WO2024066981A1 - 氘代吡唑类衍生物、药物组合物及应用与制备方法 - Google Patents

氘代吡唑类衍生物、药物组合物及应用与制备方法 Download PDF

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WO2024066981A1
WO2024066981A1 PCT/CN2023/117218 CN2023117218W WO2024066981A1 WO 2024066981 A1 WO2024066981 A1 WO 2024066981A1 CN 2023117218 W CN2023117218 W CN 2023117218W WO 2024066981 A1 WO2024066981 A1 WO 2024066981A1
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atoms
deuterated
compound
branched
cyclic
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PCT/CN2023/117218
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English (en)
French (fr)
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胡璞
陆居权
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楚浦创制（武汉）医药科技有限公司
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Publication of WO2024066981A1 publication Critical patent/WO2024066981A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • 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
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
    • C07D231/02—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
    • C07D231/10—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D231/14—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D231/38—Nitrogen atoms
    • C07D231/40—Acylated on said nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • the present invention relates to the field of medical technology, and in particular to a deuterated pyrazole derivative, a pharmaceutically acceptable salt, a stereoisomer, a pharmaceutical composition, and an application and preparation method thereof.
  • CDKs Cyclin-dependent kinases
  • CDK2 activity disorders often occur in many human cancers, so CDK2 is of interest to researchers.
  • CDK2 plays a key role in promoting G1/S transition and S phase progression.
  • CDK2 forms a complex with Cyclin E, phosphorylates retinoblastoma family members (pRb, etc.), leads to the release and activation of E2F transcription factors, promotes the transition of the cell cycle from G1 phase to S phase, and then activates CDK2/Cyclin A, promoting cell cycle DNA synthesis, replication and other processes.
  • Cyclin E1 Increased copy number and overexpression of Cyclin E1 have been identified in ovarian cancer, gastric cancer, endometrial cancer, breast cancer and other cancers, and are positively correlated with poor prognosis of the corresponding tumors.
  • high expression of Cyclin E2 is often accompanied by resistance to hormone therapy, and amplification or overexpression of Cyclin E is closely related to poor prognosis of breast cancer.
  • HER2+ breast cancer amplification of Cyclin E has also been reported to contribute to resistance to trastuzumab.
  • overexpression of Cyclin E also plays an important role in the progression of triple-negative breast cancer or inflammatory breast cancer. Therefore, CDK2 may become an important anti-tumor target.
  • the present invention provides a deuterated pyrazole derivative with high CDK2 inhibitory activity, and a pharmaceutically acceptable salt or stereoisomer thereof.
  • the present invention is achieved through the following technical solutions.
  • R is selected from linear deuterated alkyl having 1 to 20 C atoms, branched deuterated alkyl having 3 to 20 C atoms, cyclic deuterated alkyl having 3 to 20 C atoms, linear deuterated alkoxy having 1 to 20 C atoms, branched deuterated alkoxy having 3 to 20 C atoms, cyclic deuterated alkoxy having 3 to 20 C atoms, or a combination of these systems;
  • R 2 , R 3 , and R 4 are each independently selected from -H, -D, a linear alkyl group having 1 to 20 C atoms, a linear deuterated alkyl group having 1 to 20 C atoms, a branched alkyl group having 3 to 20 C atoms, a branched deuterated alkyl group having 3 to 20 C atoms, a cyclic alkyl group having 3 to 20 C atoms, a cyclic deuterated alkyl group having 3 to 20 C atoms, a linear alkoxy group having 1 to 20 C atoms, a linear deuterated alkoxy group having 1 to 20 C atoms, a branched alkoxy group having 3 to 20 C atoms, a branched deuterated alkoxy group having 3 to 20 C atoms, a cyclic alkoxy group having 3 to 20 C atoms, a cyclic deuterated alkoxy group having 3 to 20 C atoms, or a combination
  • the compound of formula (I) is a compound of formula (II):
  • R3 is selected from -H or -D.
  • R 1 is selected from a linear deuterated alkyl group having 1 to 10 C atoms, a branched deuterated alkyl group having 3 to 10 C atoms, a cyclic deuterated alkyl group having 3 to 10 C atoms, a linear deuterated alkoxy group having 1 to 10 C atoms, a branched deuterated alkoxy group having 3 to 10 C atoms, a cyclic deuterated alkoxy group having 3 to 10 C atoms, or a combination of these systems;
  • R 2 and R 4 are each independently selected from -H, -D, a straight-chain alkyl group having 1 to 10 C atoms, a straight-chain deuterated alkyl group having 1 to 10 C atoms, a branched-chain alkyl group having 3 to 10 C atoms, a branched-chain deuterated alkyl group having 3 to 10 C atoms, a cyclic alkyl group having 3 to 10 C atoms, a cyclic deuterated alkyl group having 3 to 10 C atoms, a straight-chain alkoxy group having 1 to 10 C atoms, a straight-chain deuterated alkoxy group having 1 to 10 C atoms, a branched-chain alkoxy group having 3 to 10 C atoms, a branched-chain deuterated alkoxy group having 3 to 10 C atoms, a cyclic alkoxy group having 3 to 10 C atoms, a cyclic alkoxy group having 3 to 10 C
  • R1 is selected from a linear deuterated alkyl group having 1 to 5 C atoms, a branched deuterated alkyl group having 3 to 5 C atoms, a cyclic deuterated alkyl group having 3 to 5 C atoms, a linear deuterated alkoxy group having 1 to 5 C atoms, a branched deuterated alkoxy group having 3 to 5 C atoms, a cyclic deuterated alkoxy group having 3 to 5 C atoms, or a combination of these systems;
  • R 2 and R 4 are independently selected from linear alkyl having 1 to 3 C atoms, linear deuterated alkyl having 1 to 3 C atoms, branched alkyl having 3 to 5 C atoms, branched deuterated alkyl having 3 to 5 C atoms, cyclic alkyl having 3 to 5 C atoms, cyclic deuterated alkyl having 3 to 5 C atoms, or a combination of these systems.
  • the compound of formula (I) is the following compound:
  • the present invention also provides a method for preparing the compound represented by formula (I), comprising the following steps:
  • R 1 , R 2 , R 3 and R 4 are consistent with those described above;
  • X is selected from -F, -Cl, -Br or -I.
  • the present invention also provides a use of the compound as described above, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof in the preparation of a drug for treating and/or preventing a disease associated with or mediated by CDK2 activity.
  • the disease associated with or mediated by CDK2 activity is cancer.
  • the present invention also provides a pharmaceutical composition, comprising the compound as described above, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, and a pharmaceutically acceptable carrier.
  • the compound of the present invention or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof has the following beneficial effects:
  • the deuterated pyrazole derivatives of the present invention have unexpectedly high inhibitory activity against CDK2, and can significantly improve the pharmacokinetic effect and reduce side effects. They can be used as drugs for treating and/or preventing diseases related to CDK2 activity or mediated by CDK2 activity, and can be used to treat various cancers with high expression of Cyclin E. They have potential excellent therapeutic effects on diseases mediated by CDK2 activity.
  • first and second are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, a feature defined as “first” or “second” may explicitly or implicitly include at least one of the features.
  • the meaning of “multiple” is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.
  • severeal is at least one, such as one, two, etc., unless otherwise clearly and specifically defined.
  • compositions and methods/processes of the present invention comprise, consist of, and consist essentially of the essential elements and limitations described herein, as well as any additional or optional ingredients, components, steps, or limitations described herein. No distinction is made herein between the terms “efficacy,””performance,””effect,” and “efficacy.”
  • pharmaceutically acceptable refers to those compounds, materials, compositions and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable salt refers to salts of compounds of the invention, prepared from compounds of the invention having specific substituents with relatively nontoxic acids or bases.
  • base addition salts can be obtained by contacting such compounds with a sufficient amount of base in a pure solution or a suitable inert solvent.
  • Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts.
  • acid addition salts can be obtained by contacting such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent.
  • Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, such as formic acid, acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid, and also include salts of amino acids (such as arginine, etc.), and salts of organic acids such as glucuronic acid.
  • Certain specific compounds of the present invention contain basic and acidic functional groups, and thus can
  • salts of the present invention can be synthesized by conventional chemical methods from parent compounds containing acid radicals or bases. Generally, the preparation method of such salts is: in water or an organic solvent or a mixture of the two, these compounds in free acid or base form are reacted with a stoichiometric amount of an appropriate base or acid to prepare.
  • the compounds of the present invention may exist in specific geometric or stereoisomeric forms.
  • the present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present invention.
  • Additional asymmetric carbon atoms may be present in substituents such as alkyl. All of these isomers and their mixtures are included within the scope of the present invention.
  • enantiomer or “optical isomer” refers to stereoisomers that are mirror images of one another.
  • cis-trans isomers or “geometric isomers” refer to the different Can rotate freely.
  • diastereomer refers to stereoisomers that have two or more chiral centers and that are not mirror images of each other.
  • the key is a solid wedge. and dotted wedge key To indicate the absolute configuration of a stereocenter, use a straight solid bond. and straight dashed key To indicate the relative configuration of a stereocenter, use a wavy line Denotes a solid wedge bond or dotted wedge key Or use a wavy line Represents a straight solid bond and straight dashed key
  • a compound contains a double bond structure, such as a carbon-carbon double bond, a carbon-nitrogen double bond, and a nitrogen-nitrogen double bond, and each atom on the double bond is connected to two different substituents (in a double bond containing a nitrogen atom, a lone pair of electrons on the nitrogen atom is regarded as a substituent connected to it), if a wavy line is used between the atom on the double bond and its substituent in the compound, If connected, it means the (Z) isomer, (E) isomer or a mixture of the two isomers of the compound.
  • formula (A) means that the compound exists in the form of a single isomer of formula (A-1) or formula (A-2) or in the form of a mixture of two isomers of formula (A-1) and formula (A-2);
  • formula (B) means that the compound exists in the form of a single isomer of formula (B-1) or formula (B-2) or in the form of a mixture of two isomers of formula (B-1) and formula (B-2).
  • formula (C) means that the compound exists in the form of a single isomer of formula (C-1) or formula (C-2) or in the form of a mixture of two isomers of formula (C-1) and formula (C-2).
  • tautomer or “tautomeric form” refers to isomers of different functional groups that are in dynamic equilibrium at room temperature and can readily interconvert. If tautomerism is possible (e.g., in solution), chemical equilibrium of tautomers can be achieved.
  • proton tautomers also called prototropic tautomers
  • proton migration such as keto-enol isomerization and imine-enamine isomerization.
  • Valence isomers are tautomers that undergo interconversion by reorganization of some of the bonding electrons.
  • keto-enol tautomerization is the interconversion between pentane-2,4-dione and 4-hydroxypent-3-en-2-one.
  • the terms “enriched in one isomer”, “isomerically enriched”, “enriched in one enantiomer” or “enantiomerically enriched” mean that the content of one isomer or enantiomer is less than 100%, and the content of the isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.
  • the term “isomer excess” or “enantiomeric excess” refers to the difference between the relative percentages of two isomers or two enantiomers. For example, if the content of one isomer or enantiomer is 90% and the content of the other isomer or enantiomer is 10%, the isomer or enantiomeric excess (ee value) is 80%.
  • Optically active (R)- and (S)-isomers and D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer.
  • a diastereomeric salt is formed with an appropriate optically active acid or base, and then the diastereoisomers are separated by conventional methods known in the art, and then the pure enantiomer is recovered.
  • the separation of enantiomers and diastereomers is usually accomplished by using chromatography, which uses a chiral stationary phase and is optionally combined with a chemical derivatization method (for example, a carbamate is generated from an amine).
  • the compounds of the present invention may contain non-natural proportions of atomic isotopes on one or more atoms constituting the compound.
  • the compound may be labeled with a radioactive isotope, such as tritium ( 3H ), iodine-125 ( 125I ) or C-14 ( 14C ).
  • deuterated drugs may be formed by replacing hydrogen with heavy hydrogen. The bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon. Compared with undeuterated drugs, deuterated drugs have the advantages of reducing toxic side effects, increasing drug stability, enhancing therapeutic effects, and extending the biological half-life of drugs. All isotopic composition changes of the compounds of the present invention, whether radioactive or not, are included in the scope of the present invention.
  • substituted means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, which may include deuterium and hydrogen variants, as long as the valence state of the particular atom is normal and the substituted compound is stable.
  • the type and number of substituents can be any on the basis of chemical practicability.
  • Atoms are replaced. Oxygen substitution does not occur on aromatic groups.
  • any variable e.g., R 1
  • its definition at each occurrence is independent.
  • the group may be optionally substituted with up to two R 1 s , and each occurrence of R 1 is an independent choice.
  • substituents and/or variants thereof are permitted only if such combinations result in stable compounds.
  • linking group When the number of a linking group is 0, such as -(CRR) 0 -, it means that the linking group is a single bond.
