, phosphorus, boron, selenium, silicon or sulfur atom (preferably by an oxygen, sulfur or nitrogen atom) or by a SO or a SO2 group.
  • the expression heteroalkyl furthermore refers to a carboxylic acid or to a group derived from a carboxylic acid, such as, for example, acyl, acylalkyl, alkoxycarbonyl, acyloxy, acyloxyalkyl, carboxyalkylamide or alkoxycarbonyloxy.
  • heteroalkyl refers to groups in which one or more hydrogen atoms have been replaced by a halogen atom (preferably F or Cl).
  • heteroalkyl groups are groups of formulae: R a -O-Y a -, R a -S-Y a -, R a -SO-Y a -, R a -SO 2 -Y a -, R a -N(R b )-SO 2 -Y a -, R a -SO 2 -N(R b )-Y a -, R a -N(R b )-Y a -, R a -CO-Y a -, R a -O-CO-Y a -, R a -CO-O-Y a -, R a -CO-N(R b )-Y a -, R a -N(R b )-CO-Y a -, R a -O-CO-O-Y a -, R a -CO-N(R b )-Y a -, R
  • heteroalkyl groups are methoxy, trifluoromethoxy, ethoxy, n-propyloxy, /so-propyloxy, n-butoxy, terf-butyloxy, methoxymethyl, -CH 2 CH 2 OH, -CH 2 OH, -SO 2 Me, -NHAc, -OCD 3 , -C(CH 3 ) 2 CN, methoxyethyl, ethoxymethyl, 1 -methoxyethyl, 1 -ethoxyethyl, 2-methoxyethyl or 2- ethoxyethyl, methylamino, ethylamino, propylamino, isopropylamino, dimethylamino, diethylamino, isopropylethylamino, methylamino methyl, ethylamino methyl, diisopropylamino ethyl, methylthio,
  • cycloalkyl refers to a saturated or partially unsaturated (for example, a cycloalkenyl group) cyclic group that contains one or more rings (preferably 1 or 2), and contains from 3 to 14 ring carbon atoms, preferably from 3 to 10 (especially 3, 4, 5, 6 or 7) ring carbon atoms.
  • cycloalkyl groups are a cyclopropyl, cyclobutyl, cyclopentyl, spiro[4,5]decanyl, norbornyl, cyclohexyl, cyclopentenyl, cyclohexadienyl, decalinyl, bicyclo[4.3.0]nonyl, tetraline, cyclopentylcyclohexyl, fluorocyclohexyl or cyclohex-2-enyl group.
  • cycloalkyl refers to a saturated cyclic group that contains one or more rings (preferably 1 or 2; especially preferably one), and contains from 3 to 14 ring carbon atoms, preferably from 3 to 10 (especially 3, 4, 5, 6 or 7) ring carbon atoms.
  • heterocycloalkyl refers to a cycloalkyl group as defined above in which one or more (preferably 1 , 2 or 3) ring carbon atoms have been replaced by an oxygen, nitrogen, silicon, selenium, phosphorus or sulfur atom (preferably by an oxygen, sulfur or nitrogen atom) or a SO group or a SO2 group.
  • a heterocycloalkyl group has preferably 1 or 2 ring(s) (especially preferably one) and 3 to 10 (especially 3, 4, 5, 6 or 7) ring atoms (preferably selected from C, O, N and S).
  • Examples are a piperidyl, prolinyl, imidazolidinyl, piperazinyl, morpholinyl (e.g. -N(CH2CH2)2O), urotropinyl, pyrrolidinyl, tetrahydrothiophenyl, tetrahydropyranyl, tetrahydrofuryl or 2-pyrazolinyl group and also lactames, lactones, cyclic imides and cyclic anhydrides.
  • alkylcycloalkyl refers to groups that contain both cycloalkyl and alkyl, alkenyl or alkynyl groups in accordance with the above definitions, for example alkylcycloalkyl, cycloalkylalkyl, alkylcycloalkenyl, alkenylcycloalkyl and alkynylcycloalkyl groups.
  • An alkylcycloalkyl group preferably contains a cycloalkyl group that contains one or two rings and from 3 to 10 (especially 3, 4, 5, 6 or 7) ring carbon atoms, and one or two alkyl, alkenyl or alkynyl groups (especially alkyl groups) having 1 or 2 to 6 carbon atoms.
  • heteroalkylcycloalkyl refers to alkylcycloalkyl groups as defined above in which one or more (preferably 1 , 2 or 3) carbon atoms have been replaced by an oxygen, nitrogen, silicon, selenium, phosphorus or sulfur atom (preferably by an oxygen, sulfur or nitrogen atom) or a SO group or a SO2 group.
  • a heteroalkylcycloalkyl group preferably contains 1 or 2 rings having from 3 to 10 (especially 3, 4, 5, 6 or 7) ring atoms, and one or two alkyl, alkenyl, alkynyl or heteroalkyl groups (especially alkyl or heteroalkyl groups) having from 1 or 2 to 6 carbon atoms.
