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WO2024066986A1 - 2-氨基嘧啶类化合物及其应用、药用组合物 - Google Patents

2-氨基嘧啶类化合物及其应用、药用组合物 Download PDF

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Publication number
WO2024066986A1
WO2024066986A1 PCT/CN2023/117270 CN2023117270W WO2024066986A1 WO 2024066986 A1 WO2024066986 A1 WO 2024066986A1 CN 2023117270 W CN2023117270 W CN 2023117270W WO 2024066986 A1 WO2024066986 A1 WO 2024066986A1
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Prior art keywords
pharmaceutically acceptable
stereoisomer
acceptable salt
compound
aminopyrimidine
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English (en)
French (fr)
Inventor
胡璞
陆居权
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Starg Wuhan Pharmaceutical Technology Co Ltd
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Starg Wuhan Pharmaceutical Technology Co Ltd
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Priority to CN202380063760.4A priority Critical patent/CN119790049A/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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • 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
    • 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
    • C07D403/04—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 directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems

Definitions

  • the present invention relates to the field of medical technology, and in particular to a 2-aminopyrimidine compound and an application thereof and a pharmaceutical composition.
  • Protein kinases are enzymes that catalyze protein phosphorylation. They are widely present in the human body and play an important physiological function. Protein kinases include mitogen-activated protein kinases, cell cycle-dependent kinases, and tyrosine protein kinases. Among them, cell cycle-dependent kinases (CDKs) belong to the serine/threonine kinase family and are involved in physiological processes such as cell proliferation and transcription.
  • CDKs cell cycle-dependent kinases
  • CDKs According to the different functions of CDKs, they can be divided into two categories: one type of CDK is involved in cell cycle regulation, mainly including CDK1, CDK2, CDK4, CDK6, etc.; the other type of CDK is involved in transcriptional regulation, mainly including CDK7, CDK8, CDK9, CDK12, CDK13, etc.
  • cell cycle regulation mainly including CDK1, CDK2, CDK4, CDK6, etc.
  • transcriptional regulation mainly including CDK7, CDK8, CDK9, CDK12, CDK13, etc.
  • protein kinases When protein kinases are abnormally regulated or mutated, they are often prone to hyperproliferative diseases, cardiovascular and cerebrovascular diseases, diabetes, inflammation, and immune system diseases. Therefore, it is of great significance to provide a compound that can inhibit the activity of CDK.
  • the present invention provides a 2-aminopyrimidine compound capable of inhibiting the activity of CDK4 and CDK6, and its application and pharmaceutical composition.
  • R 1 is independently selected from halogen, cyano, trihalomethyl, nitro, carboxyl or acyl, and n 1 is selected from 1 or 2;
  • R 2 , R 4 , and R 5 are independently selected from H, halogen, C1-C4 alkyl, and C1-C4 alkoxy, n 2 is selected from 1 or 2, n 4 is selected from an integer of 1 to 4, and n 5 is selected from an integer of 1 to 3;
  • R3 is selected from H, C1-C4 alkyl, C1-C4 alkoxy, (dialkylamino)methyl, acyl, hydroxy-substituted alkyl;
  • X represents O or NR 6 ;
  • R6 is selected from alkylsulfonyl
  • Y represents N or CR 7 ;
  • R7 is selected from halogen, cyano, trihalomethyl, nitro, carboxyl, and acyl.
  • the general formula (I) is the structure of formula (I-1):
  • R 1 is selected from halogen and cyano.
  • R 1 is selected from Cl, cyano.
  • R 3 is selected from C1-C4 alkyl, (dialkylamino)methyl, acyl, and hydroxy-substituted alkyl.
  • R 3 is selected from methyl, (dimethylamino)methyl, acetyl, and 2-hydroxyisopropyl.
  • R 2 is H.
  • R 4 is H.
  • R 5 is H.
  • X represents O or NR 6 ; and R 6 is methylsulfonyl.
  • Y represents N or CR 7 ; and R 7 is selected from halogen and cyano.
  • the 2-aminopyrimidine compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof In the above, Y represents N or CR 7 ; R 7 is selected from F and cyano.
  • the general formula (I) is selected from any one of the structures of formula (II-1) to (II-9):
  • the present invention provides use of the above-mentioned 2-aminopyrimidine compounds, or pharmaceutically acceptable salts thereof, or stereoisomers thereof in the preparation of protein kinase inhibitors, wherein the protein kinase inhibitors include CDK4 inhibitors and CDK6 inhibitors.
  • the 2-aminopyrimidine compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof is used in the preparation of a protein kinase inhibitor, wherein the protein kinase inhibitor is a CDK4 inhibitor.
  • the present invention provides the use of the above-mentioned 2-aminopyrimidine compounds, or pharmaceutically acceptable salts thereof, or stereoisomers thereof in the preparation of drugs for treating or preventing protein kinase-mediated diseases, wherein the protein kinases include CDK4 and CDK6.
  • 2-aminopyrimidine compounds, or pharmaceutically acceptable salts thereof, or stereoisomers thereof in the preparation of drugs for treating or preventing protein kinase-mediated diseases, wherein the protein kinase is CDK4.
  • the use of 2-aminopyrimidine compounds, or pharmaceutically acceptable salts thereof, or stereoisomers thereof in the preparation of drugs for treating or preventing protein kinase-mediated diseases wherein the protein kinase-mediated disease is cancer, and the cancer includes at least one of breast cancer, non-small cell lung cancer, colon cancer, prostate cancer, thyroid cancer, malignant melanoma, neuroblastoma and mammary secretory carcinoma.
  • the present invention provides a pharmaceutical composition comprising:
  • Active ingredients and pharmaceutically acceptable carriers wherein the active ingredients include the above-mentioned 2-aminopyrimidine compounds, or pharmaceutically acceptable salts thereof, or stereoisomers thereof.
  • the 2-aminopyrimidine compounds of the present invention have the following beneficial effects:
  • the 2-aminopyrimidine compounds of the present invention are a series of new compounds, which can inhibit the activities of CDK4 and CDK6, especially can highly selectively inhibit the activity of CDK4; they can effectively treat or prevent cancer and other transitional proliferative diseases mediated by CDK4 and CDK6, especially treat or prevent cancer and other transitional proliferative diseases mediated by CDK4.
  • 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, and the like; 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, benzene
  • the invention also includes 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 be converted into any base or acid addition salt.
  • 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” arises from the inability of a double bond or single bond forming a ring carbon atom to 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 (such as in solution), chemical equilibrium of the tautomers can be achieved.
  • proton tautomers also called prototropic tautomers
  • Valence tautomers include interconversions via reorganization of some of the bonding electrons.
  • keto-enol tautomerism is the interconversion between pentane-2,4-dione and 4-hydroxypent-3-en-2-one.
  • 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 achievable.
  • 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; It means 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 1-4 alkyl is used to represent a straight or branched saturated hydrocarbon group consisting of 1 to 4 carbon atoms. It can be monovalent (such as methyl), divalent (such as methylene) or polyvalent (such as methine).
  • Examples of C 1-4 alkyl 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 1-4 alkoxy refers to those alkyl groups containing 1 to 4 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.
  • alkylsulfonyl means a -SO2R group, where R is alkyl as defined above, for example, methylsulfonyl, ethylsulfonyl, 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.
  • Aryl and “aromatic ring” are used interchangeably and 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 can be combined with a cycloalkyl ring, a heterocycloalkyl ring, a cycloalkenyl ring, a heterocycloalkenyl ring, or a cycloalkyl ring.
  • C 6-10 aryl refers to a monocyclic or bicyclic aromatic group having 6 to 10 carbon atoms, and non-limiting examples of the aryl group 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 valence permits. Heteroaryl bicyclic ring systems may include one or more heteroatoms in one or both rings.
  • 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 when more than one When hydrogen is replaced by a substituent, the substituents may be the same or different in type, and the substituents selected are each independent in type.
  • the compound of the present invention or its pharmaceutically acceptable salt, or its stereoisomer can be used in a suitable dosage form with one or more pharmaceutical carriers.
  • 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 therapeutically effective amount of the compound of the present invention or its pharmaceutically acceptable salt, or its stereoisomer contained in the pharmaceutical composition or the pharmaceutical composition of the present invention is preferably 0.1 mg-5 g/kg (body weight).