  • substituent When a substituent is vacant, it means that the substituent does not exist. For example, when X in A-X is vacant, it means that the structure is actually A. When the listed substituent does not specify which atom it is connected to the substituted group through, the substituent can be bonded through any atom of it. For example, pyridyl as a substituent can be connected to the substituted group through any carbon atom on the pyridine ring.
  • linking group L When the linking group is listed without specifying its linking direction, its linking direction is arbitrary, for example,
  • the connecting group L is -MW-, in which case -MW- can connect ring A and ring B in the same direction as the reading order from left to right to form You can also connect ring A and ring B in the opposite direction of the reading order from left to right to form Combinations of linkers, substituents, and/or variations thereof are permissible only if such combinations result in stable compounds.
  • any one or more sites of the group can be connected to other groups through chemical bonds.
  • the chemical bond connection mode is non-positional and there are H atoms at the connectable sites, when the chemical bonds are connected, the number of H atoms at the site will decrease accordingly with the number of connected chemical bonds to become a group with a corresponding valence.
  • the chemical bond connecting the site to other groups can be a straight solid bond.
  • the straight solid bond in -OCH 3 indicates that it is connected to other groups through the oxygen atom in the group;
  • the straight dashed bond in the group indicates that the two ends of the nitrogen atom in the group are connected to other groups;
  • the wavy line in the phenyl group indicates that it is connected to other groups through the carbon atoms at positions 1 and 2 in the phenyl group; surface It indicates that any connectable site on the piperidine group can be connected to other groups through one chemical bond, including at least These four connection methods, even if the H atom is drawn on -N-, Still includes For groups connected in this way, when one chemical bond is connected, the H at that site will be reduced by one and become the corresponding monovalent piperidine group.
  • the number of atoms in a ring is generally defined as the ring member number, for example, "3-7 membered ring” refers to a “ring” having 3-7 atoms arranged around it.
  • C 1-6 alkyl is used to represent a straight or branched saturated hydrocarbon group consisting of 1 to 6 carbon atoms. Preferably, it is a C 1-4 alkyl group, which may be monovalent (such as methyl), divalent (such as methylene) or polyvalent (such as methine).
  • Examples of C 1-3 alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, tert-butyl and sec-butyl).
  • C 1-6 alkoxy refers to those alkyl groups containing 1 to 6 carbon atoms connected to the rest of the molecule through an oxygen atom. Preferably, it is C 1-3 alkoxy. Examples of C 1-3 alkoxy include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), and the like.
  • C 1-6 alkylamino refers to those alkyl groups containing 1 to 6 carbon atoms which are attached to the rest of the molecule via an amino group. Preferably, it is a C 1-3 alkylamino group.
  • Examples of C 1-3 alkylamino groups include, but are not limited to, -NHCH 3 , -N(CH 3 ) 2 , -NHCH 2 CH 3 , -N(CH 3 )CH 2 CH 3 , -NHCH 2 CH 2 CH 3 , -NHCH 2 (CH 3 ) 2 and the like.
  • halo or halogen, by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.
  • 5-membered heteroaromatic ring and “5-membered heteroaryl” are used interchangeably in the present invention.
  • the term “5-membered heteroaryl” refers to a monocyclic group with a conjugated ⁇ electron system consisting of 5 ring atoms, 1, 2, 3 or 4 of which are heteroatoms independently selected from O, S and N, and the rest are carbon atoms.
  • the nitrogen atom is optionally quaternized, and the nitrogen and sulfur heteroatoms are optionally oxidized (i.e., NO and S(O) p , p is 1 or 2).
  • the 5-membered heteroaryl can be connected to the rest of the molecule through a heteroatom or a carbon atom.
  • Examples of the 5-membered heteroaryl group include, but are not limited to, pyrrolyl (including N-pyrrolyl, 2-pyrrolyl and 3-pyrrolyl, etc.), pyrazolyl (including 2-pyrazolyl and 3-pyrazolyl, etc.), imidazolyl (including N-imidazolyl, 2-imidazolyl, 4-imidazolyl and 5-imidazolyl, etc.), oxazolyl (including 2-oxazolyl, 4-oxazolyl and 5-oxazolyl, etc.), triazolyl (1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl, 1H-1,2,4-triazolyl and 4H-1,2,4-triazolyl, etc.), tetrazolyl, isoxazolyl (3-isoxazolyl, 4-isoxazolyl and 5-isoxazolyl, etc.), thiazolyl (including 2-thiazolyl, 4-thiazo
  • Cn-n+m or Cn - Cn+m includes any specific case of n to n+m carbon atoms, for example, C1-12 includes C1 , C2 , C3 , C4 , C5 , C6 , C7 , C8 , C9 , C10 , C11 , and C12 , and also includes any range from n to n+m, for example, C1-12 includes C1-3 , C1-6 , C1-9, C3-6 , C3-9 , C3-12 , C6-9 , C6-12 , and C13 .
  • n-membered to n+m-membered means that the number of atoms in the ring is n to n+m
  • 3-12-membered ring includes 3-membered ring, 4-membered ring, 5-membered ring, 6-membered ring, 7-membered ring, 8-membered ring, 9-membered ring, 10-membered ring, 11-membered ring, and 12-membered ring, and also includes any range from n to n+m, for example, 3-12-membered ring includes 3-6-membered ring, 3-9-membered ring, 5-6-membered ring, 5-7-membered ring, 6-7-membered ring, 6-8-membered ring, and 6-10-membered ring, etc.
  • C 3-7 cycloalkyl means a saturated cyclic hydrocarbon group consisting of 3 to 7 carbon atoms, including monocyclic and bicyclic systems, wherein the bicyclic system includes spirocyclic, fused and bridged rings.
  • the C 3-7 cycloalkyl includes C 3-6 , C 4-6 , C 4-5 , C 5-7 or C 5-6 cycloalkyl, etc.; it can be monovalent, divalent or polyvalent.
  • Examples of C 3-7 cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, etc.
  • the term "3-7 membered heterocycloalkyl" by itself or in combination with other terms refers to a saturated cyclic group consisting of 3 to 7 ring atoms, 1, 2, 3 or 4 of which are heteroatoms independently selected from O, S and N, and the rest are carbon atoms, wherein the nitrogen atom is optionally quaternized, and the nitrogen and sulfur heteroatoms may be optionally oxidized (i.e., NO and S(O) p , p is 1 or 2). It includes monocyclic and bicyclic ring systems, wherein the bicyclic ring system includes spirocyclic, paracyclic and bridged rings.
  • heteroatoms may occupy the position where the heterocycloalkyl is connected to the rest of the molecule.
  • the 3-7 membered heterocycloalkyl includes 5-7 membered, 3 membered, 4 membered, 5 membered, 6 membered and 7 membered heterocycloalkyl, etc.
  • 3-7 membered heterocycloalkyl groups include, but are not limited to, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl, tetrahydrothiophenyl (including tetrahydrothiophen-2-yl and tetrahydrothiophen-3-yl, etc.), tetrahydrofuranyl (including tetrahydrofuran-2-yl, etc.), tetrahydropyranyl, piperidinyl (including 1-piperidinyl, 2-piperidinyl and 3-piperidinyl, etc.), piperazinyl (including 1-piperazinyl and 2-piperazinyl, etc.), morpholinyl (including 3-morpholinyl and 4-morpholinyl, etc.), dioxanyl, dithianyl, isoxazolidinyl, isothiazolidinyl
  • 3-7 membered nitrogen-containing heterocycloalkyl refers to a 3-7 membered heterocycloalkyl group containing at least one nitrogen atom.
  • Alicyclic refers to a saturated or partially unsaturated all-carbon ring system. Wherein “partially unsaturated” refers to a ring portion including at least one double bond or triple bond, and “partially unsaturated” is intended to cover rings with multiple unsaturated sites, but is not intended to include aryl or heteroaryl moieties as defined herein.
  • Non-limiting examples include cyclopropyl ring, cyclobutyl ring, cyclopentyl ring, cyclopentenyl ring, cyclohexyl ring, cyclohexenyl ring, cyclohexadienyl ring, cycloheptyl ring, cycloheptatrienyl ring, cyclopentanone ring, cyclopentane-1,3-dione ring, etc.
  • Alicyclic group refers to a saturated or partially unsaturated alicyclic group in which 1, 2 or 3 ring carbon atoms are replaced by heteroatoms selected from nitrogen, oxygen or S(O) t (wherein t is an integer from 0 to 2), but does not include the ring part of -OO-, -OS- or -SS-, and the remaining ring atoms are Carbon.
  • Non-limiting examples include an oxetane ring, an azetidine ring, an oxetane ring, a tetrahydrofuran ring, a tetrahydrothiophene ring, a tetrahydropyrrole ring, a piperidine ring, a pyrroline ring, an oxazolidine ring, a piperazine ring, a dioxolane ring, a dioxane ring, a morpholine ring, a thiomorpholine ring, a thiomorpholine-1,1-dioxide, a tetrahydropyran ring, an azetidine-2-one ring, an oxetane-2-one ring, a pyrrolidine-2-one ring, a pyrrolidine-2,5-dione ring, a piperidine-2-one ring, a dihydrofur
  • Non-limiting examples of partially unsaturated monocyclic heterocycles include 1,2-dihydroazetidine ring, 1,2-dihydrooxetadiene ring, 2,5-dihydro-1H-pyrrole ring, 2,5-dihydrofuran ring, 2,3-dihydrofuran ring, 2,3-dihydro-1H-pyrrole ring, 3,4-dihydro-2H-pyran ring, 1,2,3,4-tetrahydropyridine ring, 3,6-dihydro-2H-pyran ring, 1,2,3,6-tetrahydropyridine ring, 4,5-dihydro-1H-imidazole ring, 1,4,5,6-tetrahydropyrimidine ring, 3,4,7,8-tetrahydro-2H-1,4,6-oxadiazolidine ring, 1,6-dihydropyrimidine ring, 4,5,6,7-tetrahydro-1H-1,3-diazapin
  • Aryl and “aromatic ring” are used interchangeably, and both refer to an all-carbon monocyclic or fused polycyclic (i.e., rings that share adjacent pairs of carbon atoms) group with a conjugated ⁇ electron system, which group may be fused with a cycloalkyl ring, a heterocycloalkyl ring, a cycloalkenyl ring, a heterocycloalkenyl ring, or a heteroaryl group.
  • C6-10 aryl refers to a monocyclic or bicyclic aromatic group having 6 to 10 carbon atoms, and non-limiting examples of aryl include phenyl, naphthyl, and the like.
  • Heteroaryl and “heteroaryl ring” are used interchangeably and refer to a monocyclic, bicyclic or polycyclic 4n+2 aromatic ring system (e.g., having 6 or 10 ⁇ electrons shared in a cyclic arrangement) having ring carbon atoms and ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur.
  • heteroaryl also includes a ring system in which the above-mentioned heteroaryl ring is fused with one or more cycloalkyl rings, heterocycloalkyl rings, cycloalkenyl rings, heterocycloalkenyl rings or aromatic rings.
  • the heteroaryl ring may be optionally substituted.
  • “5 to 10 membered heteroaryl” refers to a monocyclic or bicyclic heteroaryl having 5 to 10 ring atoms, wherein 1, 2, 3 or 4 ring atoms are heteroatoms.
  • “5- to 6-membered heteroaryl” refers to a monocyclic heteroaryl group having 5 to 6 ring atoms, wherein 1, 2, 3 or 4 of the ring atoms are heteroatoms, non-limiting examples of which include thienyl, furanyl, thiazolyl, isothiazolyl, imidazolyl, oxazolyl, pyrrolyl, pyrazolyl, triazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl, tetrazolyl, isoxazolyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-o
  • 8- to 10-membered heteroaryl refers to a bicyclic heteroaryl group having 8 to 10 ring atoms, wherein 1, 2, 3 or 4 of the ring atoms are heteroatoms, non-limiting examples of which include indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzisothiazolyl,
  • heteroatom refers to nitrogen, oxygen or sulfur. In heteroaryl groups containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom, as valency permits.
  • Heteroaryl bicyclic ring systems may include one or more heteroatoms in one or both rings.
  • leaving group refers to a functional group or atom that can be replaced by another functional group or atom through a substitution reaction (e.g., a nucleophilic substitution reaction).
  • a substitution reaction e.g., a nucleophilic substitution reaction.
  • representative leaving groups include trifluoromethanesulfonate; chlorine, bromine, iodine; sulfonate groups, such as mesylate, tosylate, p-brosylate, p-toluenesulfonate, etc.; acyloxy groups, such as acetoxy, trifluoroacetoxy, etc.
  • protecting group includes, but is not limited to, "amino protecting group", “hydroxy protecting group” or “thiol protecting group”.