  • Examples of such groups are alkylheterocycloalkyl, alkylheterocycloalkenyl, alkenylheterocycloalkyl, alkynylheterocycloalkyl, heteroalkylcyclo-alkyl, heteroalkylheterocycloalkyl and heteroalkylheterocycloalkenyl, the cyclic groups being saturated or mono-, di- or tri-unsaturated.
  • aryl refers to an aromatic group that contains one or more rings and from 6 to 14 ring carbon atoms, preferably from 6 to 10 (especially 6) ring carbon atoms.
  • the expression aryl refers furthermore to groups that are substituted by fluorine, chlorine, bromine or iodine atoms or by OH, SH, NH2, N3 or NO2 groups. Examples are the phenyl (Ph), naphthyl, biphenyl, 2-fluorophenyl, anilinyl, 3-nitrophenyl or 4-hydroxyphenyl group.
  • heteroaryl refers to an aromatic group that contains one or more rings and from 5 to 14 ring atoms, preferably from 5 to 10 (especially 5 or 6 or 9 or 10) ring atoms, comprising one or more (preferably 1 , 2, 3 or 4) oxygen, nitrogen, phosphorus or sulfur ring atoms (preferably O, S or N).
  • heteroaryl refers furthermore to groups that are substituted by fluorine, chlorine, bromine or iodine atoms or by OH, SH, N3, NH2 or NO2 groups. Examples are pyridyl (e.g. 4-pyridyl), imidazolyl (e.g. 2- imidazolyl), phenylpyrrolyl (e.g.
  • aralkyl refers to groups containing both aryl and also alkyl, alkenyl, alkynyl and/or cycloalkyl groups in accordance with the above definitions, such as, for example, arylalkyl, arylalkenyl, arylalkynyl, arylcycloalkyl, arylcycloalkenyl, alkylarylcycloalkyl and alkylarylcycloalkenyl groups.
  • aralkyls are phenylcyclopentyl, cyclohexylphenyl as well as groups derived from toluene, xylene, mesitylene, styrene, benzyl chloride, o-fluorotoluene, 1 /-/-indene, tetraline, dihydronaphthalene, indanone, cumene, fluorene and indane.
  • An aralkyl group preferably contains one or two aromatic ring systems (especially 1 or 2 rings; especially preferably one ring), each containing from 6 to 10 carbon atoms and one or two alkyl, alkenyl and/or alkynyl groups containing from 1 or 2 to 6 carbon atoms and/or one or two cycloalkyl group containing 3, 4, 5, 6 or 7 ring carbon atoms.
  • heteroaralkyl refers to groups containing both aryl and/or heteroaryl groups and also alkyl, alkenyl, alkynyl and/or heteroalkyl and/or cycloalkyl and/or heterocycloalkyl groups in accordance with the above definitions containing at least one heteroatom, which is preferably selected from N, O and S.
  • a heteroaralkyl group preferably contains one or two aromatic ring systems (especially 1 or 2 rings; especially preferably one ring), each containing from 5 or 6 to 9 or 10 ring atoms (preferably selected from C, N, O and S) and one or two alkyl, alkenyl and/or alkynyl groups containing 1 or 2 to 6 carbon atoms and/or one or two heteroalkyl groups containing 1 to 6 carbon atoms and 1 , 2 or 3 heteroatoms selected from O, S and N and/or one or two cycloalkyl groups each containing 3, 4, 5, 6 or 7 ring carbon atoms and/or one or two heterocycloalkyl groups, each containing 3, 4, 5, 6 or 7 ring atoms comprising 1 , 2, 3 or 4 oxygen, sulfur or nitrogen atoms.
  • Examples are arylheteroalkyl, arylheterocycloalkyl, arylheterocycloalkenyl, arylalkylhetero-cycloalkyl, arylalkenylheterocycloalkyl, arylalkynylheterocycloalkyl, arylalkylheterocyclo-alkenyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heteroarylheteroalkyl, heteroarylcycloalkyl, heteroarylcycloalkenyl, heteroaryl- heterocycloalkyl, heteroaryl-heterocycloalkenyl, heteroarylalkylcycloalkyl, heteroarylalkylheterocycloalkenyl, heteroarylalkylcycloalkyl, heteroarylalkylheterocycloalkenyl, heteroaryl-heteroalkylcycloalkyl, hetero
  • all alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, aryl, heteroaryl, aralkyl and heteroaralkyl groups described herein may optionally be substituted.
  • halogen refers to F, Cl, Br or I.
  • aryl, heteroaryl, cycloalkyl, alkylcycloalkyl, heteroalkylcycloalkyl, heterocycloalkyl, aralkyl or heteroaralkyl group contains more than one ring
  • these rings may be bonded to each other via a single or double bond, or these rings may be annulated, fused or bridged.
  • optionally substituted refers to a group which is unsubstituted or substituted by one or more (especially by one, two or three; preferably by one or two) substituents.