  • 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: Pd/C represents palladium carbon; H 2 represents hydrogen; N 2 represents nitrogen; mL represents milliliter; NaNO 2 represents sodium nitrite; HCl represents; SnCl 2 .2H 2 O represents dihydrated tin dichloride; H 2 O represents water; AcOH represents acetic acid; B 2 Pin 2 represents bis-pinacol borate; Pd(dppf)Cl 2 represents 1,1'-bis(diphenylphosphino)ferrocenepalladium dichloride; KOAc represents potassium acetate; EtOAc represents ethyl acetate; Pd(PPh 3 ) 4 represents tetrakis(triphenylphosphine)palladium; K 2 CO 3 represents potassium carbonate; MeCN represents acetonitrile; DIEA represents N,N-diisopropylethylamine; MeOH represents methanol; NaBH
  • One embodiment of the present invention provides a 2-aminopyrimidine compound having a general formula (I), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof:
  • R 1 is independently selected from halogen, cyano (-CN), trihalomethyl, nitro, carboxyl or acyl, and n 1 is selected from 1 or 2;
  • R 2 , R 4 , and R 5 are independently selected from H, halogen, C1-C4 alkyl, and C1-C4 alkoxy, n 2 is selected from 1 or 2, n 4 is selected from an integer of 1 to 4, and n 5 is selected from an integer of 1 to 3;
  • R3 is selected from H, C1-C4 alkyl, C1-C4 alkoxy, (dialkylamino)methyl, acyl, hydroxy-substituted alkyl;
  • X represents O or NR 6 ;
  • R6 is selected from alkylsulfonyl
  • Y represents N or CR 7 ;
  • R7 is selected from halogen, cyano, trihalomethyl, nitro, carboxyl, and acyl.
  • connection relationship between Ar 1 and Ar 2 can be any site of the 4th, 5th, and 6th positions of Ar 1 connected to any site of the 1st, 2nd, and 6th positions of Ar 2 ;
  • R 1 can appear at any site of the 4th, 5th, and 6th positions of Ar 1 except the site connected to Ar 2
  • R 2 can appear at any site of the 1st, 2nd, and 6th positions of Ar 2 except the site connected to Ar 1 .
  • halogen includes but is not limited to F, Cl, Br; trihalomethyl includes but is not limited to trifluoromethyl, trichloromethyl, tribromomethyl; acyl includes but is not limited to formyl, acetyl, propionyl; C1-C4 alkyl includes but is not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl; C1-C4 alkoxy includes but is not limited to methoxy, ethoxy, propoxy; (dialkylamino)methyl includes but is not limited to (dimethylamino)methyl, (diethylamino)methyl; hydroxy substituted alkyl includes but Not limited to hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, 2-hydroxypropyl, 2-hydroxyisopropyl, 2-hydroxybutyl, 3-hydroxybutyl, 4-hydroxybutyl, 5-hydroxypent
  • n1 is selected from 1 or 2, which means that one or two R1s may be contained;
  • n2 is selected from 1 or 2, which means that one or two R2s may be contained;
  • n4 is selected from an integer of 1 to 4, and n4 is selected from 1, 2, 3 or 4, which means that 1, 2, 3 or 4 R4s may be contained;
  • n5 is selected from an integer of 1 to 3, and n3 is selected from 1, 2 or 3, which means that 1, 2 or 3 R5s may be contained.
  • CDK4/6 inhibitors include Pfizer's Palbociclib, Novartis' LEE011 and Eli Lilly's Abemaciclib, which are used in combination with endocrine therapy for hormone receptor-positive, human epidermal growth factor receptor 2-negative advanced or metastatic breast cancer.
  • treatment with CDK4/6 inhibitors has been shown to cause adverse reactions in clinical practice, such as gastrointestinal and hematological toxicity.
  • CDK4 is a single oncogenic driver in many breast cancers.
  • 2-aminopyrimidine compounds with the general formula (I) can inhibit the activities of CDK4 and CDK6, especially can highly selectively inhibit the activity of CDK4; can effectively treat or prevent cancer and other transitional proliferative diseases mediated by CDK4 and CDK6, especially treat or prevent cancer and other transitional proliferative diseases mediated by CDK4.
  • the 2-aminopyrimidine compounds of the general formula (I) have a highly selective inhibitory effect on CDK4 activity and can effectively reduce the side effects caused by CDK4/6 inhibitors.
  • the 2-aminopyrimidine compound having the general formula (I), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, the general formula (I) is the structure of formula (I-1):
  • R 1 is selected from halogen and cyano.
  • R 1 is selected from Cl and cyano.
  • R 3 is selected from C1-C4 alkyl, (dialkylamino)methyl, acyl, or hydroxy-substituted alkyl.
  • R3 is selected from methyl, (dimethylamino)methyl Acetyl, 2-hydroxyisopropyl
  • R 2 , R 4 , and R 5 are independently selected from H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, and tert-butyl.
  • R 2 is H.
  • R 4 is H.
  • R 5 is H.
  • X represents O or NR 6 ; and R 6 is selected from methylsulfonyl and ethylsulfonyl.
  • R 6 is methylsulfonyl; in this case, NR 6 is
  • Y represents N or CR 7 ; and R 7 is selected from halogen and cyano.
  • R7 is selected from F and cyano.
  • the 2-aminopyrimidine compound having the general formula (I), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, the general formula (I) is selected from any one of the structures of formula (II-1) to (II-9):
  • the 2-aminopyrimidine compound having the general formula (I), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof is selected from the following compounds:
  • the 2-aminopyrimidine compound having the general formula (I), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof is selected from the following compounds:
  • the 2-aminopyrimidine compound having the general formula (I), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, the general formula (I) is selected from any one of the structures of formula (II-1), (II-2), (II-5), and (II-7):
  • the pharmaceutically acceptable salt is an alkyl acid salt.
  • the pharmaceutically acceptable salt is formate.
  • One embodiment of the present invention provides the use of the above-mentioned 2-aminopyrimidine compounds, or pharmaceutically acceptable salts thereof, or stereoisomers thereof in the preparation of protein kinase inhibitors, wherein the protein kinase inhibitors include CDK inhibitors.
  • 2-aminopyrimidine compounds, or pharmaceutically acceptable salts thereof, or stereoisomers thereof are used in the preparation of protein kinase inhibitors, and the protein kinase inhibitors include CDK4 inhibitors and CDK6 inhibitors.
  • a 2-aminopyrimidine compound, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof is used in the preparation of a protein kinase inhibitor, wherein the protein kinase inhibitor is a CDK4 inhibitor.
  • One embodiment of the present invention provides the use of the above-mentioned 2-aminopyrimidine compounds, or pharmaceutically acceptable salts thereof, or stereoisomers thereof in the preparation of drugs for treating or preventing protein kinase-mediated diseases, wherein the protein kinase inhibitors include CDK inhibitors.
  • 2-aminopyrimidine compounds, or pharmaceutically acceptable salts thereof, or stereoisomers thereof are used in the preparation of drugs for treating or preventing protein kinase-mediated diseases, wherein the protein kinases include CDK4 and CDK6.
  • the protein kinase in the use of 2-aminopyrimidine compounds, or pharmaceutically acceptable salts thereof, or stereoisomers thereof for preparing a drug for treating or preventing protein kinase-mediated diseases, the protein kinase is CDK4.
  • 2-aminopyrimidine compounds, or pharmaceutically acceptable salts thereof, or stereoisomers thereof are used in the preparation of drugs for treating or preventing protein kinase-mediated diseases, wherein the protein kinase-mediated disease is cancer, and the cancer includes at least one of breast cancer, non-small cell lung cancer, colon cancer, prostate cancer, thyroid cancer, malignant melanoma, neuroblastoma and mammary secretory carcinoma.
  • 2-aminopyrimidine compounds, or pharmaceutically acceptable salts thereof, or stereoisomers thereof are used in the preparation of drugs for treating or preventing protein kinase-mediated diseases, and the drugs for treating or preventing protein kinase-mediated diseases are drugs for inhibiting breast cancer cell proliferation.
  • the breast cancer cells are MCF7 cells or T47D cells.