  • amino protecting group refers to a protecting group suitable for preventing side reactions at the amino nitrogen position.
  • Representative amino protecting groups include, but are not limited to: formyl; acyl, such as alkanoyl (such as acetyl, trichloroacetyl or trifluoroacetyl); alkoxycarbonyl, such as tert-butyloxycarbonyl (Boc); arylmethoxycarbonyl, such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); arylmethyl, such as benzyl (Bn), trityl (Tr), 1,1-bis-(4'-methoxyphenyl)methyl; silyl, such as trimethylsilyl (TMS) and tert-butyldi
  • hydroxy protecting group refers to a protecting group suitable for preventing side reactions of the hydroxyl group.
  • Representative hydroxy protecting groups include, but are not limited to, alkyl groups such as methyl, ethyl and tert-butyl; acyl groups such as alkanoyl (e.g., acetyl); arylmethyl groups such as benzyl (Bn), p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm) and diphenylmethyl (diphenylmethyl, DPM); silyl groups such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS), and the like.
  • alkyl groups such as methyl, ethyl and tert-butyl
  • acyl groups such as alkanoyl (e.g., acetyl)
  • arylmethyl groups such as benzyl (Bn), p-methoxybenzyl (
  • substituted independently selected from
  • substituents independently selected from means that when more than one hydrogen on a group is replaced by a substituent, the substituents may be the same or different in type, and the substituents selected are independently of each other.
  • the compounds of the present invention or their pharmaceutically acceptable salts, or their stereoisomers can be used in a suitable dosage form with one or more pharmaceutical carriers.
  • These dosage forms are suitable for oral, rectal, topical, oral and other parenteral administration (e.g., subcutaneous, intramuscular, intravenous, etc.).
  • dosage forms suitable for oral administration include capsules, tablets, granules and syrups.
  • the compounds of the present invention contained in these preparations can be solid powders or particles; solutions or suspensions in aqueous or non-aqueous liquids; water-in-oil or water-in-oil emulsions, etc.
  • the above dosage forms can be made from active compounds and one or more carriers or excipients via a common pharmaceutical method.
  • non-toxic carriers include but are not limited to mannitol, lactose, starch, magnesium stearate, cellulose, glucose, sucrose, etc.
  • Carriers for liquid preparations include water, saline, aqueous glucose solution, ethylene glycol and polyethylene glycol, etc.
  • the active compound can form a solution or suspension with the above carriers.
  • compositions of the present invention are formulated, dosed and administered in a manner consistent with medical practice.
  • the "therapeutically effective amount" of the compound administered is determined by factors such as the specific condition to be treated, the individual being treated, the cause of the condition, the target of the drug, and the mode of administration.
  • “Therapeutically effective amount” refers to the amount of the compound of the present invention that will elicit a biological or medical response in a subject, such as reducing or inhibiting enzyme or protein activity or improving symptoms, alleviating symptoms, slowing or delaying disease progression, or preventing disease.
  • the pharmaceutical composition of the present invention or the compound of the present invention or its pharmaceutically acceptable is preferably 0.1 mg to 5 g/kg (body weight).
  • Patient refers to an animal, preferably a mammal, more preferably a human.
  • mammal refers to warm-blooded vertebrate mammals, including, for example, cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, mice, pigs and humans.
  • Treatment means to lessen, slow the progression, attenuate, prevent, or maintain an existing disease or condition (eg, cancer). Treatment also includes curing, preventing the development of, or alleviating to some extent, one or more symptoms of a disease or condition.
  • the compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present invention.
  • the structure of the compound of the present invention can be confirmed by conventional methods known to those skilled in the art. If the present invention relates to the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the art.
  • single crystal X-ray diffraction (SXRD) is used to collect diffraction intensity data of the cultured single crystal using a Bruker D8 venture diffractometer, the light source is CuK ⁇ radiation, and the scanning mode is: After scanning and collecting relevant data, the crystal structure is further analyzed using the direct method (Shelxs97) to confirm the absolute configuration.
  • SXRD single crystal X-ray diffraction
  • the solvent used in the present invention can be obtained commercially.
  • the present invention uses the following abbreviations: DIBAL-H represents diisobutylaluminum hydride; SiO 2 represents silicon dioxide; NaH represents sodium hydride; CD 3 I represents deuterated iodomethane; NH 4 Cl represents ammonium chloride; LiOH.H 2 O represents hydrated lithium hydroxide; DMF represents N,N-dimethylformamide; HATU represents 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate; DIEA represents diisopropylethylamine; FA represents formic acid.
  • the present invention provides a compound represented by formula (I), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof:
  • R1 is selected from a linear deuterated alkyl group having 1 to 20 C atoms, a branched deuterated alkyl group having 3 to 20 C atoms, Cyclic deuterated alkyl having 3 to 20 C atoms, linear deuterated alkoxy having 1 to 20 C atoms, branched deuterated alkoxy having 3 to 20 C atoms, cyclic deuterated alkoxy having 3 to 20 C atoms, or a combination of these systems;
  • R 2 , R 3 , and R 4 are each independently selected from -H, -D, a linear alkyl group having 1 to 20 C atoms, a linear deuterated alkyl group having 1 to 20 C atoms, a branched alkyl group having 3 to 20 C atoms, a branched deuterated alkyl group having 3 to 20 C atoms, a cyclic alkyl group having 3 to 20 C atoms, a cyclic deuterated alkyl group having 3 to 20 C atoms, a linear alkoxy group having 1 to 20 C atoms, a linear deuterated alkoxy group having 1 to 20 C atoms, a branched alkoxy group having 3 to 20 C atoms, a branched deuterated alkoxy group having 3 to 20 C atoms, a cyclic alkoxy group having 3 to 20 C atoms, a cyclic deuterated alkoxy group having 3 to 20 C atoms, or a combination
  • the compound of formula (I) is a structure shown in formula (II):
  • R3 is selected from -H or -D.
  • R1 is selected from a linear deuterated alkyl group having 1 to 10 C atoms, a branched deuterated alkyl group having 3 to 10 C atoms, a cyclic deuterated alkyl group having 3 to 10 C atoms, a linear deuterated alkoxy group having 1 to 10 C atoms, a branched deuterated alkoxy group having 3 to 10 C atoms, a cyclic deuterated alkoxy group having 3 to 10 C atoms, or a combination of these systems.
  • R1 is selected from a linear deuterated alkyl group having 1 to 5 C atoms, a branched deuterated alkyl group having 3 to 5 C atoms, a cyclic deuterated alkyl group having 3 to 5 C atoms, a linear deuterated alkoxy group having 1 to 5 C atoms, a branched deuterated alkoxy group having 3 to 5 C atoms, a cyclic deuterated alkoxy group having 3 to 5 C atoms, or a combination of these systems.
  • R 1 is selected from a linear deuterated alkyl group having 1 to 3 C atoms, a branched deuterated alkyl group having 3 to 5 C atoms, a cyclic deuterated alkyl group having 3 to 5 C atoms.
  • R 2 and R 4 are independently selected from -H, -D, a straight-chain alkyl group having 1 to 10 C atoms, a straight-chain deuterated alkyl group having 1 to 10 C atoms, a branched-chain alkyl group having 3 to 10 C atoms, a branched-chain deuterated alkyl group having 3 to 10 C atoms, a cyclic alkyl group having 3 to 10 C atoms, a cyclic deuterated alkyl group having 3 to 10 C atoms, a straight-chain alkoxy group having 1 to 10 C atoms, a straight-chain deuterated alkoxy group having 1 to 10 C atoms, a branched-chain alkoxy group having 3 to 10 C atoms, a branched-chain deuterated alkoxy group having 3 to 10 C atoms, a cyclic alkyl group having 3 to 10 C atoms, a cyclic deuterated alkyl group having 3 to 10
  • R 2 and R 4 are independently selected from a straight-chain alkyl group having 1 to 3 C atoms, a straight-chain deuterated alkyl group having 1 to 3 C atoms, a branched alkyl group having 3 to 5 C atoms, a branched deuterated alkyl group having 3 to 5 C atoms, a cyclic alkyl group having 3 to 5 C atoms, a cyclic deuterated alkyl group having 3 to 5 C atoms, or a combination of these systems.
  • the compound of formula (I) is the following compound:
  • the pharmaceutically acceptable salt is an alkyl acid salt.
  • the pharmaceutically acceptable salt is a formate salt.
  • the present invention also provides a method for preparing a compound represented by formula (I), comprising the following steps:
  • R 1 , R 2 , R 3 and R 4 are consistent with those above;
  • X is selected from -F, -Cl, -Br or -I.
  • the present invention also provides a use of the above compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof in the preparation of a drug for treating and/or preventing a disease associated with CDK2 activity or mediated by CDK2 activity.
  • the disease associated with or mediated by CDK2 activity is cancer.
  • the cancer is one or more of ovarian cancer, gastric cancer, endometrial cancer, and breast cancer.
  • the present invention also provides a pharmaceutical composition, comprising the above compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, and a pharmaceutically acceptable carrier.
  • deuterated pyrazole derivatives and the preparation method thereof of the present invention are further described in detail below in conjunction with specific examples.
  • the raw materials used in the following examples are all commercially available products unless otherwise specified.
  • This embodiment provides compound 1, whose structural formula is as follows:
  • the reaction route is as follows:
  • the preparation process is as follows:
  • step 1
  • compound 1-3 200 mg, 993 ⁇ mol, 1.00 eq was dissolved in tetrahydrofuran (1.00 mL) and water (1.00 mL), and LiOH.H 2 O (43.7 mg, 1.04 mmol, 1.05 eq) was added. After reacting at 25°C for 10 hours, the reaction mixture was concentrated under reduced pressure to remove tetrahydrofuran, and then the pH was adjusted to 1.0 with hydrochloric acid, and then extracted with ethyl acetate (2.00 mL) for 3 times.
  • compound 1-4 168 mg, 972 ⁇ mol, 1.00 eq
  • compound 1-5 300 mg, 972 ⁇ mol, 1.00 eq
  • DMF 3.00 mL
  • HATU 406 mg, 1.07 mmol, 1.10 eq
  • DIEA 377 mg, 2.92 mmol, 508 ⁇ L, 3.00 eq
  • the reaction solution was diluted with water (9.00 mL) and extracted with ethyl acetate (10.0 mL) for 3 times.
  • CDK2/CyclinE 1 was purchased from Syngenecon.
  • Ulight-4E-BP1 peptide, Eu-anti-phospho-tyrosine antibody, and 1X detection buffer were purchased from PerkinElmer.
  • High-purity ATP was purchased from Promega.
  • EDTA was purchased from Sigma. Nivo multi-label analyzer (PerkinElmer).
  • Kinase buffer contains 50 mM HEPES, 1 mM EDTA, 10 mM MgCl 2 , 0.01% Brij-35, pH 7.4. Add 2.38 g HEPES, 58 mg EDTA, 406 mg MgCl 2 , 20 mg Brij-35 to 200 ml buffer and adjust the pH to 7.4.
  • stop solution Use 100 ⁇ L 1M EDTA stock solution, add 0.625 uL 1X detection buffer and 1725 uL distilled water to mix to prepare the stop solution.
  • the final compound concentration gradient is 1 ⁇ M diluted to 0.0128Nm, and the final concentrations of ATP and substrate are 1mM and 25nM.
  • the reaction system is placed at 25 degrees for 120 minutes. After the reaction, 5 ⁇ L of stop solution was added to each well, and the reaction was continued at 25 degrees for 5 minutes. After the reaction was completed, 5 uL of Eu-anti-phospho-tyrosine antibody dilution was added to each well. After the reaction was completed at 25 degrees for 60 minutes, the PerkinElmer Nivo multi-label analyzer TR-FRET mode was used for data acquisition (excitation wavelength was 320 nm, emission wavelengths were 615 nm and 665 nm).
  • Table 1 provides the inhibitory activity of the compounds of the present invention on CDK2/CyclinE1 enzyme.
  • CDK1/CyclinB 1 was purchased from CARNA.
  • Ulight-4E-BP1 peptide, Eu-anti-phospho-tyrosine antibody, and 1X detection buffer were purchased from PerkinElmer.
  • High-purity ATP was purchased from Promega.
  • EDTA was purchased from Sigma. Nivo multi-label analyzer (PerkinElmer).
  • Kinase buffer contains 50 mM HEPES, 1 mM EDTA, 10 mM MgCl 2 , 0.01% Brij-35, pH 7.4. Add 2.38 g HEPES, 58 mg EDTA, 406 mg MgCl 2 , 20 mg Brij-35 to 200 ml buffer and adjust the pH to 7.4.
  • stop solution Use 100 ⁇ L 1M EDTA stock solution, add 0.625 uL 1X detection buffer and 1725 uL distilled water to mix to prepare the stop solution.