  • a group comprises more than one substituent, these substituents are independently selected, i.e. they may be the same or different. If a group is substituted by a cyclic group, such as e.g., a cycloalkyl group or a heterocycloalkyl group, this cyclic group may be bonded to said group via a single or double bond or this cyclic group may be annulated or fused to said group.
  • a cyclic group such as e.g., a cycloalkyl group or a heterocycloalkyl group
  • substituents are fluorine, chlorine, bromine and iodine and OH, SH, NH2, -SO3H, -SO2NH2, C1-4 alkyl (e.g., methyl), C1-4 heteroalkyl, -COOH, -COOMe, - COMe (Ac), -NHSO 2 Me, -SO 2 NMe 2 , -CH2NH2, -NHAc, -SO 2 Me, -CONH2, -CN, - NHCONH2, -NHC(NH)NH 2 , -NOHCH3, -N 3 and -NO2 groups.
  • substituents are fluorine, chlorine, bromine and iodine and OH, SH, NH2, -SO3H, -SO2NH2, C1-4 alkyl (e.g., methyl), C1-4 heteroalkyl, -COOH, -COOMe, - COMe (Ac), -NHSO 2 Me, -SO 2 NM
  • substituents are C1-C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C1-C10 heteroalkyl, C3-C18 cycloalkyl, C1-C17 heterocycloalkyl, C4-C20 alkylcycloalkyl, C1-C19 heteroalkylcycloalkyl, Ce-C aryl, C1-C17 heteroaryl, C7-C20 aralkyl and C1-C19 heteroaralkyl groups; especially C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Ci-Ce heteroalkyl, C3-C10 cycloalkyl, C1-C9 heterocycloalkyl, C4-C12 alkylcycloalkyl, C1-C11 heteroalkylcycloalkyl, C6-C10 aryl, C1-C9 heteroaryl, C7-C12 aralky
  • the present invention further provides pharmaceutical compositions comprising one or more compounds of formula (I), (la) or (II) or a salt thereof as defined herein or a pharmaceutically acceptable ester, prodrug, hydrate or solvate thereof, optionally in combination with a pharmaceutically acceptable carrier and/or adjuvant.
  • the compounds of the present invention may be used for the treatment and/or prevention of diseases that are associated with CDK activity, such as cancer and inflammation.
  • a therapeutically effective amount of a compound in accordance with this invention means an amount of compound that is effective to prevent, alleviate or ameliorate symptoms of a disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is within the skill in the art.
  • the therapeutically effective amount or dosage of a compound according to this invention can vary within wide limits and may be determined in a manner known in the art. Such dosage may be adjusted to the individual requirements in each particular case including the specific compound being administered, the route of administration, the condition being treated, as well as the patient being treated.
  • the salt of a compound of formula (I), (la) or (II) is preferably a pharmacologically acceptable salt.
  • pharmacologically acceptable salts of sufficiently basic compounds of formula (I), (la) or (II) are salts of physiologically acceptable mineral acids like hydrochloric, hydrobromic, sulfuric and phosphoric acid; or salts of organic acids like methanesulfonic, p-toluenesulfonic, lactic, acetic, trifluoroacetic, citric, succinic, fumaric, maleic and salicylic acid.
  • a sufficiently acidic compound of formula (I), (la) or (II) may form alkali or earth alkali metal salts, for example sodium, potassium, lithium, calcium or magnesium salts; ammonium salts; or organic base salts, for example methylamine, dimethylamine, trimethylamine, triethylamine, ethylenediamine, ethanolamine, choline hydroxide, meglumin, piperidine, morpholine, tris-(2-hydroxyethyl)amine, lysine or arginine salts; all of which are also further examples of salts of formula (I), (la) or (II).
  • alkali or earth alkali metal salts for example sodium, potassium, lithium, calcium or magnesium salts; ammonium salts; or organic base salts, for example methylamine, dimethylamine, trimethylamine, triethylamine, ethylenediamine, ethanolamine, choline hydroxide, meglumin, piperidine, morpholine, tris-(2-hydroxy
  • Compounds of formula (I), (la) or (II) may be solvated, especially hydrated.
  • the hydratization/hydration may occur during the process of production or as a consequence of the hygroscopic nature of the initially water free compounds of formula (I), (la) or (II).
  • the solvates and/or hydrates may e.g. be present in solid or liquid form.
  • the compounds of formula (I), (la) or (II) may contain asymmetric C-atoms, they may be present either as achiral compounds, mixtures of diastereomers, mixtures of enantiomers or as optically pure compounds.
  • the present invention comprises both all pure enantiomers and all pure diastereomers, and also the mixtures thereof in any mixing ratio.
  • one or more hydrogen atoms of the compounds of the present invention may be replaced by deuterium.
  • Deuterium modification improves the metabolic properties of a drug with little or no change in its intrinsic pharmacology.
  • Deuterium substitution at specific molecular positions improves metabolic stability, reduces formation of toxic metabolites and/or increases the formation of desired active metabolites.