  • One embodiment of the present invention provides a pharmaceutical composition, comprising:
  • Active ingredients and pharmaceutically acceptable carriers wherein the active ingredients include the above-mentioned 2-aminopyrimidine compounds, or pharmaceutically acceptable salts thereof, or stereoisomers thereof.
  • pharmaceutically acceptable carriers include, but are not limited to, sustained-release agents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, adsorption carriers, surfactants and lubricants.
  • the synthetic route is as follows:
  • step 1
  • compound 1-2 (2.00 g, 9.75 mmol, 1.00 eq) and compound 1-3 (1.20 g, 10.7 mmol, 1.10 eq) were added to AcOH (10.0 mL) solution, stirred at 120°C for 5 hours; 10.0 mL H 2 O was added to the reaction system for dilution, extracted three times with 45.0 mL (15.0 mL*3) of ethyl acetate, then washed three times with 45.0 mL (15.0 mL*3) of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product compound 1-4.
  • compound 1-4 500 mg, 1.77 mmol, 1.00 eq
  • B 2 Pin 2 585 mg, 2.30 mmol, 1.30 eq
  • Pd(dppf)Cl 2 144 mg, 177 umol, 0.10 eq
  • KOAc 521 mg, 5.32 mmol, 3.00 eq
  • compound 1-5 400 mg, 1.22 mmol, 1.00 eq
  • compound 1-6 222 mg, 1.22 mmol, 1.00 eq
  • Pd(PPh 3 ) 4 140 mg, 121 umol, 0.100 eq
  • K 2 CO 3 503 mg, 3.65 mmol, 3.00 eq
  • 10.0 mL H 2 O was added to the reaction system for dilution, extracted three times with 15.0 mL (5.00 mL*3) of ethyl acetate, and then washed three times with 15.0 mL (5.00 mL*3) of saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain The crude product was obtained.
  • compound 1-7 400 mg, 1.14 mmol, 1.00 eq
  • compound 1-8 (267 mg, 2.28 mmol, 2.00 eq) were dissolved in MeCN (4.00 mL), DIEA (147 mg, 1.14 mmol, 198 uL, 1.00 eq) was added, and the mixture was stirred at 80°C for 12 hours.
  • the synthetic route is as follows:
  • step 1
  • compound 1-2 (5.00 g, 24.3 mmol, 1.00 eq) and compound 2-1 (4.15 g, 24.3 mmol, 1.00 eq) were added to AcOH (10.0 mL) solution and stirred at 120°C for 5 hours.
  • 300 mL H 2 O was added to the reaction system for dilution, extracted three times with EtOAc 300 mL (100 mL*3), then washed three times with saturated sodium chloride solution 300 mL (100 mL*3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product.
  • Boc 2 O (6.38 g, 29.2 mmol, 6.71 mL, 10.0 eq) and DMAP (35.7 mg, 292 umol, 0.100 eq) were added to a solution of compound 2-3 (1.00 g, 2.92 mmol, 1.00 eq) in THF (1.00 mL), and stirred at 25°C for 4 hours.
  • compound 2-4 400 mg, 904 umol, 1.00 eq was added to a solution of THF (4.00 mL) with compound 2-5 (185 mg, 1.90 mmol, 2.10 eq), and then isopropylmagnesium chloride (2.00 M, 2.03 mL, 4.50 eq) was slowly added dropwise, and stirred at -20°C for 1 hour.
  • compound 2-10 23.0 mg, 64.3 umol, 1.00 eq
  • compound 1-6 (11.8 mg, 64.3 umol, 1.0 eq) were dissolved in a mixed solution of dioxane (0.500 mL) and water (0.200 mL), and then Pd(PPh 3 ) 4 (7.44 mg, 6.44 umol, 0.10 eq) and K 2 CO 3 (26.7 mg, 193 umol, 3.00 eq) were added, and stirred at 80°C for 3 hours.
  • the synthetic route is as follows:
  • step 1
  • compound 3-3 (150 mg, 523 umol, 1.00 eq, HCl) was dissolved in ethyl acetate (0.50 mL) and saturated aqueous sodium bicarbonate solution (0.50 mL). After replacing N 2 three times, MsCl (180 mg, 1.57 mmol, 121 uL, 3.00 eq) was added dropwise to the reaction solution at 0°C. After the addition was completed, the mixture was reacted at 0°C for 2 hours. The reaction solution was slowly poured into water (1.00 mL) at 0°C.
  • the synthetic route is as follows:
  • step 1
  • compound 1-5 ((200 mg, 607 umol, 1.00 eq), compound 4-1 (112 mg, 607 umol, 1.00 eq), K 2 CO 3 (251 mg, 1.82 mmol, 3.00 eq) and Pd(Amphos) 2 Cl 2 (43.0mg, 60.7umol, 43.0uL, 0.100eq) was dissolved in dioxane (2.00mL) and water (0.500mL), and replaced with nitrogen 3 times. Reacted for 12 hours under a nitrogen atmosphere at 90°C. Water (2.00mL) was added to the reaction system, and then extracted 3 times with ethyl acetate (5.00mL*3).
  • compound 4-3 (50.0 mg, 146 umol, 1.00 eq), compound 4-4 (27.0 mg, 176 umol, 1.20 eq), DIEA (56.8 mg, 440 umol, 76.6 uL, 3.00 eq) were dissolved in acetonitrile (0.500 mL) and reacted at 80 ° C for 12 hours. Water (1.00 mL) was added to the reaction system, and then extracted with ethyl acetate (2.00 mL*3) for 3 times.
  • CDK4/CyclinD1 was purchased from Invitrogen; Ulight-4E-BP1 peptide, 1X detection buffer, and Eu-anti-phospho-tyrosine antibody were purchased from PerkinElmer; high-purity ATP was purchased from Promega; EDTA was purchased from Sigma; Nivo multi-label analyzer (PerkinElmer).
  • Kinase buffer contained: 50 mM HEPES, 1 mM EDTA, 10 mM MgCl 2 , 0.01% Brij-35, pH 7.4;
  • kinase buffer Add 2.38 g HEPES, 58 mg EDTA, 406 mg MgCl 2 , and 20 mg Brij-35 to 200 mL buffer and adjust the pH to 7.4.
  • kinase buffer to dilute enzyme, Ulight-4E-BP1 peptide, ATP and inhibitor; use detection buffer to dilute Eu-anti-phospho-tyrosine antibody to 8nM/L concentration; dilute the test compound 5 times to the 8th concentration with a gun, that is, from 8 ⁇ M to 0.102nM, the final DMSO concentration is 4%, and set up a double-well experiment.
  • the final concentration gradient of the compound is 2 ⁇ M diluted to 0.0256nM, and the final concentrations of ATP and substrate are 1mM and 25nM.
  • the reaction system is placed at 25°C for 180 minutes. After the reaction, 5 ⁇ L of stop solution was added to each well, and the reaction was continued at 25°C for 5 minutes. After the reaction was completed, 5 uL of Eu-anti-phospho-tyrosine antibody dilution was added to each well, and the reaction was carried out at 25°C for 60 minutes, and the data were collected using the TR-FRET mode of the PerkinElmer Nivo multi-label analyzer (excitation wavelength was 320 nm, and emission wavelengths were 615 nm and 665 nm).
  • CDK6/CyclinD1 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 contained: 50 mM HEPES, 1 mM EDTA, 10 mM MgCl 2 , 0.01% Brij-35, pH 7.4;
  • kinase buffer Add 2.38 g HEPES, 58 mg EDTA, 406 mg MgCl 2 , and 20 mg Brij-35 to 200 mL buffer and adjust the pH to 7.4.
  • kinase buffer to dilute enzyme, Ulight-4E-BP1 peptide, ATP and inhibitor; use detection buffer to dilute Eu-anti-phospho-tyrosine antibody to 8nM/L concentration; dilute the test compound 5 times to the 8th concentration with a gun, that is, from 8 ⁇ M to 0.102nM, the final DMSO concentration is 4%, and set up a double-well experiment.
  • Add 2.5 ⁇ L of inhibitor concentration gradients, 5 ⁇ L CDK6/CyclinD1 enzyme (2ng), 2.5 ⁇ L of substrate and ATP mixture (4mMATP, 50nM Ulight-4E-BP1 peptide) to the microplate.