  • the compound to be tested was diluted 5-fold to the 8th concentration using a gun, i.e., from 4 ⁇ M to 0.0512 nM, with a final DMSO concentration of 4%, and a double-well experiment was set up.
  • 2.5 ⁇ L of inhibitor concentration gradients, 5 ⁇ L of LCDK1/CyclinB1 enzyme (0.5 ng), and 2.5 ⁇ L of a mixture of substrate and ATP (4 mM ATP, 200 nM Ulight-4E-BP1 peptide) were added to the microplate.
  • the final compound concentration gradient was 1 ⁇ M diluted to 0.0128 nM, and the final concentrations of ATP and substrate were 1 mM and 50 nM.
  • the reaction system was placed at 25 degrees for 60 minutes.
  • Table 1 provides the inhibitory activity of the compounds of the present invention on CDK1/CyclinB1 enzyme.
  • GSK3 ⁇ Active was purchased from SignalChem; GSK3 Substrate was purchased from SignalChem; ADP-Glo Kinase Assay was purchased from Promega; Kinase assay buffer III was purchased from SignalChem; Nivo multi-label analyzer (PerkinElmer).
  • the compound to be tested was diluted to 100 ⁇ M with 100% DMSO as the first concentration, and then diluted 5 times with a gun to the 8th concentration, that is, from 100 ⁇ M to 0.0013 ⁇ M.
  • Each concentration point of the compound was diluted 20 times with 1X kinase buffer to prepare a compound working solution containing 5% DMSO, and 1 ⁇ L of each concentration gradient working solution of the compound was added to the microplate, and double wells were set.
  • 2 ⁇ l substrate and ATP mixture (62.5 ⁇ M ATP, 0.5 ⁇ g/ ⁇ l GSK3 Substrate) were added to the microplate.
  • the final concentration gradient of the compound was diluted from 1 ⁇ M to 0.013 nM, and the final concentrations of ATP and substrate were 25 ⁇ M and 0.2 ⁇ g/ ⁇ l.
  • the reaction system was placed at 25 degrees for 60 minutes. After the reaction, add 5 ⁇ l ADP-Glo reagent to each well and continue the reaction at 25 degrees for 40 minutes. After the reaction is finished, add 10 uL kinase detection The reagents were reacted at 25°C for 30 minutes and the chemiluminescence was read using a PerkinElmer Nivo multi-label analyzer with an integration time of 0.5 seconds.
  • the raw data was converted into inhibition rate using the equation (Sample-Min)/(Max-Min)*100%, and the IC50 value was obtained by four-parameter curve fitting (obtained by log(inhibitor) vs.response--Variable slope mode in GraphPad Prism).
  • Table 1 provides the inhibitory activity of the compounds of the present invention on GSK3 ⁇ enzymatic activity.
  • Max well positive control well reading value, blank well without enzyme
  • Negative control wells contain 1% DMSO solvent.
  • 1640 culture medium, fetal bovine serum, and penicillin/streptomycin antibiotics were purchased from Vicente.
  • CellTiter-Glo (cell viability chemiluminescence detection reagent) reagent was purchased from Promega.
  • OVCAR3 cell line was purchased from Nanjing Kebai Biotechnology Co., Ltd. Envision multi-label analyzer (PerkinElmer).
  • OVCAR3 cells were seeded in a white 384-well plate, with 40 ⁇ L of cell suspension per well, containing 300 OVCAR3 cells. The cell plate was placed in a carbon dioxide incubator for overnight culture.
  • the compound to be tested was diluted 5-fold to the 8th concentration, that is, from 2000 ⁇ M to 0.00512 ⁇ M, using a dispenser, and a double-well experiment was set up. 78 ⁇ L of culture medium was added to the middle plate, and then 2 ⁇ L of the gradient diluted compound per well was transferred to the middle plate according to the corresponding position, and 10 ⁇ L of each well was transferred to the cell plate after mixing. The concentration range of the compound transferred to the cell plate is 10 ⁇ M to 0.026nM.
  • the cell plate was placed in a carbon dioxide incubator and cultured for 7 days. Prepare another cell plate, and read the signal value on the day of drug addition as the maximum value (Max value in the equation below) for data analysis. Add 10 ⁇ L of cell viability chemiluminescence detection reagent to each well of this cell plate and incubate at room temperature for 10 minutes to stabilize the luminescence signal. Readings were taken using a multi-label analyzer.
  • Table 1 provides the inhibitory activity of the compounds of the present invention on OVCAR3 cell proliferation.
  • 1640 medium, fetal bovine serum, and penicillin/streptomycin antibiotics were purchased from Vicente.
  • CellTiter-Glo cell viability Chemiluminescence detection reagent (chemiluminescence detection reagent) was purchased from Promega.
  • T-47D cell line was purchased from Nanjing Kebai Biotechnology Co., Ltd. Envision multi-label analyzer (PerkinElmer).
  • T-47D cells were seeded in a white 384-well plate, with 40 ⁇ L of cell suspension per well, containing 300 T-47D cells. The cell plate was placed in a carbon dioxide incubator for overnight culture.
  • the compound to be tested was diluted 5-fold to the 8th concentration, that is, from 2000 ⁇ M to 0.00512 ⁇ M, using a dispenser, and a double-well experiment was set up. 78 ⁇ L of culture medium was added to the middle plate, and then 2 ⁇ L of the gradient diluted compound per well was transferred to the middle plate according to the corresponding position, and 10 ⁇ L of each well was transferred to the cell plate after mixing. The concentration range of the compound transferred to the cell plate is 10 ⁇ M to 0.026nM.
  • the cell plate was placed in a carbon dioxide incubator and cultured for 7 days. Prepare another cell plate, and read the signal value on the day of drug addition as the maximum value (Max value in the equation below) for data analysis. Add 10 ⁇ L of cell viability chemiluminescence detection reagent to each well of this cell plate and incubate at room temperature for 10 minutes to stabilize the luminescence signal. Readings were taken using a multi-label analyzer.