  • the present invention also encompasses the partially and fully deuterated compounds of formula (I), (la) or (II).
  • the term hydrogen also encompasses deuterium.
  • the present invention also relates to pro-drugs which are composed of a compound of formula (I), (la) or (II) and at least one pharmacologically acceptable protective group which will be cleaved off under physiological conditions, such as an alkoxy-, arylalkyloxy- , acyl-, acyloxymethyl group (e.g. pivaloyloxymethyl), an 2-alkyl-, 2-aryl- or 2-arylalkyl- oxycarbonyl-2-alkylidene ethyl group or an acyloxy group as defined herein, e.g.
  • ester especially refers to esters which hydrolyze in vivo and include those that break down readily in the human body to leave the parent compound or a salt thereof.
  • Suitable ester groups include, for example, those derived from pharmaceutically acceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, in which each alkyl or alkenyl moiety advantageously has not more than 6 carbon atoms.
  • esters include, but are not limited to, formates, acetates, propionates, butyrates, acrylates and ethylsuccinates.
  • therapeutically useful agents that contain compounds of formula (I), (la) or (II), their solvates, salts or formulations are also comprised in the scope of the present invention.
  • compounds of formula (I), (la) or (II) will be administered by using the known and acceptable modes known in the art, either alone or in combination with any other therapeutic agent.
  • such therapeutically useful agents can be administered by one of the following routes: oral, e.g. as tablets, dragees, coated tablets, pills, semisolids, soft or hard capsules, for example soft and hard gelatine capsules, aqueous or oily solutions, emulsions, suspensions or syrups, parenteral including intravenous, intramuscular and subcutaneous injection, e.g. as an injectable solution or suspension, rectal as suppositories, by inhalation or insufflation, e.g. as a powder formulation, as microcrystals or as a spray (e.g.
  • liquid aerosol transdermal
  • TDS transdermal delivery system
  • the therapeutically useful product may be mixed with pharmaceutically inert, inorganic or organic excipients as are e.g. lactose, sucrose, glucose, gelatine, malt, silica gel, starch or derivatives thereof, talc, stearinic acid or their salts, dried skim milk, and the like.
  • pharmaceutically inert, inorganic or organic excipients as are e.g. lactose, sucrose, glucose, gelatine, malt, silica gel, starch or derivatives thereof, talc, stearinic acid or their salts, dried skim milk, and the like.
  • excipients are e.g.
  • excipients e.g. water, alcohols, aqueous saline, aqueous dextrose, polyols, glycerin, lipids, phospholipids, cyclodextrins, vegetable, petroleum, animal or synthetic oils.
  • lipids and more preferred are phospholipids (preferred of natural origin; especially preferred with a particle size between 300 to 350 nm) preferred in phosphate buffered saline (pH 7 to 8, preferred 7.4).
  • excipients as are e.g.
  • the pharmaceutically useful agents may also contain additives for conservation, stabilization, e.g. UV stabilizers, emulsifiers, sweetener, aromatizers, salts to change the osmotic pressure, buffers, coating additives and antioxidants.
  • stabilization e.g. UV stabilizers, emulsifiers, sweetener, aromatizers, salts to change the osmotic pressure, buffers, coating additives and antioxidants.
  • the daily dosage can be administered as a single dose or in divided doses, or for parenteral administration, it may be given as continuous infusion or subcutaneous injection.
  • the present invention provides a method for treating one or more diseases specified herein which comprises administering to a subject in need of such treatment a therapeutically effective amount of a compound of formula (I), (la) or (II), or a pharmaceutically acceptable salt or solvate thereof.
  • the present invention provides a method for treating one or more diseases specified herein which comprises administering to a subject in need of such treatment a pharmaceutical composition comprising a compound of formula (I), (la) or (II), or a pharmaceutically acceptable salt or solvate thereof.
  • Example 1 Synthesis of 6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2- methyl-1 H-benzo[d]imidazole (1)
  • 2,4-dichloro-5-fluoropyrimidine 1.1 equiv, 22 mmol, 3.6 g
  • EtOH a mixture toluene: EtOH
  • a saturated solution of NaHCOs (30 ml)
  • 4-fluoro-1-isopropyl-2-methyl-6-(4,4,5,5-tetramethyl-1 ,3,2- dioxaborolan-2-yl)-1H-benzo[d]imidazole (1 equiv, 20 mmol, 6.3 g) were added.
  • reaction mixture was subjected to microwave irradiation for 1 h at 110°C. Then, the dark brown reaction mixture was filtered over silica under vacuum with DCM. The solvent was removed, and the obtained residue was purified by column chromatography DCM - MeOH - NH3 (85: 10: 5) to obtain the pure product.
  • the microwave vial was sealed, and it was stirred for 10min at room temperature under N2.