  • the final compound concentration gradient is 2 ⁇ M diluted to 0.0256nM, and the final concentrations of ATP and substrate are 1mM and 12.5nM.
  • the reaction system is placed at 25°C for 60 minutes. After the reaction, 5 ⁇ L of stop solution was added to each well, and the reaction was continued at 25°C for 5 minutes. After the reaction was completed, 5 uL of Eu-anti-phospho-tyrosine antibody dilution was added to each well, and the reaction was carried out at 25°C for 60 minutes, and the data were collected using the TR-FRET mode of the PerkinElmer Nivo multi-label analyzer (excitation wavelength was 320 nm, and emission wavelengths were 615 nm and 665 nm).
  • EMEM culture medium was from Vicente; fetal bovine serum was from Biosera; penicillin/streptomycin antibiotics were purchased from Yuanpei; CellTiter-Glo (cell viability chemiluminescence detection reagent) reagent was purchased from Promega; MCF-7 cell line was purchased from Pronose; Envision multi-label analyzer (PerkinElmer).
  • MCF-7 cells were seeded in a white 96-well plate, with 80 ⁇ L of cell suspension per well, containing 2000 MCF-7 cells, and the cell plate was placed in a carbon dioxide incubator for overnight culture.
  • the compound to be tested was diluted 5 times to the 8th concentration, i.e., from 2000 ⁇ M to 0.0256 nM, 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 dilution compound per well was transferred to the middle plate according to the corresponding position. After mixing, 20 ⁇ L of each well was transferred to the cell plate.
  • the concentration range of the compound transferred to the cell plate was 10 ⁇ M to 0.128 nM, and the cell plate was placed in a carbon dioxide incubator 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 25 ⁇ L of cell viability chemiluminescent detection reagent to each well of this cell plate, and incubate at room temperature for 10 minutes to stabilize the luminescent signal. Reading was performed using a multi-label analyzer. Add 25 ⁇ L of cell viability chemiluminescent detection reagent to each well of the cell plate and incubate at room temperature for 10 minutes to stabilize the luminescent signal. Read using a multi-label analyzer.
  • 1640 culture medium was from Vivacell; fetal bovine serum was from Biosera; penicillin/streptomycin antibiotics were purchased from Yuanpei; CellTiter-Glo (cell viability chemiluminescence detection reagent) 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, and the cell plate was placed in a carbon dioxide incubator for overnight culture.
  • the compound to be tested was diluted 5 times to the 8th concentration, i.e., from 2000 ⁇ M to 0.0256 ⁇ 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 dilution compound per well was transferred to the middle plate according to the corresponding position. After mixing, 10 ⁇ L of each well was transferred to the cell plate.
  • the concentration range of the compound transferred to the cell plate was 10 ⁇ M to 0.128 nM, and the cell plate was placed in a carbon dioxide incubator 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. Reading was performed using a multi-label analyzer. Add 10 ⁇ L of cell viability chemiluminescent detection reagent to each well of the cell plate and incubate at room temperature for 10 minutes to stabilize the luminescent signal. Read using a multi-label analyzer.
  • the compounds prepared in Examples 1 to 4 have different degrees of inhibitory effects on CDK4 and CDK6, especially highly selective inhibition of CDK4; and can effectively inhibit the proliferation of MCF7 cells and T47D cells.

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Abstract

本发明涉及一种2-氨基嘧啶类化合物及其应用、药用组合物，该类化合物能够抑制CDK4和CDK6活性，尤其是可高选择性抑制CDK4活性；可有治疗或预防由CDK4和CDK6介导的癌症等过渡增殖性疾病，尤其是治疗或预防由CDK4介导的癌症等过渡增殖性疾病。

Description

2-氨基嘧啶类化合物及其应用、药用组合物 技术领域
本发明涉及医药技术领域，特别涉及一种2-氨基嘧啶类化合物及其应用、药用组合物。
背景技术
蛋白激酶(protein kinases，简称PK)，是催化蛋白质磷酸化过程的酶，在人体中广泛存在并发挥着重要的生理功能。蛋白激酶包括分裂原激活的蛋白激酶、细胞周期依赖性激酶和酪氨酸蛋白激酶等。其中，细胞周期依赖性激酶(CDK)属于丝氨酸/苏氨酸激酶家族，参与细胞的增殖和转录等生理过程。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。当蛋白激酶出现异常调节或突变后，往往易引发过度增殖性疾病、心脑血管疾病、糖尿病、炎症和免疫系统疾病等。因此，提供一种能够抑制CDK活性的化合物具有重要意义。
发明内容
基于此，本发明提供了一种能够抑制CDK4和CDK6活性的2-氨基嘧啶类化合物及其应用、药用组合物。
本发明解决上述技术问题的技术方案如下。
一种具有通式(I)结构的2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体：
其中：
R1分别独立地选自卤素、氰基、三卤甲基、硝基、羧基或酰基，n1选自1或2；
R2、R4、R5分别独立地选自H、卤素、C1-C4烷基、C1-C4烷氧基，n2选自1或2，n4选自1～4的整数，n5选自1～3的整数；
R3选自H、C1-C4烷基、C1-C4烷氧基、(二烷氨基)甲基、酰基、羟基取代的烷基；