  • Table 1 provides the inhibitory activity of the compounds of the present invention on T-47D cell proliferation.

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Abstract

本发明提供了一种氘代吡唑类衍生物，其结构如式(I)所示。此外本发明还公开了该衍生物的药学上可接受的盐、其立体异构体、药物组合物以及其应用。本发明的化合物具有显著的CDK2抑制活性，十分具有实用价值。

Description

氘代吡唑类衍生物、药物组合物及应用与制备方法 技术领域
本发明涉及医药技术领域，特别是涉及一种氘代吡唑类衍生物、其药学上可接受的盐、立体异构体、药物组合物以及应用与制备方法。
背景技术
细胞周期依赖性激酶(CDK)属于丝氨酸/苏氨酸激酶家族。它们参与细胞的增殖、转录等生理过程。根据CDK功能的不同，可以将其分为两大类：一类CDK参与细胞周期调控，主要包括CDK1、CDK2、CDK4、CDK6等；另一类CDK参与转录调节，主要包括CDK7、CDK8、CDK9、CDK12、CDK13等。
CDK2活性失调经常发生在多种人类癌症中，故CDK2令科研人员感兴趣。CDK2在促进G1/S转换及S期进程中起关键作用。CDK2与细胞周期蛋白E(Cyclin E)形成复合物，磷酸化视网膜母细胞瘤家族成员(pRb等)，导致E2F转录因子释放并活化，推动细胞周期自G1期至S期转换，进而使得CDK2/Cyclin A活化，促进细胞周期DNA合成、复制等过程。
Cyclin E1拷贝数增加及过表达已经在卵巢癌、胃癌、子宫内膜癌、乳腺癌及其他癌症中得到鉴定，且与对应的肿瘤不良预后正相关。在ER+乳腺癌细胞中，Cyclin E2的高表达常伴随着激素治疗耐药，且Cyclin E的扩增或过表达与乳腺癌的不良预后有着密切关系。在HER2+乳腺癌中，Cyclin E的扩增也被报道出对于曲妥珠单抗的耐药有一定的贡献。更有报告称，Cyclin E的过表达在三阴性乳腺癌或发炎性乳腺癌进展中也起到重要作用。因此，CDK2可能成为一个重要抗肿瘤靶点。
目前处于临床研究阶段的具有CDK2活性的小分子抑制剂较少，辉瑞研制出了一款PF-4091，其结构式为：然而PF-4091临床前动物药效模型的给药剂量较高，并且须1天给2次，说明PF-4091的活性和代谢均有提升的空间。因此，为了更好地开发出治疗癌症等相关疾病的新药，迫切需要开发活性高、代谢更稳定的、能够治疗多种癌症的CDK2抑制剂，特别是选择性靶向CDK2的小分子抑制剂，其可能会具有更好的安全性。
发明内容
基于此，本发明提供了一种对CDK2抑制剂活性高的氘代吡唑类衍生物、其药学上可接受的盐或立体异构体。
本发明通过如下技术方案实现。
一种式(I)所示的化合物、或其药学上可接受的盐、或其立体异构体：
其中：
R1选自具有1至20个C原子的直链氘代烷基、具有3至20个C原子的支链氘代烷基、具有3至20个C原子的环状的氘代烷基、具有1至20个C原子的直链氘代烷氧基、具有3至20个C原子的支链氘代烷氧基、具有3至20个C原子的环状氘代烷氧基，或这些体系的组合；
R2、R3、R4分别独立地选自-H、-D、具有1至20个C原子的直链烷基、具有1至20个C原子的直链氘代烷基、具有3至20个C原子的支链烷基、具有3至20个C原子的支链氘代烷基、具有3至20个C原子的环状的烷基、具有3至20个C原子的环状的氘代烷基、具有1至20个C原子的直链烷氧基、具有1至20个C原子的直链氘代烷氧基、具有3至20个C原子的支链烷氧基、具有3至20个C原子的支链氘代烷氧基、具有3至20个C原子的环状烷氧基、具有3至20个C原子的环状氘代烷氧基，或这些体系的组合。
在其中一个实施例中，式(I)化合物为式(II)所示结构：
在其中一个实施例中，R3选自-H或-D。
在其中一个实施例中，R1选自具有1至10个C原子的直链氘代烷基、具有3至10个C原子的支链氘代烷基、具有3至10个C原子的环状的氘代烷基、具有1至10个C原子的直链氘代烷氧基、具有3至10个C原子的支链氘代烷氧基、具有3至10个C原子的环状氘代烷氧基，或这些体系的组合；
R2、R4分别独立地选自-H、-D、具有1至10个C原子的直链烷基、具有1至10个C原子的直链氘代烷基、具有3至10个C原子的支链烷基、具有3至10个C原子的支链氘代烷基、具有3至10个C原子的环状的烷基、具有3至10个C原子的环状的氘代烷基、具有1至10个C原子的直链烷氧基、具有1至10个C原子的直链氘代烷氧基、具有3至10个C原子的支链烷氧基、具有3至10个C原子的支链氘代烷氧基、具有3至10个C原子的环状烷氧基、具有3至10个C原子的环状氘代烷氧基，或这些体系的组合。
在其中一个实施例中，R1选自具有1至5个C原子的直链氘代烷基、具有3至5个C原子的支链氘代烷基、具有3至5个C原子的环状的氘代烷基、具有1至5个C原子的直链氘代烷氧基、具有3至5个C原子的支链氘代烷氧基、具有3至5个C原子的环状氘代烷氧基，或这些体系的组合；
R2、R4分别独立地选自具有1至3个C原子的直链烷基、具有1至3个C原子的直链氘代烷基、具有3至5个C原子的支链烷基、具有3至5个C原子的支链氘代烷基、具有3至5个C原子的环状的烷基、具有3至5个C原子的环状的氘代烷基，或这些体系的组合。
在其中一个实施例中，式(I)化合物为下列化合物：
本发明还提供一种式(I)所示的化合物的制备方法，包括如下步骤：
将化合物A经还原反应制备化合物B：
将化合物B与化合物C经亲核取代反应制备化合物D：
将化合物D进行水解反应制备化合物E：
将化合物E与化合物F经酰化反应制备化合物G：
将化合物G经取代反应制备式(I)所述化合物：
其中，R1、R2、R3、R4的定义与如上所述一致；X选自-F、-Cl、-Br或-I。
本发明还提供一种如上所述的化合物、或其药学上可接受的盐、或其立体异构体在制备治疗和/或预防与CDK2活性相关的或由CDK2活性介导的疾病的药物中的应用。
在其中一个实施例中，所述与CDK2活性相关的或由CDK2活性介导的疾病为癌症。
本发明还提供一种药物组合物，包括如上所述的化合物、或其药学上可接受的盐、或其立体异构体，以及药学上可接受的载体。
与现有技术相比较，本发明的化合物、或其药学上可接受的盐、或其立体异构体具有如下有益效果：
本发明人经过长期而深入的研究，意外地发现了一种新颖的氘代吡唑类衍生物，药物分 子中的一部分位点除的氢元素用氘取代，这种细微的调整会对药物的活性与代谢产生很大的影响。本发明的氘代吡唑类衍生物对CDK2具有出乎意料的较高的抑制活性，同时能够明显提高药代效果并减轻副作用，可用作治疗和/或预防与CDK2活性相关的或由CDK2活性介导的疾病的药物，可用于治疗Cyclin E高表达的各类癌症，对于CDK2活性介导的疾病具有潜在的优异治疗效果。
具体实施方式
为了便于理解本发明，下面将参照相关实施例对本发明进行更全面的描述。实施例中给出了本发明的较佳实施方式。但是，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施方式。相反地，提供这些实施方式的目的是使对本发明的公开内容的理解更加透彻全面。
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。在本发明的描述中，“若干”的含义是至少一个，例如一个，两个等，除非另有明确具体的限定。
本发明中的词语“优选地”、“更优选地”等是指，在某些情况下可提供某些有益效果的本发明实施方案。然而，在相同的情况下或其他情况下，其他实施方案也可能是优选的。此外，对一个或多个优选实施方案的表述并不暗示其他实施方案不可用，也并非旨在将其他实施方案排除在本发明的范围之外。
当本文中公开一个数值范围时，上述范围视为连续，且包括该范围的最小值及最大值，以及这种最小值与最大值之间的每一个值。进一步地，当范围是指整数时，包括该范围的最小值与最大值之间的每一个整数。此外，当提供多个范围描述特征或特性时，可以合并该范围。换言之，除非另有指明，否则本文中所公开之所有范围应理解为包括其中所归入的任何及所有的子范围。
除非另外指明，所有百分比、分数和比率都是按本发明组合物的总质量计算的。除非另外指明，有关所列成分的所有质量均给予活性物质的含量，因此它们不包括在可商购获得的材料中可能包含的溶剂或副产物。本文术语“质量百分比含量”可用符号“％”表示。除非另外指明，在本文中所有的分子量都是以道尔顿为单位表示的重均分子量。除非另外指明，在本文中所有配制和测试发生在25℃的环境。本文中“包括”、“包含”、“含”、“含有”、“具有”或其它变体意在涵盖非封闭式包括，这些术语之间不作区分。术语“包含”是指可加入不影响最终 结果的其它步骤和成分。本发明的组合物和方法/工艺包含、由其组成和基本上由本文描述的必要元素和限制项以及本文描述的任一的附加的或任选的成分、组份、步骤或限制项组成。本文中术语“效能”、“性能”、“效果”、“功效”之间不作区分。
除非另有说明，本文所用的下列术语和短语旨在具有下列含义。一个特定的术语或短语在没有特别定义的情况下不应该被认为是不确定的或不清楚的，而应该按照普通的含义去理解。当本文中出现商品名时，意在指代其对应的商品或其活性成分。
术语“药学上可接受的”，是针对那些化合物、材料、组合物和/或剂型而言，它们在可靠的医学判断的范围之内，适用于与人类和动物的组织接触使用，而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症，与合理的利益/风险比相称。
术语“药学上可接受的盐”是指本发明化合物的盐，由本发明发现的具有特定取代基的化合物与相对无毒的酸或碱制备。当本发明的化合物中含有相对酸性的功能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的碱与这类化合物接触的方式获得碱加成盐。药学上可接受的碱加成盐包括钠、钾、钙、铵、有机胺或镁盐或类似的盐。当本发明的化合物中含有相对碱性的官能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的酸与这类化合物接触的方式获得酸加成盐。药学上可接受的酸加成盐的实例包括无机酸盐，所述无机酸包括例如盐酸、氢溴酸、硝酸、碳酸，碳酸氢根，磷酸、磷酸一氢根、磷酸二氢根、硫酸、硫酸氢根、氢碘酸、亚磷酸等；以及有机酸盐，所述有机酸包括如甲酸、乙酸、丙酸、异丁酸、马来酸、丙二酸、苯甲酸、琥珀酸、辛二酸、反丁烯二酸、乳酸、扁桃酸、邻苯二甲酸、苯磺酸、对甲苯磺酸、柠檬酸、酒石酸和甲磺酸等类似的酸；还包括氨基酸(如精氨酸等)的盐，以及如葡糖醛酸等有机酸的盐。本发明的某些特定的化合物含有碱性和酸性的官能团，从而可以被转换成任一碱或酸加成盐。
本发明的药学上可接受的盐可由含有酸根或碱基的母体化合物通过常规化学方法合成。一般情况下，这样的盐的制备方法是：在水或有机溶剂或两者的混合物中，经由游离酸或碱形式的这些化合物与化学计量的适当的碱或酸反应来制备。
本发明的化合物可以存在特定的几何或立体异构体形式。本发明设想所有的这类化合物，包括顺式和反式异构体、(-)-和(+)-对映体、(R)-和(S)-对映体、非对映异构体、(D)-异构体、(L)-异构体，及其外消旋混合物和其他混合物，例如对映异构体或非对映体富集的混合物，所有这些混合物都属于本发明的范围之内。烷基等取代基中可存在另外的不对称碳原子。所有这些异构体以及它们的混合物，均包括在本发明的范围之内。
除非另有说明，术语“对映异构体”或者“旋光异构体”是指互为镜像关系的立体异构体。
除非另有说明，术语“顺反异构体”或者“几何异构体”系由因双键或者成环碳原子单键不 能自由旋转而引起。
除非另有说明，术语“非对映异构体”是指分子具有两个或多个手性中心，并且分子间为非镜像的关系的立体异构体。
除非另有说明，“(+)”表示右旋，“(-)”表示左旋，“(±)”表示外消旋。
除非另有说明，用楔形实线键和楔形虚线键表示一个立体中心的绝对构型，用直形实线键和直形虚线键表示立体中心的相对构型，用波浪线表示楔形实线键或楔形虚线键或用波浪线表示直形实线键和直形虚线键
除非另有说明，当化合物中存在双键结构，如碳碳双键、碳氮双键和氮氮双键，且双键上的各个原子均连接有两个不同的取代基时(包含氮原子的双键中，氮原子上的一对孤对电子视为其连接的一个取代基)，如果该化合物中双键上的原子与其取代基之间用波浪线连接，则表示该化合物的(Z)型异构体、(E)型异构体或两种异构体的混合物。例如下式(A)表示该化合物以式(A-1)或式(A-2)的单一异构体形式存在或以式(A-1)和式(A-2)两种异构体的混合物形式存在；下式(B)表示该化合物以式(B-1)或式(B-2)的单一异构体形式存在或以式(B-1)和式(B-2)两种异构体的混合物形式存在。下式(C)表示该化合物以式(C-1)或式(C-2)的单一异构体形式存在或以式(C-1)和式(C-2)两种异构体的混合物形式存在。
除非另有说明，术语“互变异构体”或“互变异构体形式”是指在室温下，不同官能团异构体处于动态平衡，并能很快的相互转化。若互变异构体是可能的(如在溶液中)，则可以达到互变异构体的化学平衡。例如，质子互变异构体(proton tautomer)(也称质子转移互变异构体(prototropic tautomer))包括通过质子迁移来进行的互相转化，如酮-烯醇异构化和亚胺-烯胺异 构化。价键异构体(valence tautomer)包括一些成键电子的重组来进行的相互转化。其中酮-烯醇互变异构化的具体实例是戊烷-2,4-二酮与4-羟基戊-3-烯-2-酮两个互变异构体之间的互变。