  • the reaction mixture was subjected to microwave irradiation for 1 h at 110°C. Then, the dark brown reaction mixture was filtered over silica under vacuum with DCM. Solvents were removed under reduced pressure and the crude was purified by column chromatography with (DCM - MeOH, 0 - 5% MeOH in DCM).
  • Example 11 Synthesis of tert-butyl 4-(2-(2,6-dioxopiperidin-3-yl)-1 ,3-dioxoisoindolin-4- yl) piperazine-1 -carboxylate (11) 1-Boc-piperazine (11 mmol, 1.1 equiv.) was added to a stirred solution of 2-(2,6- dioxopiperidin-3-yl)-4-fluoroisoindoline-1 , 3-dione (5) (10 mmol, 1 equiv) and DIPEA (20 mmol, 2 equiv.) in DMF (1 M). The reaction mixture was heated at reflux overnight.
  • Te/Y-butyl 4-(2-(2,6-dioxopiperidin-3-yl)-1 ,3-dioxoisoindolin-4-yl)piperazine-1 -carboxylate (11) was deprotected with 4 N HCI in dioxane (3 ml for 0.5 mmol scale). Stirring rt overnight. The reaction mixture was dried under reduced pressure. Diethylether was added (x2) and was removed under reduced pressure. The HCI salt of 2-(2,6- dioxopiperidin-3-yl)-4-(piperazin-1-yl) isoindoline-1 , 3-dione (0.5 mmol, 1 equiv.) was suspended in 3 ml DCM.
  • Example 23 Synthesis of 5-(4-(2-(2,6-dioxopiperidin-3-yl)-1 ,3-dioxoisoindolin-4-yl)-1 ,4- diazepan-1-yl)-3,3-dimethyl-5-oxopentanoic acid (23) (2,6-dioxopiperidin-3-yl)-1 ,3-dioxoisoindolin-4-yl)-1 ,4-diazepane-1- carboxylate (22) (1 equiv., 3 mmol), was deprotected with 4N HCI in dioxane (15 ml). Stirring at rt overnight. Solvent was removed under reduced pressure.
  • the aqua phase was acidified with 2N HCI and it was extracted with DCM (x3).
  • the combined organic phases were dried over MgSC , filtered and the solvent was removed under reduced pressure. 3.0 mmol scale; 822 mg, 1.65 mmol, yield 55 %, yellow solid.
  • Example 26 Synthesis of N-(2-(2,6-dioxopiperidin-3-yl)-1 ,3-dioxoisoindolin-4-yl)-5-(4-(6- ((5-fluoro-4-(4-fluoro-1 -isopropyl-2-methyl-1 H-benzo[d]imidazol-6-yl) pyrimidin-2-yl) amino) pyridin-3-yl) piperazin-1 -yl)-5-oxopentanamide (26)
  • Example 28 Synthesis of 2-(2,6-dioxopiperidin-3-yl)-4-(4-(5-(4-(6-((5-fluoro-4-(4-fluoro- 1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl) pyrimidin-2-yl) amino) pyridin-3-yl) piperazin-1 -yl)-5-oxopentanoyl) piperazin-1 -yl) isoindoline-1 ,3-dione (28) [lot 1 , lot 2]
  • Example 29 Synthesis of 2-(2,6-dioxopiperidin-3-yl)-4-(4-(5-(4-(6-((5-fluoro-4-(4-fluoro- 1-isopropyl-2-methyl-1 H-benzo[d]imidazol-6-yl) pyrimidin-2-yl) amino) pyridin-3-yl) piperazin-1 -yl)-3,3-dimethyl-5-oxopentanoyl) piperazin-1 -yl) isoindoline-1 ,3-dione (29)
  • Example 34 Synthesis of 2-(2,6-dioxopiperidin-3-yl)-4-(4-(4-(4-(6-((5-fluoro-4-(4-fluoro- 1-isopropyl-2-methyl-1 H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3- yl)piperazin-1 -yl)-4-oxobutanoyl)piperazin-1 -yl)isoindoline-1 ,3-dione (34)
  • Example 36 Synthesis of 2-(2,6-dioxopiperidin-3-yl)-4-(4-(2-(4-(6-((5-fluoro-4-(4-fluoro- 1-isopropyl-2-methyl-1 H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3- yl)piperazine-1-carbonyl)cyclopropane-1-carbonyl)piperazin-1-yl)isoindoline-1 , 3-dione
  • Example 37 Synthesis of 2-(2,6-dioxopiperidin-3-yl)-4-(4-(3-(4-(6-((5-fluoro-4-(4-fluoro- 1-isopropyl-2-methyl-1 H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3- yl)piperazine-1 -carbonyl)-2,2-dimethylcyclopropane-1 -carbonyl)piperazin-1 - yl)isoindoline-1, 3-dione (37)
  • Example 38 Synthesis of 2-(2,6-dioxopiperidin-3-yl)-4-(4-(5-(4-(6-((5-fluoro-4-(4-fluoro- 1-isopropyl-2-methyl-1 H-benzo[d]imidazol-6-yl) pyrimidin-2-yl) amino) pyridin-3-yl) piperazin-1 -yl)-3,3-dimethyl-5-oxopentanoyl)-1 ,4-diazepan-1-yl) isoindoline-1 ,3-dione
  • Example 39 Synthesis of N-(1-(2-(2,6-dioxopiperidin-3-yl)-1 ,3-dioxoisoindolin-4-yl) piperidin-4-yl)-5-(4-(6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1 H-benzo[d]imidazol-6- yl) pyrimidin-2-yl) amino) pyridin-3-yl) piperazin-1 -yl)-3,3-dimethyl-5-oxopentanamide
  • Example 42 Synthesis of 2-(2,6-dioxopiperidin-3-yl)-4-(4-(4-(6-(4-(6-((5-fluoro-4-(4- fluoro-1-isopropyl-2-methyl-1 H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3- yl)piperazin-1 -yl)-6-oxohex-1 -yn-1 -yl)benzoyl)piperazin-1 -yl)isoindoline-1 ,3-dione (42)
  • the aqua phase was acidified with 2N HCI and it was extracted with DCM (x3).