X表示O或NR6；
R6选自烷基磺酰基；
Y表示N或CR7；
R7选自卤素、氰基、三卤甲基、硝基、羧基、酰基。
在其中一些实施例中，2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体中，所述通式(I)为式(I-1)结构：
在其中一些实施例中，2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体中，R1选自卤素、氰基。
在其中一些实施例中，2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体中，R1选自Cl、氰基。
在其中一些实施例中，2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体中，R3选自C1-C4烷基、(二烷氨基)甲基、酰基、羟基取代的烷基。
在其中一些实施例中，2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体中，R3选自甲基、(二甲氨基)甲基、乙酰基、2-羟基异丙基。
在其中一些实施例中，2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体中，R2为H。
在其中一些实施例中，2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体中，R4为H。
在其中一些实施例中，2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体中，R5为H。
在其中一些实施例中，2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体中，X表示O或NR6；，R6为甲基磺酰基。
在其中一些实施例中，2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体中，Y表示N或CR7；R7选自卤素、氰基。
在其中一些实施例中，2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体 中，Y表示N或CR7；R7选自F、氰基。
在其中一些实施例中，2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体中，所述通式(I)选自式(II-1)～(II-9)中任一结构：
本发明提供了上述2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体在制备蛋白激酶抑制剂中的应用，所述蛋白激酶抑制剂包括CDK4抑制剂和CDK6抑制剂。
在其中一些实施例中，2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体在制备蛋白激酶抑制剂中的应用中，所述蛋白激酶抑制剂为CDK4抑制剂。
本发明提供了上述2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体在制备治疗或预防蛋白激酶介导疾病药物中的应用，所述蛋白激酶包括CDK4和CDK6。
在其中一些实施例中，2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体在制备治疗或预防蛋白激酶介导疾病药物中的应用中，所述蛋白激酶为CDK4。
在其中一些实施例中，2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体在制备治疗或预防蛋白激酶介导疾病药物中的应用中，所述蛋白激酶介导疾病为癌症，所述癌症包括乳腺癌、非小细胞肺癌、结肠癌、前列腺癌、甲状腺癌、恶性黑色素瘤、神经母细胞瘤和乳腺样分泌癌中的至少一种。
本发明提供了一种药用组合物，包括：
活性成分和药学上可接受的载体，所述活性成分包括上述的2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体。
与现有技术相比较，本发明的2-氨基嘧啶类化合物具有如下有益效果：
本发明的2-氨基嘧啶类化合物，是一系列新的化合物，该类化合物能够抑制CDK4和CDK6活性，尤其是可高选择性抑制CDK4活性；可有效治疗或预防由CDK4和CDK6介导的癌症等过渡增殖性疾病，尤其是治疗或预防由CDK4介导的癌症等过渡增殖性疾病。
具体实施方式
以下结合具体实施例对本发明的技术方案作进一步详细的说明。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。应当理解，提供这些实施方式的目的是使对本发明公开内容理解更加透彻全面。
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例的目的，不是旨在于限制本发明。
术语定义
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，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。
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“C1-4烷基”用于表示直链或支链的由1至4个碳原子组成的饱和碳氢基团。其可以是一价(如甲基)、二价(如亚甲基)或者多价(如次甲基)。C1-4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“C1-4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4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“烷基磺酰基”是指-SO2R基团，其中R是如上定义的烷基，例如，甲基磺酰基、乙基磺酰基等。
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。
“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“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)。
“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：Pd/C代表钯碳；H2代表氢气；N2代表氮气；mL代表毫升；NaNO2代表亚硝酸钠；HCl代表；SnCl2.2H2O代表2水合二氯化锡；H2O代表水；AcOH代表醋酸；B2Pin2代表双联频哪醇硼酸酯；Pd(dppf)Cl2代表1,1'-双二苯基膦二茂铁二氯化钯；KOAc代表醋酸钾；EtOAc代表乙酸乙酯；Pd(PPh3)4代表四(三苯基膦)钯；K2CO3代表碳酸钾；MeCN代表乙腈；DIEA代表N,N-二异丙基乙胺；MeOH 代表甲醇；NaBH3CN代表氰基硼氢化钠；THF代表四氢呋喃；Boc2O代表碳酸酐二叔丁酯；DMAP代表4-二甲氨基吡啶；HCl/EtOAc代表盐酸乙酸乙酯；MnO2代表二氧化锰；Cbz-Cl代表氯甲酸苄酯；MsCl代表甲烷磺酰氯；Pd(Amphos)2Cl2代表二氯二叔丁基-(4-二甲基氨基苯基)磷钯(II)；SO2Cl2代表二氯化砜；NaHCO3代表碳酸氢钠。
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。
本发明一实施方式提供了一种具有通式(I)结构的2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体：
其中：
R1分别独立地选自卤素、氰基(-CN)、三卤甲基、硝基、羧基或酰基，n1选自1或2；
R2、R4、R5分别独立地选自H、卤素、C1-C4烷基、C1-C4烷氧基，n2选自1或2，n4选自1～4的整数，n5选自1～3的整数；
R3选自H、C1-C4烷基、C1-C4烷氧基、(二烷氨基)甲基、酰基、羟基取代的烷基；
X表示O或NR6；
R6选自烷基磺酰基；
Y表示N或CR7；
R7选自卤素、氰基、三卤甲基、硝基、羧基、酰基。
可以理解，如式(I-A)结构所示，通式(I)中，Ar1与Ar2的连接关系可以是Ar1的4位、5位、6位的任一位点与Ar2的1位、2位、6位的任一位点连接；R1可以出现在Ar1的4位、5位、6位除与Ar2连接的位点以外的任意位点，R2可以出现在Ar2的1位、2位、6位除与Ar1连接的位点以外的任意位点。
进一步可理解，卤素包括但不限于F、Cl、Br；三卤甲基包括但不限于三氟甲基、三氯甲基、三溴甲基；酰基包括但不限于甲酰基、乙酰基、丙酰基；C1-C4烷基包括但不限于甲基、乙基、正丙基、异丙基、正丁基、叔丁基、异丁基；C1-C4烷氧基包括但不限于甲氧基、乙氧基、丙氧基；(二烷氨基)甲基包括但不限于(二甲氨基)甲基、(二乙氨基)甲基；羟基取代的烷基包括但不限于羟甲基、2-羟基乙基、3-羟基丙基、2-羟基丙基、2-羟基异丙基、2-羟基丁基、3-羟基丁基、4-羟基丁基、5-羟基戊基、2-羟基戊基、3-羟基戊基、4-羟基戊基、6-羟基己基、2-羟己基、3-羟己基、4-羟己基、1-甲基-2-羟乙基、1，1-二甲基-2-羟乙基、1，2-二羟乙基、2，2-二羟乙基；烷基磺酰基包括但不限于甲基磺酰基、乙基磺酰基。
还可理解，n1选自1或2，代表可包含一个或者两个R1；n2选自1或2，代表可包含一个或者两个R2；n4选自1～4的整数，n4选自1、2、3或4，代表可包含1、2、3或4个R4；n5选自1～3的整数，n3选自1、2或3，代表可包含1、2、3个R5。
当前，获批上市的CDK4/6抑制剂有辉瑞的Palbociclib，诺华的LEE011和礼来的Abemaciclib，联用内分泌治疗在激素受体阳性、人类表皮生长因子受体2阴性晚期或转移性乳腺癌。但用CDK4/6抑制剂治疗在临床被证明导致不良反应，例如胃肠道和血液学毒性。
研究发现，CDK4是许多乳腺癌中的单一致癌驱动因子。
研究证明，通式(I)结构的2-氨基嘧啶类化合物，能够抑制CDK4和CDK6活性，尤其是可高选择性抑制CDK4活性；可有效治疗或预防由CDK4和CDK6介导的癌症等过渡增殖性疾病，尤其是治疗或预防由CDK4介导的癌症等过渡增殖性疾病。