除非另有说明，术语“富含一种异构体”、“异构体富集”、“富含一种对映体”或者“对映体富集”指其中一种异构体或对映体的含量小于100％，并且，该异构体或对映体的含量大于等于60％，或者大于等于70％，或者大于等于80％，或者大于等于90％，或者大于等于95％，或者大于等于96％，或者大于等于97％，或者大于等于98％，或者大于等于99％，或者大于等于99.5％，或者大于等于99.6％，或者大于等于99.7％，或者大于等于99.8％，或者大于等于99.9％。
除非另有说明，术语“异构体过量”或“对映体过量”指两种异构体或两种对映体相对百分数之间的差值。例如，其中一种异构体或对映体的含量为90％，另一种异构体或对映体的含量为10％，则异构体或对映体过量(ee值)为80％。
可以通过的手性合成或手性试剂或者其他常规技术制备光学活性的(R)-和(S)-异构体以及D和L异构体。如果想得到本发明某化合物的一种对映体，可以通过不对称合成或者具有手性助剂的衍生作用来制备，其中将所得非对映体混合物分离，并且辅助基团裂开以提供纯的所需对映异构体。或者，当分子中含有碱性官能团(如氨基)或酸性官能团(如羧基)时，与适当的光学活性的酸或碱形成非对映异构体的盐，然后通过本领域所公知的常规方法进行非对映异构体拆分，然后回收得到纯的对映体。此外，对映异构体和非对映异构体的分离通常是通过使用色谱法完成的，所述色谱法采用手性固定相，并任选地与化学衍生法相结合(例如由胺生成氨基甲酸盐)。
本发明的化合物可以在一个或多个构成该化合物的原子上包含非天然比例的原子同位素。例如，可用放射性同位素标记化合物，比如氚(3H)，碘-125(125I)或C-14(14C)。又例如，可用重氢取代氢形成氘代药物，氘与碳构成的键比普通氢与碳构成的键更坚固，相比于未氘化药物，氘代药物有降低毒副作用、增加药物稳定性、增强疗效、延长药物生物半衰期等优势。本发明的化合物的所有同位素组成的变换，无论放射性与否，都包括在本发明的范围之内。
“任选”或“任选地”指的是随后描述的事件或状况可能但不是必需出现的，并且该描述包括其中所述事件或状况发生的情况以及所述事件或状况不发生的情况。
术语“取代”是指特定原子上的任意一个或多个氢原子被取代基取代，取代基可以包括重氢和氢的变体，只要特定原子的价态是正常的并且取代后的化合物是稳定的。取代基的种类和数目在化学上可以实现的基础上可以是任意的。当取代基为氧(即＝O)时，意味着两个氢 原子被取代。氧取代不会发生在芳香基上。
当任何变量(例如R1)在化合物的组成或结构中出现一次以上时，其在每一种情况下的定义都是独立的。因此，例如，如果一个基团被0-2个R1所取代，则所述基团可以任选地至多被两个R1所取代，并且每种情况下的R1都有独立的选项。此外，取代基和/或其变体的组合只有在这样的组合会产生稳定的化合物的情况下才是被允许的。
当一个连接基团的数量为0时，比如-(CRR)0-，表示该连接基团为单键。
当其中一个变量选自单键时，表示其连接的两个基团直接相连，比如A-L-Z中L代表单键时表示该结构实际上是A-Z。
当一个取代基为空缺时，表示该取代基是不存在的，比如A-X中X为空缺时表示该结构实际上是A。当所列举的取代基中没有指明其通过哪一个原子连接到被取代的基团上时，这种取代基可以通过其任何原子相键合，例如，吡啶基作为取代基可以通过吡啶环上任意一个碳原子连接到被取代的基团上。
当所列举的连接基团没有指明其连接方向，其连接方向是任意的，例如，中连接基团L为-M-W-，此时-M-W-既可以按与从左往右的读取顺序相同的方向连接环A和环B构成也可以按照与从左往右的读取顺序相反的方向连接环A和环B构成所述连接基团、取代基和/或其变体的组合只有在这样的组合会产生稳定的化合物的情况下才是被允许的。
除非另有规定，当某一基团具有一个或多个可连接位点时，该基团的任意一个或多个位点可以通过化学键与其他基团相连。当该化学键的连接方式是不定位的，且可连接位点存在H原子时，则连接化学键时，该位点的H原子的个数会随所连接化学键的个数而对应减少变成相应价数的基团。所述位点与其他基团连接的化学键可以用直形实线键直形虚线键或波浪线表示。例如-OCH3中的直形实线键表示通过该基团中的氧原子与其他基团相连；中的直形虚线键表示通过该基团中的氮原子的两端与其他基团相连；中的波浪线表示通过该苯基基团中的1和2位碳原子与其他基团相连；表 示该哌啶基上的任意可连接位点可以通过1个化学键与其他基团相连，至少包括 这4种连接方式，即使-N-上画出了H原子，但是仍包括这种连接方式的基团，只是在连接1个化学键时，该位点的的H会对应减少1个变成相应的一价哌啶基。
除非另有规定，环上原子的数目通常被定义为环的元数，例如，“3-7元环”是指环绕排列3-7个原子的“环”。
除非另有规定，术语“C1-6烷基”用于表示直链或支链的由1至6个碳原子组成的饱和碳氢基团。优选为C1-4烷基，其可以是一价(如甲基)、二价(如亚甲基)或者多价(如次甲基)。C1-3烷基的实例包括但不限于甲基(Me)、乙基(Et)、丙基(包括n-丙基和异丙基)、丁基(包括正丁基、异丁基、叔丁基和仲丁基)。
除非另有规定，术语“C1-6烷氧基”表示通过一个氧原子连接到分子的其余部分的那些包含1至6个碳原子的烷基基团。优选为C1-3烷氧基。C1-3烷氧基的实例包括但不限于甲氧基、乙氧基、丙氧基(包括正丙氧基和异丙氧基)等。
除非另有规定，术语“C1-6烷氨基”表示通过氨基连接到分子的其余部分的那些包含1至6个碳原子的烷基基团。优选为C1-3烷氨基。C1-3烷氨基的实例包括但不限于-NHCH3、-N(CH3)2、-NHCH2CH3、-N(CH3)CH2CH3、-NHCH2CH2CH3、-NHCH2(CH3)2等。
除非另有规定，术语“卤代素”或“卤素”本身或作为另一取代基的一部分表示氟、氯、溴或碘原子。
除非另有规定，本发明术语“5杂芳环”和“5元杂芳基”可以互换使用，术语“5元杂芳基”表示由5环原子组成的具有共轭π电子体系的单环基团，其1、2、3或4个环原子为独立选自O、S和N的杂原子，其余为碳原子。其中氮原子任选地被季铵化，氮和硫杂原子可任选被氧化(即NO和S(O)p，p是1或2)。5元杂芳基可通过杂原子或碳原子连接到分子的其余部分。所述5元杂芳基的实例包括但不限于吡咯基(包括N-吡咯基、2-吡咯基和3-吡咯基等)、吡唑基(包括2-吡唑基和3-吡唑基等)、咪唑基(包括N-咪唑基、2-咪唑基、4-咪唑基和5-咪唑基等)、噁唑基(包括2-噁唑基、4-噁唑基和5-噁唑基等)、三唑基(1H-1,2,3-三唑基、2H-1,2,3-三唑基、1H-1,2,4-三唑基和4H-1,2,4-三唑基等)、四唑基、异噁唑基(3-异噁唑基、4-异噁唑基和5-异噁唑基等)、噻唑基(包括2-噻唑基、4-噻唑基和5-噻唑基等)、呋喃基(包 括2-呋喃基和3-呋喃基等)、噻吩基(包括2-噻吩基和3-噻吩基等)。
除非另有规定，Cn-n+m或Cn-Cn+m包括n至n+m个碳的任何一种具体情况，例如C1-12包括C1、C2、C3、C4、C5、C6、C7、C8、C9、C10、C11、和C12，也包括n至n+m中的任何一个范围，例如C1-12包括C1-3、C1-6、C1-9、C3-6、C3-9、C3-12、C6-9、C6-12、和C9-12等；同理，n元至n+m元表示环上原子数为n至n+m个，例如3-12元环包括3元环、4元环、5元环、6元环、7元环、8元环、9元环、10元环、11元环、和12元环，也包括n至n+m中的任何一个范围，例如3-12元环包括3-6元环、3-9元环、5-6元环、5-7元环、6-7元环、6-8元环、和6-10元环等。
除非另有规定，“C3-7环烷基”表示由3至7个碳原子组成的饱和环状碳氢基团，其包括单环和双环体系，其中双环体系包括螺环、并环和桥环。所述C3-7环烷基包括C3-6、C4-6、C4-5、C5-7或C5-6环烷基等；其可以是一价、二价或者多价。C3-7环烷基的实例包括，但不限于，环丙基、环丁基、环戊基、环己基、环庚基等。
除非另有规定，术语“3-7元杂环烷基”本身或者与其他术语联合分别表示由3至7个环原子组成的饱和环状基团，其1、2、3或4个环原子为独立选自O、S和N的杂原子，其余为碳原子，其中氮原子任选地被季铵化，氮和硫杂原子可任选被氧化(即NO和S(O)p，p是1或2)。其包括单环和双环体系，其中双环体系包括螺环、并环和桥环。此外，就该“3-7元杂环烷基”而言，杂原子可以占据杂环烷基与分子其余部分的连接位置。所述3-7元杂环烷基包括5-7元、3元、4元、5元、6元和7元杂环烷基等。3-7元杂环烷基的实例包括但不限于氮杂环丁基、氧杂环丁基、硫杂环丁基、吡咯烷基、吡唑烷基、咪唑烷基、四氢噻吩基(包括四氢噻吩-2-基和四氢噻吩-3-基等)、四氢呋喃基(包括四氢呋喃-2-基等)、四氢吡喃基、哌啶基(包括1-哌啶基、2-哌啶基和3-哌啶基等)、哌嗪基(包括1-哌嗪基和2-哌嗪基等)、吗啉基(包括3-吗啉基和4-吗啉基等)、二噁烷基、二噻烷基、异噁唑烷基、异噻唑烷基、1,2-噁嗪基、1,2-噻嗪基或六氢哒嗪基等。
除非另有规定，术语“3-7元含氮杂环烷基”表示为至少含有一个N原子的3-7元杂环烷基。
脂环基是指饱和或部分不饱和的全碳环系统。其中“部分不饱和”是指包括至少一个双键或三键的环部分，“部分不饱和”意图涵盖具有多个不饱和位点的环，但并不意图包括如本文所定义的芳基或杂芳基部分。非限制性实施例包括环丙基环、环丁基环、环戊基环、环戊烯基环、环己基环、环己烯基环、环己二烯基环、环庚基环、环庚三烯基环、环戊酮环、环戊烷-1,3-二酮环等。
脂杂环基是指饱和或部分不饱和脂环基中的1、2或3个环碳原子被选自氮、氧或S(O)t(其中t是整数0至2)的杂原子所取代，但不包括-O-O-、-O-S-或-S-S-的环部分，其余环原子为 碳。非限制性实施例包括环氧丙烷环、氮杂环丁烷环、氧杂环丁烷环、四氢呋喃环、四氢噻吩环、四氢吡咯环、哌啶环、吡咯啉环、噁唑烷环、哌嗪环、二氧戊环、二氧六环、吗啉环、硫代吗啉环、硫代吗啉-1,1-二氧化物、四氢吡喃环、氮杂环丁烷-2-酮环、氧杂环丁烷-2-酮环、吡咯烷-2-酮环、吡咯烷-2,5-二酮环、哌啶-2-酮环、二氢呋喃-2(3H)-酮环、二氢呋喃-2,5-二酮环、四氢-2H-吡喃-2-酮环、哌嗪-2-酮环、吗啉-3-酮环。部分不饱和单杂环的非限制性实施例包括1,2-二氢氮杂环丁二烯环、1,2-二氢氧杂环丁二烯环、2,5-二氢-1H-吡咯环、2,5-二氢呋喃环、2,3-二氢呋喃环、2,3-二氢-1H-吡咯环、3,4-二氢-2H-吡喃环、1,2,3,4-四氢吡啶环、3,6-二氢-2H-吡喃环、1,2,3,6-四氢吡啶环、4,5-二氢-1H-咪唑环、1,4,5,6-四氢嘧啶环、3,4,7,8-四氢-2H-1,4,6-噁二唑嗪环、1,6-二氢嘧啶环、4,5,6,7-四氢-1H-1,3-二氮杂环、2,5,6,7-四氢-1,3,5-噁二氮杂环等。
“芳基”和“芳环”可互换使用，均指具有共轭的π电子体系的全碳单环或稠合多环(也就是共享毗邻碳原子对的环)基团，该基团可以与环烷基环、杂环烷基环、环烯基环、杂环烯基环或杂芳基稠合。“C6-10芳基”指具有6至10个碳原子的单环或双环芳基，芳基的非限制性实施例包括苯基、萘基等。
“杂芳基”和“杂芳基环”可互换使用，均指具有环碳原子和环杂原子的单环、双环或多环的4n+2芳族环体系(例如，具有以环状排列共享的6或10个π电子)的基团，其中每个杂原子独立地选自氮、氧和硫。本发明中，杂芳基还包括其中上述杂芳基环与一个或多个环烷基环、杂环烷基环、环烯基环、杂环烯基环或芳环稠合的环系统。杂芳基环可以任选地被取代。“5至10元杂芳基”是指具有5至10个环原子，其中1、2、3或4个环原子为杂原子的单环或双环杂芳基。“5至6元杂芳基”是指具有5至6个环原子，其中1、2、3或4个环原子为杂原子的单环杂芳基，非限制性实施例包括噻吩基、呋喃基、噻唑基、异噻唑基、咪唑基、噁唑基、吡咯基、吡唑基、三唑基、1,2,3-三唑基、1,2,4-三唑基、1,2,5-三唑基、1,3,4-三唑基、四唑基、异噁唑基、噁二唑基、1,2,3-噁二唑基、1,2,4-噁二唑基、1,2,5-噁二唑基、1,3,4-恶二唑基、噻二唑基、吡啶基、哒嗪基、嘧啶基、吡嗪基、三嗪基、四嗪基。“8至10元杂芳基”是指具有8至10个环原子，其中1、2、3或4个环原子为杂原子的双环杂芳基，非限制性实施例包括吲哚基、异吲哚基、吲唑基、苯并三唑基、苯并噻吩基、异苯并噻吩基、苯并呋喃基、苯并异呋喃基、苯并咪唑基、苯并噁唑基、苯并异噁唑基、苯并噁二唑基、苯并噻唑基、苯并异噻唑基、苯并噻二唑基、茚嗪基、嘌呤基、吡啶并[3,2-d]嘧啶基、吡啶并[2,3-d]嘧啶基、吡啶并[3,4-d]嘧啶基、吡啶并[4,3-d]嘧啶基、1,8-萘啶基、1,7-萘啶基、1,6-萘啶基、1,5-萘啶基、喋啶基、喹啉基、异喹啉基、噌琳基、喹喔啉基、酞嗪基和喹唑啉基。“杂原子”是指氮、氧或硫。在含有一个或多个氮原子的杂芳基中，只要化合价允许，连接点可以是碳或氮原子。 杂芳基双环系统在一个或两个环中可以包括一个或多个杂原子。
术语“离去基团”是指可以被另一种官能团或原子通过取代反应(例如亲核取代反应)所取代的官能团或原子。例如，代表性的离去基团包括三氟甲磺酸酯；氯、溴、碘；磺酸酯基，如甲磺酸酯、甲苯磺酸酯、对溴苯磺酸酯、对甲苯磺酸酯等；酰氧基，如乙酰氧基、三氟乙酰氧基等。