  • the combined organic phases were dried over MgSC , filtered and the solvent was removed under reduced pressure.
  • the obtained intermediate (44) was used directly in the next step, due to low solubility.
  • Example 45 Synthesis of 3-(4-(4-(((2-(2,6-dioxopiperidin-3-yl)-1 ,3-dioxoisoindolin-4- yl)oxy)methyl)benzyl)piperazine-1-carbonyl)-2,2-dimethylcyclopropane-1 -carboxylic acid 2-(2,6-dioxopiperidin-3-yl)-4-hydroxyisoindoline-1 ,3-dione (6) (1.02 mmol, 1 equiv.), tertbu y 4-(4-(chloromethyl)benzyl)piperazine-1 -carboxylate (7) (1.02 mmol, 1 equiv.), potassium carbonate (2.04 mmol, 2 equiv.), and sodium iodide (1 .02 mmol, 1 equiv.) were suspended in acetonitrile (10 ml).
  • the reaction mixture was heated at 40 °C for 1 h.
  • the reaction was allowed to reach rt and it was extracted x2 (DCM - H2O).
  • the aqua phase was acidified with 2N HCI and it was extracted with DCM (x3).
  • the combined organic phases were dried over MgSO4, filtered and the solvent was removed under reduced pressure.
  • the obtained intermediate (45) was used directly in the next step, due to low solubility.
  • Example 48 Synthesis of 4-fluoro-2-(1-methyl-2,6-dioxopiperidin-3-yl)isoindoline-1 ,3- dione (48) 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1 , 3-dione (5) (2 mmol, 1.0 equiv.) and K2CO3 (2.4 mmol, 1.2 equiv.) were dissolved in DMF (8 ml). Then, iodomethane (2.4 mmol, 1.2 equiv.) was carefully added into the reaction mixture. The reaction mixture was stirred at 60 °C for 3 hours.
  • Example 51 Synthesis of 4-(4-(5-(4-(6-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1 H- benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)pyridin-3-yl)piperazin-1-yl)-5- oxopentanoyl)piperazin-1 -y l)-2-( 1 -methyl-2,6-dioxopiperidin-3-yl)isoindoline-1 ,3-dione
  • the neuroblastoma cell lines CHP-134, GI-M-EN, NGP, SJNB-1, SJNB-6 and TR14 and the breast cancer cell lines SK-BR-3 and MDA-MB-231 were acquired from the American Type Culture Collection or via historic collaborations.
  • All cells, except MDA-MB-231 were cultured in DMEM high glucose (41965, Life Technologies), supplemented with 10% (v/v) FBS (F0804, Sigma-Aldrich), 2 mM L-glutamine (25030081 , Life Technologies), 1x MEM- NEAA (11140050, Life Technologies) and 100 u/mL penicillin and 100 mg/mL streptomycin (15140122, Life Technologies) at 37°C in a humidified environment containing 5% CO2.
  • DMEM high glucose 41965, Life Technologies
  • F0804 Sigma-Aldrich
  • 2 mM L-glutamine 25030081 , Life Technologies
  • 1x MEM- NEAA 11140050, Life Technologies
  • penicillin and 100 mg/mL streptomycin 15140122, Life Technologies
  • MDA-MB-231 was cultured in Leibovitz L15 (11415, Life Technologies), supplemented with 10% (v/v) FBS (F0804, Sigma-Aldrich) and 100 u/mL penicillin and 100 mg/mL streptomycin (15140122, Life Technologies) at 37°C in a humidified environment containing 0% CO2. The identity of all cell lines was verified with short tandem repeat (STR) profiling and the cultures were regularly checked for mycoplasma contamination.
  • STR short tandem repeat
  • the KINOMEscan was performed using the scanMAX kinase assay panel by Eurofins Discovery. Images were generated using TREEspotTM Software Tool and reprinted with permission from KINOMEscan®, a division of DiscoveRx Corporation, ⁇ DISCOVERX CORPORATION 2010.