通式(I)结构的2-氨基嘧啶类化合物，对CDK4活性具有高选择性抑制作用，可有效减少CDK4/6抑制剂带来的副作用。
在其中一些示例中，具有通式(I)结构的2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体中，通式(I)为式(I-1)结构：
进一步地，n1、n2、n4、n5均为1时，通式(I)为式(I-2)结构：
在其中一些示例中，具有通式(I)结构的2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体中，R1选自卤素、氰基。
进一步地，具有通式(I)结构的2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体中，R1选自Cl、氰基。
在其中一些示例中，具有通式(I)结构的2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体中，R3选自C1-C4烷基、(二烷氨基)甲基、酰基、羟基取代的烷基。
在其中一些示例中，具有通式(I)结构的2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体中，R3选自甲基、(二甲氨基)甲基乙酰基、2-羟基异丙基
在其中一些示例中，具有通式(I)结构的2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体中，R2、R4、R5分别独立地选自H、甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基。
在其中一些示例中，具有通式(I)结构的2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体中，R2为H。
在其中一些示例中，具有通式(I)结构的2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体中，R4为H。
在其中一些示例中，具有通式(I)结构的2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体中，R5为H。
在其中一些示例中，具有通式(I)结构的2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体中，X表示O或NR6；，R6选自甲基磺酰基、乙基磺酰基。
进一步地，R6为甲基磺酰基；此时NR6为
在其中一些示例中，具有通式(I)结构的2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体中，Y表示N或CR7；R7选自卤素、氰基。
进一步地，R7选自F、氰基。
在其中一些示例中，具有通式(I)结构的2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体中，通式(I)选自式(II-1)～(II-9)中任一结构：
相应地，具有通式(I)结构的2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体中，2-氨基嘧啶类化合物选自如下化合物：
(3S,4R)-4-((5-氯-4-(7'-氟-2'-甲基螺[环戊烷-1,3'-吲哚]-5'-基)嘧啶-2-基)氨基)四氢-2H-吡喃-3-醇、
1-(5'-(5-氯-2-(((3S,4R)-3-羟基四氢-2H-吡喃-4-基)氨基)嘧啶-4-基)-7'-氟螺[环戊烷-1,3'-吲哚]-2'-基)乙烷-1-酮、
(3S,4R)-4-((5-氯-4-(2'-((二甲氨基)甲基)-7'-氟螺[环戊烷-1,3'-吲哚]-5'-基)嘧啶-2-基)氨基)四氢-2H-吡喃-3-醇、
(3S,4R)-4-((5-氯-4-(7'-氟-2'-(2-羟基丙烷-2-基螺[环戊烷-1,3'-吲哚]-5'-基))嘧啶-2-基)氨基)四氢-2H-吡喃-3-醇、
4-(7'-氟-2'-甲基螺[环戊烷-1,3'-吲哚]-5'-基)-2-(((3S,4R)-3-羟基四氢-2H-吡喃-4-基)氨基)嘧啶-5-腈、
5'-(5-氰基-2-(((3S,4R)-3-羟基四氢-2H-吡喃-4-基)氨基)嘧啶-4-基)-2'-甲基螺[环戊烷-1,3'-吲哚]-7'-腈、
(3R,4R)-4-((5-氯-4-(7'-氟-2'-甲基螺[环戊烷-1,3'-吲哚]-5'-基)嘧啶-2-基)氨基)-1-(甲基磺酰 基)哌啶-3-醇、
4-(7'-氟-2'-甲基螺[环戊烷-1,3'-吲哚]-5'-基)-2-(((3R,4R)-3-羟基-1-(甲基磺酰基)哌啶-4-基)氨基)嘧啶-5-腈、
(3S,4R)-4-((5-氯-4-(2'-甲基螺[环戊烷-1,3'-吡咯并[2,3-b]吡啶]-5'-基)嘧啶-2-基)氨基)四氢-2H-吡喃-3-醇。
在其中一些示例中，具有通式(I)结构的2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体中，2-氨基嘧啶类化合物选自如下化合物：
(3S,4R)-4-((5-chloro-4-(7'-fluoro-2'-methylspiro[cyclopentane-1,3'-indol]-5'-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol、
1-(5'-(5-chloro-2-(((3S,4R)-3-hydroxytetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)-7'-fluorospiro[cyclopentane-1,3'-indol]-2'-yl)ethan-1-one、
(3S,4R)-4-((5-chloro-4-(2'-((dimethylamino)methyl)-7'-fluorospiro[cyclopentane-1,3'-indol]-5'-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol、
(3S,4R)-4-((5-chloro-4-(7'-fluoro-2'-(2-hydroxypropan-2-yl)spiro[cyclopentane-1,3'-indol]-5'-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol、
4-(7'-fluoro-2'-methylspiro[cyclopentane-1,3'-indol]-5'-yl)-2-(((3S,4R)-3-hydroxytetrahydro-2H-pyran-4-yl)amino)pyrimidine-5-carbonitrile、
5'-(5-cyano-2-(((3S,4R)-3-hydroxytetrahydro-2H-pyran-4-yl)amino)pyrimidin-4-yl)-2'-methylspiro[cyclopentane-1,3'-indole]-7'-carbonitrile、
(3R,4R)-4-((5-chloro-4-(7'-fluoro-2'-methylspiro[cyclopentane-1,3'-indol]-5'-yl)pyrimidin-2-yl)amino)-1-(methylsulfonyl)piperidin-3-ol、
4-(7'-fluoro-2'-methylspiro[cyclopentane-1,3'-indol]-5'-yl)-2-(((3R,4R)-3-hydroxy-1-(methylsulfonyl)piperidin-4-yl)amino)pyrimidine-5-carbonitrile、
(3S,4R)-4-((5-chloro-4-(2'-methylspiro[cyclopentane-1,3'-pyrrolo[2,3-b]pyridin]-5'-yl)pyrimidin-2-yl)amino)tetrahydro-2H-pyran-3-ol。
在其中一些具体的示例中，具有通式(I)结构的2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体中，通式(I)选自式(II-1)、(II-2)、(II-5)、(II-7)中任一结构：
在其中一些示例中，具有通式(I)结构的2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体中，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。
本发明一实施方式提供了上述2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体在制备蛋白激酶抑制剂中的应用，所述蛋白激酶抑制剂包括CDK抑制剂。
在其中一些示例中，2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体在制备蛋白激酶抑制剂中的应用中，蛋白激酶抑制剂包括CDK4抑制剂和CDK6抑制剂。
在其中一些示例中，2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体在制备蛋白激酶抑制剂中的应用中，蛋白激酶抑制剂为CDK4抑制剂。
本发明一实施方式提供了上述2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体在制备治疗或预防蛋白激酶介导疾病药物中的应用，蛋白激酶抑制剂包括CDK抑制剂。
在其中一些示例中，2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体在制备治疗或预防蛋白激酶介导疾病药物中的应用中，蛋白激酶包括CDK4和CDK6。
在其中一些示例中，2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体在制备治疗或预防蛋白激酶介导疾病药物中的应用中，蛋白激酶为CDK4。
在其中一些示例中，2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体在制备治疗或预防蛋白激酶介导疾病药物中的应用中，蛋白激酶介导疾病为癌症，所述癌症包括乳腺癌、非小细胞肺癌、结肠癌、前列腺癌、甲状腺癌、恶性黑色素瘤、神经母细胞瘤和乳腺样分泌癌中的至少一种。