术语“保护基”包括但不限于“氨基保护基”、“羟基保护基”或“巯基保护基”。术语“氨基保护基”是指适合用于阻止氨基氮位上副反应的保护基团。代表性的氨基保护基包括但不限于：甲酰基；酰基，例如链烷酰基(如乙酰基、三氯乙酰基或三氟乙酰基)；烷氧基羰基，如叔丁氧基羰基(Boc)；芳基甲氧羰基，如苄氧羰基(Cbz)和9-芴甲氧羰基(Fmoc)；芳基甲基，如苄基(Bn)、三苯甲基(Tr)、1,1-二-(4'-甲氧基苯基)甲基；甲硅烷基，如三甲基甲硅烷基(TMS)和叔丁基二甲基甲硅烷基(TBS)等等。术语“羟基保护基”是指适合用于阻止羟基副反应的保护基。代表性羟基保护基包括但不限于：烷基，如甲基、乙基和叔丁基；酰基，例如链烷酰基(如乙酰基)；芳基甲基，如苄基(Bn)，对甲氧基苄基(PMB)、9-芴基甲基(Fm)和二苯基甲基(二苯甲基，DPM)；甲硅烷基，如三甲基甲硅烷基(TMS)和叔丁基二甲基甲硅烷基(TBS)等等。
除非另有定义，本发明所述“各自独立地选自……的取代基”是指当基团上的一个以上的氢被取代基取代时，所述的取代基种类可以相同或不同，所选自的取代基为各自独立的种类。
通常本发明化合物或其药学可接受的盐、或其立体异构体可以与一种或多种药用载体形成适合的剂型施用。这些剂型适用于口服、直肠给药、局部给药、口内给药以及其他非胃肠道施用(例如，皮下、肌肉、静脉等)。例如，适合口服给药的剂型包括胶囊、片剂、颗粒剂以及糖浆等。这些制剂中包含的本发明的化合物可以是固体粉末或颗粒；水性或非水性液体中的溶液或是混悬液；油包水或水包油的乳剂等。上述剂型可由活性化合物与一种或多种载体或辅料经由通用的药剂学方法制成。上述的载体需要与活性化合物或其他辅料兼容。对于固体制剂，常用的无毒载体包括但不限于甘露醇、乳糖、淀粉、硬脂酸镁、纤维素、葡萄糖、蔗糖等。用于液体制剂的载体包括水、生理盐水、葡萄糖水溶液、乙二醇和聚乙二醇等。活性化合物可与上述载体形成溶液或是混悬液。
本发明的组合物以符合医学实践规范的方式配制，定量和给药。给予化合物的“治疗有效量”由要治疗的具体病症、治疗的个体、病症的起因、药物的靶点以及给药方式等因素决定。
“治疗有效量”是指将引起个体的生物学或医学响应，例如降低或抑制酶或蛋白质活性或改善症状、缓解病症、减缓或延迟疾病进程或预防疾病等的本发明化合物的量。
本发明的所述药物组合物或所述药用组合物中含有的本发明化合物或其药学上可接受的 盐、或其立体异构体的治疗有效量优选为0.1mg-5g/kg(体重)。
“患者”是指一种动物，最好为哺乳动物，更好的为人。术语“哺乳动物”是指温血脊椎类哺乳动物，包括如猫、狗、兔、熊、狐狸、狼、猴子、鹿、鼠、猪和人类。
“治疗”是指减轻、延缓进展、衰减、预防，或维持现有疾病或病症(例如癌症)。治疗还包括将疾病或病症的一个或多个症状治愈、预防其发展或减轻到某种程度。
本发明的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本发明的实施例。
本发明的化合物可以通过本领域技术人员所熟知的常规方法来确认结构，如果本发明涉及化合物的绝对构型，则该绝对构型可以通过本领域常规技术手段予以确证。例如单晶X射线衍射法(SXRD)，把培养出的单晶用Bruker D8 venture衍射仪收集衍射强度数据，光源为CuKα辐射，扫描方式：扫描，收集相关数据后，进一步采用直接法(Shelxs97)解析晶体结构，便可以确证绝对构型。
本发明所使用的溶剂可经市售获得。本发明采用下述缩略词：DIBAL-H代表二异丁基氢化铝；SiO2代表二氧化硅；NaH代表钠氢；CD3I代表氘代碘甲烷；NH4Cl代表氯化铵；LiOH.H2O代表水合氢氧化锂；DMF代表N,N-二甲基甲酰胺；HATU代表2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯；DIEA代表二异丙基乙胺；FA代表甲酸。
化合物依据本领域常规命名原则或者使用软件命名，市售化合物采用供应商目录名称。
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施方式的目的，不是旨在于限制本发明。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
本发明提供了一种式(I)所示的化合物、或其药学上可接受的盐、或其立体异构体：
其中：
R1选自具有1至20个C原子的直链氘代烷基、具有3至20个C原子的支链氘代烷基、 具有3至20个C原子的环状的氘代烷基、具有1至20个C原子的直链氘代烷氧基、具有3至20个C原子的支链氘代烷氧基、具有3至20个C原子的环状氘代烷氧基，或这些体系的组合；
R2、R3、R4分别独立地选自-H、-D、具有1至20个C原子的直链烷基、具有1至20个C原子的直链氘代烷基、具有3至20个C原子的支链烷基、具有3至20个C原子的支链氘代烷基、具有3至20个C原子的环状的烷基、具有3至20个C原子的环状的氘代烷基、具有1至20个C原子的直链烷氧基、具有1至20个C原子的直链氘代烷氧基、具有3至20个C原子的支链烷氧基、具有3至20个C原子的支链氘代烷氧基、具有3至20个C原子的环状烷氧基、具有3至20个C原子的环状氘代烷氧基，或这些体系的组合。
在一个具体的示例中，式(I)化合物为式(II)所示结构：
在一个具体的示例中，R3选自-H或-D。
在一个具体的示例中，R1选自具有1至10个C原子的直链氘代烷基、具有3至10个C原子的支链氘代烷基、具有3至10个C原子的环状的氘代烷基、具有1至10个C原子的直链氘代烷氧基、具有3至10个C原子的支链氘代烷氧基、具有3至10个C原子的环状氘代烷氧基，或这些体系的组合。
在一个具体的示例中，R1选自具有1至5个C原子的直链氘代烷基、具有3至5个C原子的支链氘代烷基、具有3至5个C原子的环状的氘代烷基、具有1至5个C原子的直链氘代烷氧基、具有3至5个C原子的支链氘代烷氧基、具有3至5个C原子的环状氘代烷氧基，或这些体系的组合。
更具体地，R1选自R1选自具有1至3个C原子的直链氘代烷基、具有3至5个C原子的支链氘代烷基、具有3至5个C原子的环状的氘代烷基。
在一个具体的示例中，R2、R4分别独立地选自-H、-D、具有1至10个C原子的直链烷基、具有1至10个C原子的直链氘代烷基、具有3至10个C原子的支链烷基、具有3至10个C原子的支链氘代烷基、具有3至10个C原子的环状的烷基、具有3至10个C原子的环状的氘代烷基、具有1至10个C原子的直链烷氧基、具有1至10个C原子的直链氘代烷氧基、具有3至10个C原子的支链烷氧基、具有3至10个C原子的支链氘代烷氧基、具有3 至10个C原子的环状烷氧基、具有3至10个C原子的环状氘代烷氧基，或这些体系的组合。
在一个具体的示例中，R2、R4分别独立地选自具有1至3个C原子的直链烷基、具有1至3个C原子的直链氘代烷基、具有3至5个C原子的支链烷基、具有3至5个C原子的支链氘代烷基、具有3至5个C原子的环状的烷基、具有3至5个C原子的环状的氘代烷基，或这些体系的组合。
在一个具体的示例中，式(I)化合物为下列化合物：
在一个具体的示例中，药学上可接受的盐为烷基酸盐。进一步地，药学上可接受的盐为甲酸盐。
在一个具体的示例中，本发明还提供一种式(I)所示的化合物的制备方法，包括如下步骤：
将化合物A经还原反应制备化合物B：
将化合物B与化合物C经亲核取代反应制备化合物D：
将化合物D进行水解反应制备化合物E：
将化合物E与化合物F经酰化反应制备化合物G：
将化合物G经取代反应制备式(I)所述化合物：
其中，R1、R2、R3、R4的定义与上述一致；X选自-F、-Cl、-Br或-I。
本发明还提供一种上述化合物、或其药学上可接受的盐、或其立体异构体在制备治疗和/或预防与CDK2活性相关的或由CDK2活性介导的疾病的药物中的应用。
在一个具体的示例中，与CDK2活性相关的或由CDK2活性介导的疾病为癌症。
在一个具体的示例中，癌症为卵巢癌、胃癌、子宫内膜癌与乳腺癌中的一种或多种。
本发明还提供一种药物组合物，包括上述化合物、或其药学上可接受的盐、或其立体异构体，以及药学上可接受的载体。
可以理解地，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
以下结合具体实施例对本发明的氘代吡唑类衍生物及其制备方法做进一步详细的说明。以下实施例中所用的原料，如无特别说明，均为市售产品。
实施例1
本实施例提供化合物1，其结构式如下：
反应路线如下所示：
制备过程具体如下：
第1步：
在-60℃的温度下，将化合物1-1(2.00g,8.84mmol,1.00eq)溶于四氢呋喃(15.0mL)中，缓慢滴加DIBAL-H(1.00M,17.6mL,2.00eq)后，滴完后升温至-20℃，反应3小时，在0℃下向反应体系加入甲醇(20.0mL)淬灭反应，在20℃下搅拌2小时，然后减压浓缩得到粗品，粗品经柱层析法(SiO2，石油醚/乙酸乙酯＝1/0～0/1)进行纯化后得到化合物1-2。1HNMR:(400MHz,CDCl3)δ6.82(s,1H),4.67(s,2H),4.39-4.32(m,2H),4.15(s,3H),2.02(br s,1H),1.38(t,J＝7.2Hz,3H)。
第2步：
在0℃的温度下，将NaH(52.1mg,1.30mmol,60％purity,1.20eq)溶于四氢呋喃(1.00mL) 中，再将化合物1-2(200mg,1.09mmol,1.00eq)溶于四氢呋喃(1.00mL)后滴加到反应液中。在25℃下反应1小时后降温到0℃，滴加CD3I(770mg,5.43mmol,330μL,5.00eq)，在25℃下反应9小时后，在0℃下缓慢加入NH4Cl溶液2.0mL，再用乙酸乙酯(3.00mL)萃取3次。合并有机相，用饱和氯化钠水溶液(4.00mL)洗涤3次，无水硫酸钠干燥，过滤，浓缩滤液得到化合物1-3。LCMS:RT＝0.310min,MS(ESI)m/z＝167.0[M-34]+1HNMR:(400MHz,CDCl3)δ＝6.84(s,1H),4.44(s,2H),4.34(q,J＝7.2Hz,2H),4.16(s,3H),1.37(t,J＝7.2Hz,3H)。
第3步：
在25℃的温度下，将化合物1-3(200mg,993μmol,1.00eq)溶于四氢呋喃(1.00mL)和水(1.00mL)中，加入LiOH.H2O(43.7mg,1.04mmol,1.05eq)，25℃反应10小时后，将反应混合物减压浓缩以去除四氢呋喃后用盐酸调节pH到1.0，再用乙酸乙酯(2.00mL)萃取3次。合并有机相，用饱和氯化钠水溶液(3.00mL)洗涤3次，无水硫酸钠干燥，过滤，浓缩滤液得到化合物1-4。LCMS:MS(ESI)m/z＝139.0[M-34]+1HNMR:(400MHz,CDCl3)δ＝6.97(s,1H),4.48(s,2H),4.19(s,3H)。
第4步：
在25℃的温度下，将化合物1-4(168mg,972μmol,1.00eq)，化合物1-5(300mg,972μmol,1.00eq)溶于DMF(3.00mL)，加入HATU(406mg,1.07mmol,1.10eq)and DIEA(377mg,2.92mmol,508μL,3.00eq)，25℃下搅拌10小时后，将反应液加水(9.00mL)稀释，用乙酸乙酯(10.0mL)萃取3次。合并有机相，饱和氯化钠水溶液(15.0mL)洗涤3次，无水硫酸钠干燥，过滤，浓缩滤液得到化合物1-6。LCMS:m/z＝464.4(M+H)+1HNMR:EC6454-331-P1N1(400MHz,DMSO-d6)δ＝9.92(s,1H),7.95(s,1H),7.00(s,1H),6.02(s,1H),5.22(s,1H),4.70(s,1H),4.36(s,1H),4.03(s,3H),3.57(brdd,J＝6.4,13.2Hz,1H),3.05-2.94(m,1H),1.81-1.60(m,6H),1.51(s,9H),1.03(br d,J＝6.4Hz,6H)。
第5步：
室温下，将化合物1-6(273mg,588μmol,1.00eq)溶于甲酸(2.50mL)，在100℃反应1小时。将反应混合物减压浓缩以去除甲酸得到粗产品，粗产品经prep-HPLC(FA condition)纯化后得到化合物1。LCMS:m/z＝408.3(M+H)+1H NMR:(400MHz,DMSO-d6)δ12.22(br s,1H),10.72(s,1H),7.11(s,1H),6.95(br d,J＝7.2Hz,1H),6.41(br s,1H),5.01(br s,1H),4.33(s,2H),4.05(s,3H),3.58(qd,J＝6.8,13.6Hz,1H),3.14-3.03(m,1H),2.10-1.55(m,6H),1.03(d,J＝6.4Hz,6H)。
体外活性测试
实验例一：体外CDK2/CyclinE酶活性测试
实验材料：
CDK2/CyclinE 1购自新格诺康。Ulight-4E-BP1多肽，Eu-anti-phospho-tyrosine抗体，1X检测缓冲液购自PerkinElmer公司。高纯度ATP购自Promega公司。EDTA购自Sigma。Nivo多标记分析仪(PerkinElmer)。
实验方法：
激酶缓冲液配制：激酶缓冲液包含50mM HEPES，1mM EDTA，10mM MgCl2，0.01％Brij-35，pH7.4 200ml缓冲液中加入2.38g HEPES，58mg EDTA，406mg MgCl2，20mg Brij-35，调整pH到7.4。
终止液配制：使用100μL1MEDTA原液加上0.625uL的1X检测缓冲液与1725uL蒸馏水混合配置成终止液。