  • Cells were lysed in 1x Laemmli buffer (100 mM Tris-HCI, 4% (w/v) SDS, 20% (v/v) glycerol, pH 6.8) supplemented with complete Protease Inhibitor Cocktail (11836153001 , Roche Diagnostics).
  • the cell lysates were passed through 23g and 27g Microlance needles (300800, 300635 (respectively), BD Biosciences) consecutively and heated at 50°C for 10 minutes. Protein concentration was determined using the DC protein assay (5000112, Bio-Rad) according to manufacturer’s protocol.
  • Protein samples were prepared by adding 5x loading buffer (250 mM Tris-HCI, 10% (w/v) SDS, 50% (v/v) glycerol, 500 mM DTT, 0.25% (w/v) bromophenol blue, pH 6.8), after which samples were boiled at 95°C for 5 minutes. 10 ug protein was loaded on 10% Mini-PROTEAN TGX stain-free gels (4568036, Bio-Rad), which were run in 1x running buffer (5 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3).
  • 5x loading buffer 250 mM Tris-HCI, 10% (w/v) SDS, 50% (v/v) glycerol, 500 mM DTT, 0.25% (w/v) bromophenol blue, pH 6.8
  • 10 ug protein was loaded on 10% Mini-PROTEAN TGX stain-free gels (4568036, Bio-Rad), which were run in 1x running buffer
  • Proteins were transferred onto PVDF membranes (1704273, Trans-Blot Turbo RTA Transfer kit, Bio-Rad) using the Trans-Blot Turbo Transfer System (Bio-Rad) according to manufacturer’s protocol (Mixed MW protocol).
  • Membranes were blocked in 1x TBS-T (20 mM Tris-HCI, 150 mM NaCI, 0.1 % (v/v) Tween20) supplemented with 2% (w/v) Amersham ECL Prime Blocking Reagent (RPN418, GE Healthcare Life Sciences) for 1 hour. Next, the membranes were incubated with primary antibody overnight at 4°C, followed by incubation with secondary antibody for 1 hour at RT. Membranes were incubated with Amersham ECL Prime Detection Reagent (RPN2232, GE Healthcare Life Sciences) and visualized by chemiluminescence on the ChemiDoc imager (Bio-Rad).
  • the data was normalization to the DMSO-treated cells (defined as 100% viability) and the empty controls (0% viability) and curves were fit using the extension package drc in the statistic environment of R Studio (version 4.0.2) 2 .
  • the half maximal concentration that inhibits the viability (IC50) and the area under the curve (AUC) were determined.
  • HTS High-throughput screening
  • Abemaciclib-based PROTACs exhibit differential efficacy depending on the neuroblastoma cell line.
  • AUC area-under-the-curve
  • three sets of abemaciclib-based PROTACs (numbered compounds) could be identified based on their efficacy (highly efficacious (bottom), moderately efficacious (middle), and non-efficacious (top).
  • Each set shows an increased effect in cell lines with high CRBN E3 ligase expression (NGP, SJNB6, CHP134 (left)) compared to low CRBN E3 ligase expressing cell lines (SJNB1, GIMEN, TR14 (right)), except in case of the non-efficacious set.
  • CDK4/6 inhibitors (abemaciclib (2), palbociclib, ribociclib), commercially available CDK4/6 PROTACs (compounds starting with BSJ and CP-10), CRBN inhibitors (pomalidomide, thalidomide(-4-OH)) and an inactive PROTAC (51) (inactive variant of (28)) were taken along for comparison.
  • the CDK4/6 inhibitors are either highly or moderately efficacious, whereas the latter two are non-efficacious. (51) is moderately efficacious but is outperformed by its active counterpart (28).
  • FIG. 1 Abemaciclib-based PROTACs reduce cell viability more effectively than CDK4/6 inhibitors and commercially available CDK4/6 PROTACs in neuroblastoma cell lines.
  • A Differential effect on cell viability upon (29) treatment in 6 neuroblastoma cell lines. NGP, SJNB6 and CHP134 are highly responsive, whereas SJNB1 has a moderate response and TR14 and GIMEN are relatively unresponsive.
  • B. Top Top.
  • FIG. 3 Degradation of CDK4/6/9 upon treatment with abemaciclib-based PROTACs in a concentration- and time-dependent manner.
  • A. (29) (top) and (28, lot 1) (bottom) induce CDK4 and CDK6 degradation after 24 h treatment at concentrations as low as 0.1 and 10 nM, respectively.
  • B. CDK9 is degraded upon treatment with (29) and (28, lot 1) for 24 h at 10 and 1000 nM, respectively, whilst CDK2 is unaffected.
  • C Time series showing differential, but persistent CDK4/6/9 degradation patterns after (29) treatment at 100 nM.
  • FIG. 1 Abemaciclib-based PROTACs reduce cell viability more effectively than CDK4/6 inhibitors and commercially available CDK4/6 PROTACs in HER2-positive (HER2+) and triple negative (TNBC) breast cancer cell lines.