在其中一些示例中，2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体在制备治疗或预防蛋白激酶介导疾病药物中的应用中，治疗或预防蛋白激酶介导疾病药物为抑制乳腺癌细胞增殖药物。
进一步地，乳腺癌细胞为MCF7细胞或T47D细胞。
本发明一实施方式提供了一种药用组合物，包括：
活性成分和药学上可接受的载体，活性成分包括上述的2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体。
可以理解，药学上可接受的载体包括但不限于缓释剂、赋形剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、吸附载体、表面活性剂和润滑剂。
具体实施例
以下按照本发明的2-氨基嘧啶类化合物及其应用、药用组合物举例，可理解，本发明的2-氨基嘧啶类化合物及其应用、药用组合物并不局限于下述实施例。
实施例1
(3S,4R)-4-((5-氯-4-(7'-氟-2'-甲基螺[环戊烷-1,3'-吲哚]-5'-基)嘧啶-2-基)氨基)四氢-2H-吡喃-3-醇(II-1)
合成路线如下：
第1步：
在-20℃下，将NaNO2(1.90g,27.5mmol,1.05eq)滴加到化合物1-1(5.00g,26.3mmol,1.00eq)的HCl(25.0mL)溶液中，在此温度下搅拌30分钟；然后将混合物在-20℃下滴加至SnCl2.2H2O(19.0g,84.2mmol,3.20eq)的HCl(25.0mL)溶液中，将得到的混合物在0℃下搅拌1.5小时；向反应体系中加入10.0mL H2O稀释，用乙酸乙酯15.0mL(5.00mL*3)萃取三次，然后用饱和氯化钠溶液15.0mL(5.00mL*3)水洗三次，无水硫酸钠干燥，过滤，减压浓缩得到粗产物化合物1-2。LCMS:MS(ESI)m/z＝206.8[M+H]+。1H NMR:CDCl3,400MHzδ＝7.11(dd,J＝1.2,8.6Hz,1H),7.04(dd,J＝2.0,11.0Hz,1H),6.94(t,J＝8.8Hz,1H),5.34(br s,1H),3.48(br s,2H)。
第2步：
在室温下，将化合物1-2(2.00g,9.75mmol,1.00eq)和化合物1-3(1.20g,10.7mmol,1.10eq)分别加入到AcOH(10.0mL)溶液中，在120℃下搅拌5小时；向反应体系中加入10.0mL H2O稀释，用乙酸乙酯45.0mL(15.0mL*3)萃取三次，然后用饱和氯化钠溶液45.0mL(15.0mL*3)水洗三次，无水硫酸钠干燥，过滤，减压浓缩得到粗产物化合物1-4。LCMS:MS(ESI)m/z＝283.7[M+H]+。1H NMR:CDCl3,400MHzδ＝7.15(d,J＝1.6Hz,1H),7.12(dd,J＝1.6,9.0Hz,1H),2.23(s,3H),1.99-1.90(m,6H),1.76-1.70(m,2H)。
第3步：
在室温下，将化合物1-4(500mg,1.77mmol,1.00eq)，B2Pin2(585mg,2.30mmol,1.30eq)，Pd(dppf)Cl2(144mg,177umol,0.10eq)、KOAc(521mg,5.32mmol,3.00eq)分别加入到二氧六环(5.00mL)溶液中，在N2氛围下置换气3次，然后在90℃下搅拌12小时；向反应体系中加入10.0mL H2O稀释，用EtOAc 15.0mL(5.00mL*3)萃取三次，然后用饱和氯化钠溶液15.0mL(5.00mL*3)水洗三次，无水硫酸钠干燥，过滤，减压浓缩得到粗产物化合物1-5(500mg)为白色固体。LCMS:MS(ESI)m/z＝329.8[M+H]+。1H NMR:CDCl3,400MHzδ＝7.41(s,1H),7.38(d,J＝10.4Hz,1H),2.26(s,3H),2.05(br d,J＝3.2Hz,2H),1.96-1.88(m,5H),1.82-1.75(m,2H),1.28(s,13H),1.17(s,6H)。
第4步：
在室温下，将化合物1-5(400mg,1.22mmol,1.00eq)、化合物1-6(222mg,1.22mmol,1.00eq)、Pd(PPh3)4(140mg,121umol,0.100eq)、K2CO3(503mg,3.65mmol,3.00eq)分别加入二氧六环(3.00mL)和水(1.00mL)的混合溶液中，在N2氛围下置换3次，然后在90℃下搅拌12小时；向反应体系中加入10.0mL H2O稀释，用乙酸乙酯15.0mL(5.00mL*3)萃取三次，然后用饱和氯化钠溶液15.0mL(5.00mL*3)水洗三次，无水硫酸钠干燥，过滤，减压浓缩得 到粗产物。采用制备-高效液相色谱法(FA条件)对其进行纯化获得的化合物1-7。LCMS:MS(ESI)m/z＝349.8[M+H]+。1H NMR:CDCl3,400MHzδ＝8.58(s,1H),7.61-7.55(m,2H),2.31(s,3H),2.06-1.96(m,6H),1.86-1.78(m,2H)。
第5步：
室温下，将化合物1-7(400mg,1.14mmol,1.00eq)和化合物1-8(267mg,2.28mmol,2.00eq)溶解于MeCN(4.00mL)中，加入DIEA(147mg,1.14mmol,198uL,1.00eq)，在80℃下搅拌12小时。向反应体系中加入10.0mL H2O稀释，用乙酸乙酯15.0mL(5.00mL*3)萃取三次，然后用饱和氯化钠溶液15.0mL(5.00mL*3)水洗三次，无水硫酸钠干燥，过滤，减压浓缩得到粗产物。采用制备-高效液相色谱法(FA条件:Phenomenexluna C18 150*25.0mm*10.0um；mobile phase:[water(FA)-ACN]；B％:36.0％-66.0％,15.0min)对其进行纯化获得的化合物II-1。LCMS:MS(ESI)m/z＝432.1[M+H]+。1H NMR:DMSO-d6,400MHzδ＝8.41(s,1H),7.64(brdd,J＝1.6,5.1Hz,1H),7.58-7.46(m,2H),4.94-4.89(m,1H),3.94-3.70(m,4H),3.56-3.43(m,1H),3.04(br t,J＝10.4Hz,1H),2.32(s,3H),2.06-1.91(m,7H),1.73(br d,J＝4.0Hz,2H),1.55-1.45(m,1H)。
实施例2
1-(5'-(5-氯-2-(((3S,4R)-3-羟基四氢-2H-吡喃-4-基)氨基)嘧啶-4-基)-7'-氟螺[环戊烷-1,3'-吲哚]-2'-基)乙烷-1-酮(II-2)
合成路线如下：
第1步：
在室温下，将化合物1-2(5.00g,24.3mmol,1.00eq)和化合物2-1(4.15g,24.3mmol,1.00eq)分别加入到AcOH(10.0mL)溶液中在120℃下搅拌5小时。向反应体系中加入300mL H2O稀释，用EtOAc 300mL(100mL*3)萃取三次，然后用饱和氯化钠溶液300mL(100mL*3)水洗三次，无水硫酸钠干燥，过滤，减压浓缩得到粗产物。采用柱层析法(SiO2，石油醚/乙酸乙酯＝1/0～0/1)对粗产物进行纯化，获得化合物2-2。LCMS:MS(ESI)m/z＝342.0[M+H]+。1H NMR:CDCl3,400MHzδ＝7.34-7.30(m,1H),7.28(s,1H),4.46(q,J＝7.2Hz,2H),2.57-2.45(m,2H),2.27-2.01(m,5H),1.79(td,J＝6.1,12.2Hz,3H),1.49-1.42(m,3H)。
第2步：
在室温下，将化合物2-2(1.00g,2.94mmol,1.00eq)加入到AcOH(6.00mL)和MeOH(8.00mL)的混合溶液中加入NaBH3CN(554mg,8.82mmol,3.00eq)，在25℃下搅拌5小时。向反应体系中加入30.0mL H2O稀释，用EtOAc 30.0mL(10.0mL*3)萃取三次，然后用饱和氯化钠溶液30.0mL(10.0mL*3)水洗三次，无水硫酸钠干燥，过滤，减压浓缩得到粗产物化合物2-3。LCMS:MS(ESI)m/z＝343.7[M+H]+。
第3步：
在室温下，将化合物2-3(1.00g,2.92mmol,1.00eq)的THF(1.00mL)溶液中加入Boc2O(6.38g,29.2mmol,6.71mL,10.0eq)和DMAP(35.7mg,292umol,0.100eq)，在25℃下搅拌4小时。向反应体系中加入30.0mL H2O稀释，用EtOAc 30.0mL(10.0mL*3)萃取三次，然后 用饱和氯化钠溶液30.0mL(10.0mL*3)水洗三次，无水硫酸钠干燥，过滤，减压浓缩得到粗产物化合物2-4。LCMS:MS(ESI)m/z＝341.8[M+H]+。1H NMR:CDCl3,400MHzδ＝7.07-7.00(m,1H),6.88(d,J＝1.8Hz,1H),4.53(s,1H),4.14-4.06(m,2H),1.82-1.76(m,4H),1.73-1.54(m,4H),1.44(s,10H),1.18(d,J＝7.1Hz,3H)。
第4步：
在-20℃下，将化合物2-4(400mg,904umol,1.00eq)的THF(4.00mL)溶液中加入化合物2-5(185mg,1.90mmol,2.10eq)，然后缓慢的滴加异丙基氯化镁(2.00M,2.03mL,4.50eq)，在-20℃下搅拌1小时。0℃下向反应体系中加入30.0mL H2O稀释，用EtOAc 30.0mL(10.0mL*3)萃取三次，然后用饱和氯化钠溶液30.0mL(10.0mL*3)水洗三次，无水硫酸钠干燥，过滤，减压浓缩得到粗产物化合物2-6。LCMS:MS(ESI)m/z＝341.9[M+H]+。
第5步：
在-30℃下，将化合物2-6(400mg,874umol,1.00eq)的THF(4.00mL)溶液中缓慢的滴加甲基溴化镁(2.50M,1.05mL,3.00eq)，在-30℃下搅拌1小时。0℃下向反应体系中加入10.0mL H2O稀释，用EtOAc 9.00mL(3.00mL*3)萃取三次，然后用饱和氯化钠溶液9.00mL(3.00mL*3)水洗三次，无水硫酸钠干燥，过滤，减压浓缩得到粗产物化合物2-7。LCMS:MS(ESI)m/z＝314.0[M+H]+。