使用激酶缓冲液稀释酶，Ulight-4E-BP1多肽，ATP和抑制剂。使用检测缓冲液稀释Eu-anti-phospho-tyrosine抗体稀释至8nM/L浓度。将待测化合物用排枪进行5倍稀释至第8个浓度，即从4μM稀释至0.0512nM，DMSO终浓度为4％，设置双复孔实验。向微孔板中加入2.5μL抑制剂各浓度梯度，5μLCDK2/CyclinE 1酶(10ng)，2.5μL底物和ATP的混合物(4mMATP，100nM Ulight-4E-BP1多肽)，此时化合物终浓度梯度为1μM稀释至0.0128Nm,ATP和底物终浓度为1mM和25nM。反应体系置于25度反应120分钟。反应结束后，每孔加入5μL终止液，25度继续反应5分钟，结束反应后每孔加入5uL的Eu-anti-phospho-tyrosine抗体稀释液，25度反应60分钟后采用PerkinElmerNivo多标记分析仪TR-FRET模式进行数据采集(激发波长为320nm发射波长为615nm和665nm)。
数据分析：
利用方程式(Sample-Min)/(Max-Min)*100％将原始数据换算成抑制率，IC50的值即可通过四参数进行曲线拟合得出(GraphPad Prism中log(inhibitor)vs.response--Variable slope模式得出)。表1提供了本发明的化合物对CDK2/CyclinE 1酶学抑制活性。
实验例二：体外CDK1/CyclinB1酶活性测试
实验材料：
CDK1/CyclinB 1购自CARNA。Ulight-4E-BP1多肽，Eu-anti-phospho-tyrosine抗体，1X检测缓冲液购自PerkinElmer公司。高纯度ATP购自Promega公司。EDTA购自Sigma。Nivo多标记分析仪(PerkinElmer)。
实验方法：
激酶缓冲液配制：激酶缓冲液包含50mM HEPES，1mM EDTA，10mM MgCl2，0.01％Brij-35，PH7.4 200ml缓冲液中加入2.38g HEPES，58mg EDTA，406mg MgCl2，20mg Brij-35，调整pH到7.4。
终止液配制：使用100μL1MEDTA原液加上0.625uL的1X检测缓冲液与1725uL蒸馏水混合配置成终止液。
使用激酶缓冲液稀释酶，Ulight-4E-BP1多肽，ATP和抑制剂。
使用检测缓冲液稀释Eu-anti-phospho-tyrosine抗体稀释至8nM/L浓度。
将待测化合物用排枪进行5倍稀释至第8个浓度，即从4μM稀释至0.0512nM，DMSO终浓度为4％，设置双复孔实验。向微孔板中加入2.5μL抑制剂各浓度梯度，5μLCDK1/CyclinB1酶(0.5ng)，2.5μL底物和ATP的混合物(4mMATP，200nM Ulight-4E-BP1多肽)，此时化合物终浓度梯度为1μM稀释至0.0128nM，ATP和底物终浓度为1mM和50nM。反应体系置于25度反应60分钟。反应结束后，每孔加入5μL终止液，25度继续反应5分钟，结束反应后每孔加入5uL的Eu-anti-phospho-tyrosine抗体稀释液，25度反应60分钟后采用PerkinElmerNivo多标记分析仪TR-FRET模式进行数据采集(激发波长为320nm发射波长为615nm和665nm)。
数据分析：
利用方程式(Sample-Min)/(Max-Min)*100％将原始数据换算成抑制率，IC50的值即可通过四参数进行曲线拟合得出(GraphPad Prism中log(inhibitor)vs.response--Variable slope模式得出)。表1提供了本发明的化合物对CDK1/CyclinB 1酶学抑制活性。
实验例三：体外GSK3β酶活性测试
实验材料：
GSK3βActive购自SignalChem；GSK3 Substrate购自SignalChem；ADP-Glo Kinase Assay购自Promega；Kinase assay buffer III购自SignalChem；Nivo多标记分析仪(PerkinElmer)。
实验方法：
将待测化合物用100％DMSO稀释到100μM作为第一个浓度，然后再用排枪进行5倍稀释至第8个浓度，即从100μM稀释至0.0013μM。用1X kinasebuffer将化合物各浓度点进行20倍稀释，配制成含有5％DMSO的化合物工作液，向微孔板中加入1μL化合物各浓度梯度工作液，设置双复孔。向微孔板中加入2μl GSK3β酶(1ng)，2μl底物和ATP的混合物(62.5μMATP，0.5μg/μlGSK3 Substrate)，此时化合物终浓度梯度为1μM稀释至0.013nM，ATP和底物终浓度为25μM和0.2μg/μl。反应体系置于25度反应60分钟。反应结束后，每孔加入5μlADP-Glo试剂，25度继续反应40分钟，结束反应后每孔加入10uL的kinasedetection 试剂，25度反应30分钟后采用PerkinElmer Nivo多标记分析仪读数化学发光，积分时间0.5秒。
数据分析：
利用方程式(Sample-Min)/(Max-Min)*100％将原始数据换算成抑制率，IC50的值即可通过四参数进行曲线拟合得出(GraphPad Prism中log(inhibitor)vs.response--Variable slope模式得出)。表1提供了本发明的化合物对GSK3β酶学抑制活性。
Max孔:阳性对照孔读值无酶空白孔；
Min孔：阴性对照孔读值为含有1％DMSO溶剂孔。
实验例四：体外OVCAR3细胞活性测试
实验材料：
1640培养基，胎牛血清，盘尼西林/链霉素抗生素购自维森特。CellTiter-Glo(细胞活率化学发光检测试剂)试剂购自Promega。OVCAR3细胞系购自南京科佰生物科技有限公司。Envision多标记分析仪(PerkinElmer)。
实验方法：
将OVCAR3细胞种于白色384孔板中，40μL细胞悬液每孔，其中包含300个OVCAR3细胞。细胞板置于二氧化碳培养箱中过夜培养。
将待测化合物用排枪进行5倍稀释至第8个浓度，即从2000μM稀释至0.00512μM，设置双复孔实验。向中间板中加入78μL培养基，再按照对应位置，转移2μL每孔的梯度稀释化合物至中间板，混匀后转移10μL每孔到细胞板中。转移到细胞板中的化合物浓度范围是10μM至0.026nM。细胞板置于二氧化碳培养箱中培养7天。另准备一块细胞板，在加药当天读取信号值作为最大值(下面方程式中Max值)参与数据分析。向此细胞板每孔加入10μL细胞活率化学发光检测试剂，室温孵育10分钟使发光信号稳定。采用多标记分析仪读数。
向细胞板中加入每孔10μL的细胞活率化学发光检测试剂，室温孵育10分钟使发光信号稳定。采用多标记分析仪读数。
数据分析：
利用方程式(Sample-Min)/(Max-Min)*100％将原始数据换算成抑制率，IC50的值即可通过四参数进行曲线拟合得出(GraphPad Prism中"log(inhibitor)vs.response--Variable slope"模式得出)。表1提供了本发明的化合物对OVCAR3细胞增殖的抑制活性。
实验例五：体外T47D细胞活性测试
实验材料：
1640培养基，胎牛血清，盘尼西林/链霉素抗生素购自维森特。CellTiter-Glo(细胞活率 化学发光检测试剂)试剂购自Promega。T-47D细胞系购自南京科佰生物科技有限公司。Envision多标记分析仪(PerkinElmer)。
实验方法：
将T-47D细胞种于白色384孔板中，40μL细胞悬液每孔，其中包含300个T-47D细胞。细胞板置于二氧化碳培养箱中过夜培养。
将待测化合物用排枪进行5倍稀释至第8个浓度，即从2000μM稀释至0.00512μM，设置双复孔实验。向中间板中加入78μL培养基，再按照对应位置，转移2μL每孔的梯度稀释化合物至中间板，混匀后转移10μL每孔到细胞板中。转移到细胞板中的化合物浓度范围是10μM至0.026nM。细胞板置于二氧化碳培养箱中培养7天。另准备一块细胞板，在加药当天读取信号值作为最大值(下面方程式中Max值)参与数据分析。向此细胞板每孔加入10μL细胞活率化学发光检测试剂，室温孵育10分钟使发光信号稳定。采用多标记分析仪读数。
向细胞板中加入每孔10μL的细胞活率化学发光检测试剂，室温孵育10分钟使发光信号稳定。采用多标记分析仪读数。
数据分析：
利用方程式(Sample-Min)/(Max-Min)*100％将原始数据换算成抑制率，IC50的值即可通过四参数进行曲线拟合得出(GraphPad Prism中"log(inhibitor)vs.response--Variable slope"模式得出)。表1提供了本发明的化合物对T-47D细胞增殖的抑制活性。
表1
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，便于具体和详细地理解本发明的技术方案，但并不能因此而理解为对发明专利保护范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。应当理解，本领域技术人员在本发明提供的技术方案的基础上，通过合乎逻辑的分析、推理或者有限的试验得到的技术方案，均在本发明所附权利要求的保护范围 内。因此，本发明专利的保护范围应以所附权利要求的内容为准，说明书可以用于解释权利要求的内容。

Claims (10)

  1. 一种式(I)所示的化合物、或其药学上可接受的盐、或其立体异构体：
    其中：
    R1选自具有1至20个C原子的直链氘代烷基、具有3至20个C原子的支链氘代烷基、具有3至20个C原子的环状的氘代烷基、具有1至20个C原子的直链氘代烷氧基、具有3至20个C原子的支链氘代烷氧基、具有3至20个C原子的环状氘代烷氧基，或这些体系的组合；
    R2、R3、R4分别独立地选自-H、-D、具有1至20个C原子的直链烷基、具有1至20个C原子的直链氘代烷基、具有3至20个C原子的支链烷基、具有3至20个C原子的支链氘代烷基、具有3至20个C原子的环状的烷基、具有3至20个C原子的环状的氘代烷基、具有1至20个C原子的直链烷氧基、具有1至20个C原子的直链氘代烷氧基、具有3至20个C原子的支链烷氧基、具有3至20个C原子的支链氘代烷氧基、具有3至20个C原子的环状烷氧基、具有3至20个C原子的环状氘代烷氧基，或这些体系的组合。
  2. 根据权利要求1所述的化合物、或其药学上可接受的盐、或其立体异构体，其特征在于，式(I)化合物为式(II)所示结构：
  3. 根据权利要求1所述的化合物、或其药学上可接受的盐、或其立体异构体，其特征在于，R3选自-H或-D。
  4. 根据权利要求1所述的化合物、或其药学上可接受的盐、或其立体异构体，其特征在于，R1选自具有1至10个C原子的直链氘代烷基、具有3至10个C原子的支链氘代烷基、具有3至10个C原子的环状的氘代烷基、具有1至10个C原子的直链氘代烷氧基、具有3至10个C原子的支链氘代烷氧基、具有3至10个C原子的环状氘代烷氧基，或这些体系的 组合；
    R2、R4分别独立地选自-H、-D、具有1至10个C原子的直链烷基、具有1至10个C原子的直链氘代烷基、具有3至10个C原子的支链烷基、具有3至10个C原子的支链氘代烷基、具有3至10个C原子的环状的烷基、具有3至10个C原子的环状的氘代烷基、具有1至10个C原子的直链烷氧基、具有1至10个C原子的直链氘代烷氧基、具有3至10个C原子的支链烷氧基、具有3至10个C原子的支链氘代烷氧基、具有3至10个C原子的环状烷氧基、具有3至10个C原子的环状氘代烷氧基，或这些体系的组合。
  5. 根据权利要求4所述的化合物、或其药学上可接受的盐、或其立体异构体，其特征在于，R1选自具有1至5个C原子的直链氘代烷基、具有3至5个C原子的支链氘代烷基、具有3至5个C原子的环状的氘代烷基、具有1至5个C原子的直链氘代烷氧基、具有3至5个C原子的支链氘代烷氧基、具有3至5个C原子的环状氘代烷氧基，或这些体系的组合；
    R2、R4分别独立地选自具有1至3个C原子的直链烷基、具有1至3个C原子的直链氘代烷基、具有3至5个C原子的支链烷基、具有3至5个C原子的支链氘代烷基、具有3至5个C原子的环状的烷基、具有3至5个C原子的环状的氘代烷基，或这些体系的组合。
  6. 根据权利要求1所述的化合物、或其药学上可接受的盐、或其立体异构体，其特征在于，式(I)化合物为下列化合物：
  7. 一种式(I)所述化合物的制备方法，其特征在于，包括如下步骤：
    将化合物A经还原反应制备化合物B：
    将化合物B与化合物C经亲核取代反应制备化合物D：
    将化合物D进行水解反应制备化合物E：
    将化合物E与化合物F经酰化反应制备化合物G：
    将化合物G经取代反应制备式(I)所述化合物：
    其中，R1、R2、R3、R4的定义与权利要求1～6任一项所述一致；X选自-F、-Cl、-Br或-I。
  8. 权利要求1～6任一项所述的化合物、或其药学上可接受的盐、或其立体异构体在制备治疗和/或预防与CDK2活性相关的或由CDK2活性介导的疾病的药物中的应用。
  9. 根据权利要求8所述的应用，其特征在于，所述与CDK2活性相关的或由CDK2活性介导的疾病为癌症。
  10. 一种药物组合物，其特征在于，包括权利要求1～6任一项所述的化合物、或其药学上可接受的盐、或其立体异构体，以及药学上可接受的载体。
PCT/CN2023/117218 2022-09-30 2023-09-06 氘代吡唑类衍生物、药物组合物及应用与制备方法 WO2024066981A1 (zh)

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