  • CDKs are generally involved in regulation of the cell cycle (e.g. CDK4/6) or transcription regulation (e.g. CDK9).
  • deregulated CDK signaling is often implicated in cancer to allow for uncontrolled proliferation (Roskoski. Cyclin-Dependent Protein Serine/Threonine Kinase Inhibitors as Anticancer Drugs. Pharmacol. Res. 2019. 139. 471-488.; Chou et al. Transcription-Associated Cyclin-Dependent Kinases as Targets and Biomarkers for Cancer Therapy. Cancer Discov. 2020. 10. 351-370.). Therefore, hampering CDK signaling shows great promise for cancer therapy, as illustrated by the FDA approval of CDK4/6 inhibitors to treat hormone receptor-positive, human epidermal growth factor receptor 2-negative advanced breast cancer.
  • CDK4 and CDK6 are directly involved in cell cycle progression and as such, they are the regulators with the most direct effect on cell division (Roskoski. Cyclin-Dependent Protein Serine/Threonine Kinase Inhibitors as Anticancer Drugs. Pharmacol. Res. 2019. 139. 471-488.).
  • CDK9 regulates transcription elongation and shows particular importance in cancers dependent on oncogenic transcriptional networks (e.g. MYC(N)-driven cancers) that drive cancer proliferation (Chou et al. Transcription-Associated Cyclin-Dependent Kinases as Targets and Biomarkers for Cancer Therapy. Cancer Discov. 2020. 10. 351- 370.).
  • CDK9 also regulates the expression of anti-apoptotic proteins, contributing to resistance to cell death (Whittaker et al. Molecular Profiling and Combinatorial Activity of CCT068127: a Potent CDK2 and CDK9 Inhibitor. Mol. Oncol. 2018. 12. 287-304.).
  • CDK inhibitors demand further development of treatment modalities, such as PROTAC degraders. Additionally, degradation of CDK9 was shown to have greater impact on hampering the MYC-dependent oncogenic transcriptional network and subsequently, cancer cell proliferation than CDK9 inhibition (Toure et al. Targeted Degradation of CDK9 Potently Disrupts the MYC Transcriptional Network. bioRxiv. 2024.).

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Abstract

La présente invention concerne de nouveaux agents de dégradation de CDK de formule générale (I). Ces nouveaux composés sont utiles dans le traitement de maladies qui sont associées à l'activité de CDK, telles que le cancer et l'inflammation.
PCT/EP2024/066424 2023-06-13 2024-06-13 Nouveaux agents de dégradation de cdk Ceased WO2024256569A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018106870A1 (fr) * 2016-12-08 2018-06-14 Icahn School Of Medicine At Mount Sinai Compositions et méthodes pour le traitement du cancer à médiation par cdk4/6
WO2020023480A1 (fr) * 2018-07-23 2020-01-30 Dana-Farber Cancer Institute, Inc. Dégradation de la kinase 4/6 dépendante de la cycline (cdk4/6) par conjugaison d'inhibiteurs de cdk4/6 avec un ligand de type ligase e3 et procédés d'utilisation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018106870A1 (fr) * 2016-12-08 2018-06-14 Icahn School Of Medicine At Mount Sinai Compositions et méthodes pour le traitement du cancer à médiation par cdk4/6
WO2020023480A1 (fr) * 2018-07-23 2020-01-30 Dana-Farber Cancer Institute, Inc. Dégradation de la kinase 4/6 dépendante de la cycline (cdk4/6) par conjugaison d'inhibiteurs de cdk4/6 avec un ligand de type ligase e3 et procédés d'utilisation

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CHOU ET AL.: "Transcription-Associated Cyclin-Dependent Kinases as Targets and Biomarkers for Cancer Therapy", CANCER DISCOV, vol. 10, 2020, pages 351 - 370
M.JIANG, B.ERB, M. A ET AL.: "Pharmacological Perturbation of CDK9 Using Selective CDK9 Inhibition or Degradation", NAT. CHEM. BIOL, vol. 14, 2018, pages 163 - 170, XP055820762, DOI: 10.1038/nchembio.2538
RITZ, C.BATY, F.STREIBIG, J. C.GERHARD, D: "Dose-Response Analysis Using R", PLOS ONE, vol. 10, 2015, pages 0146021
ROSKOSKI: "Cyclin-Dependent Protein Serine/Threonine Kinase Inhibitors as Anticancer Drugs", PHARMACOL. RES., vol. 139, 2019, pages 471 - 488
TOURE ET AL.: "Targeted Degradation of CDK9 Potently Disrupts the MYC Transcriptional Network", BIORXIV, 2024
WHITTAKER ET AL.: "Molecular Profiling and Combinatorial Activity of CCT068127: a Potent CDK2 and CDK9 Inhibitor", MOL. ONCOL., vol. 12, 2018, pages 287 - 304

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