第6步：
在室温下，将化合物2-7(300mg,727umol,1.00eq)溶解于HCl/EtOAc(3.00mL)中，在25℃下搅拌1小时。将反应混合物减压浓缩，去除溶剂，获得粗产物化合物2-8。LCMS:MS(ESI)m/z＝313.8[M+H]+。
第7步：
在室温下，将化合物2-8(220mg,704umol,1.00eq)的二氯甲烷(2.00mL)溶液中加入MnO2(306mg,3.52mmol,5.00eq)，在25℃下搅拌3小时。0℃下向反应体系中加入10.0mL H2O稀释，用EtOAc 9.00mL(3.00mL*3)萃取三次，然后用饱和氯化钠溶液9.00mL(3.00mL*3)水洗三次，无水硫酸钠干燥，过滤，减压浓缩得到粗产物化合物2-9。LCMS:MS(ESI)m/z＝309.8[M+H]+。1H NMR:CDCl3,400MHzδ＝4.10-4.00(m,1H),2.60(s,3H),2.37-2.27(m,2H),2.21-2.08(m,2H),1.99-1.92(m,3H),1.67-1.57(m,3H)。
第8步：
在室温下，将化合物2-9(20.0mg,64.4umol,1.00eq)和B2Pin2(81.8mg,322umol,5.00eq)溶解于二氧六环(0.500mL)溶液中，然后加入Pd(dppf)Cl2(4.72mg,6.45umol,0.10eq)和KOAc(18.9mg,193umol,3.0eq)，在80℃下搅拌12小时。0℃下向反应体系中加入10.0mL H2O稀 释，用EtOAc 9.00mL(3.00mL*3)萃取三次，然后用饱和氯化钠溶液9.00mL(3.00mL*3)水洗三次，无水硫酸钠干燥，过滤，减压浓缩得到粗产物化合物2-10。LCMS:MS(ESI)m/z＝358.0[M+H]+。
第9步：
在室温下，将化合物2-10(23.0mg,64.3umol,1.00eq)和化合物1-6(11.8mg,64.3umol,1.0eq)溶解于二氧六环(0.500mL)和水(0.200mL)的混合溶液中，然后加入Pd(PPh3)4(7.44mg,6.44umol,0.10eq)和K2CO3(26.7mg,193umol,3.00eq)，在80℃下搅拌3小时。0℃下向反应体系中加入10.0mL H2O稀释，用EtOAc 9.00mL(3.00mL*3)萃取三次，然后用饱和氯化钠溶液9.00mL(3.00mL*3)水洗三次，无水硫酸钠干燥，过滤，减压浓缩得到粗产物。采用制备-高效液相色谱法(FA条件)对其进行纯化获得化合物2-11。LCMS:MS(ESI)m/z＝335.8[M+H]+。
第10步：
室温下，将化合物2-11(13.0g,34.3umol,1.00eq)和化合物1-8(8.05mg,68.7umol,2.00eq)溶解于MeCN(0.500mL)溶液中，加入DIEA(13.3mg,103umol,17.9uL,3.00eq)，在80℃下搅拌12小时。0℃下向反应体系中加入10.0mL H2O稀释，用EtOAc 9.00mL(3.00mL*3)萃取三次，然后用饱和氯化钠溶液9.00mL(3.00mL*3)水洗三次，无水硫酸钠干燥，过滤，减压浓缩得到粗产物。采用制备-高效液相色谱法(中性条:Waters xbridge 150*25.0mm10um；mobile phase:[water(NH4HCO3)-ACN]；B％:42％-72％,8.0min)对其进行纯化获得化合物2。LCMS:MS(ESI)m/z＝459.0[M+H]+。1H NMR:DMSO-d6,400MHzδ＝8.44(s,1H),7.81-7.52(m,3H),4.93(d,J＝5.6Hz,1H),3.90-3.78(m,3H),3.57-3.44(m,2H),3.03(br t,J＝10.4Hz,1H),2.62(s,3H),2.42-2.30(m,2H),2.19-1.86(m,6H),1.70(brdd,J＝5.8,11.3Hz,2H)。
实施例3
(3R,4R)-4-((5-氯-4-(7'-氟-2'-甲基螺[环戊烷-1,3'-吲哚]-5'-基)嘧啶-2-基)氨基)-1-(甲基磺酰基)哌啶-3-醇(II-7)
合成路线如下：
第1步：
室温下，将化合物3-1(500mg,2.31mmol,1.00eq)溶于二氯甲烷(5.00mL)和碳酸钠饱和水溶液(5.00mL)，0℃下滴加Cbz-Cl(473mg,2.77mmol,394uL,1.20eq)。滴加完毕后，将混合物在0℃下反应3小时后监测。在0℃下将反应液缓慢倒入水中(5.00mL)，用二氯甲烷(5.00mL*3)萃取3次。合并有机相，用饱和氯化钠水溶液(5.00mL)洗涤，无水硫酸钠干燥，过滤，浓缩滤液得到粗产品。粗产品使用柱层析纯化(SiO2,石油醚:乙酸乙酯＝50:1～3:1,产物:Rf＝0.50)，得到化合物3-2。LCMS:m/z＝251.1[M+H]+。1H NMR:(400MHz DMSO-d6)δ7.37-7.28(m,4H),7.15(br d,J＝8.0Hz,1H),5.01(s,2H),3.76(br d,J＝13.2Hz,1H),3.31-3.26(m,1H),3.20(tt,J＝4.8,9.2Hz,1H),1.81-1.69(m,1H),1.38(s,9H),1.29-1.19(m,4H)。
第2步：
室温下，将化合物3-2(770mg,2.20mmol,1.00eq)溶于二氯甲烷(5.00mL)中，然后向该混合物中加入HCl/EtOAc(4.00M,5.50mL,10.0eq)。25℃反应2小时。将反应液直接浓缩得到化合物3-3的盐酸盐。LCMS:m/z＝251.0[M+H]+。1H NMR:(400MHz DMSO-d6)δ8.42(br s,1H),7.54-7.41(m,1H),7.38-7.28(m,4H),5.09-4.97(m,2H),3.65(td,J＝4.0,7.6Hz,1H),3.55-3.46(m,1H),3.21-3.09(m,2H),2.90(br d,J＝6.4Hz,1H),2.81-2.71(m,1H),2.07-1.94(m,1H),1.69-1.51(m,1H)。
第3步：
室温下，将化合物3-3(150mg,523umol,1.00eq,HCl)溶于乙酸乙酯(0.50mL)和碳酸氢钠饱和水溶液(0.50mL)，置换N2三次后，0℃下向反应液中滴加MsCl(180mg,1.57mmol,121uL,3.00eq)。滴加完毕后，混合物在0℃下反应2小时。在0℃下将反应液缓慢倒入水(1.00mL) 中，用乙酸乙酯(1.00mL*3)萃取3次。合并有机相，用饱和氯化钠水溶液(1.00mL)洗涤，无水硫酸钠干燥，过滤，浓缩滤液得到粗产品。粗产品使用制备-高效液相色谱法(制备柱:column:Welch Ultimate C18 150*25.0mm*5.0um；mobile phase:[water(TFA)-ACN]；B％:16.0％-46.0％,10.0min)制备纯化冻干，得到化合物3-4。LCMS:m/z＝329.1[M+H]+。1H NMR:(400MHz DMSO-d6)δ7.38-7.31(m,4H),7.25(br d,J＝8.4Hz,1H),5.18(br d,J＝3.2Hz,1H),5.02(s,2H),3.56-3.48(m,1H),3.45-3.35(m,2H),3.31-3.24(m,1H),2.86(s,3H),2.80(dt,J＝2.4,11.7Hz,1H),2.59(dd,J＝9.2,11.2Hz,1H),1.93-1.83(m,1H),1.50-1.36(m,1H)。
第4步：
室温下，将化合物3-4(80.0mg,243umol,1.00eq)溶于二氯甲烷(1.00mL)和甲醇(0.80mL)，使用H2置换空气3次后加入Pd/C(20.0mg,487umol,10％纯度，2.00eq)。使用H2置换空气3次，在15psi，H2条件下，20℃反应1小时。将反应液过滤，浓缩滤液得到化合物3-5。LCMS:EC6457-348-P1B1,产物:RT＝0.124min,m/z＝195.1[M+H]+。
第5步：
室温下，在化合物1-7(20.0mg,57.1umol,1.00eq)和化合物3-5(20.0mg,102umol,1.80eq)的MeCN(0.50mL)溶液中加入DIEA(22.1mg,171umol,29.8uL,3.00eq)。混合物在80℃下搅拌72小时。将反应液缓慢倒入水中(1.00mL)，用乙酸乙酯(1.00mL*3)萃取3次。合并有机相，用饱和氯化钠水溶液(1.00mL)洗涤，无水硫酸钠干燥，过滤，浓缩滤液得到粗产品。粗产品使用制备-高效液相色谱法(制备柱:column:Welch Ultimate C18 150*25.0mm*5.0um；mobile phase:[water(TFA)-ACN]；B％:30％-60％,10.0min)制备纯化冻干，得到化合物3。LCMS:m/z＝508.3[M+H]+。1H NMR:(400MHz DMSO-d6)δ8.41(s,1H),7.71-7.59(m,1H),7.58-7.42(m,2H),5.30-5.09(m,1H),3.86-3.73(m,1H),3.67-3.55(m,2H),3.51-3.45(m,1H),2.89(s,3H),2.87-2.79(m,1H),2.69-2.60(m,1H),2.35-2.27(m,3H),2.10-1.95(m,7H),1.73(br dd,J＝4.5,6.8Hz,2H),1.59-1.45(m,1H)。
实施例4
4-(7'-氟-2'-甲基螺[环戊烷-1,3'-吲哚]-5'-基)-2-(((3S,4R)-3-羟基四氢-2H-吡喃-4-基)氨基)嘧啶-5-腈(II-5)
合成路线如下：
第1步：
室温下，将化合物1-5((200mg,607umol,1.00eq)，化合物4-1(112mg,607umol,1.00eq)，K2CO3(251mg,1.82mmol,3.00eq)和Pd(Amphos)2Cl2(43.0mg,60.7umol,43.0uL,0.100eq)溶于二氧六环(2.00mL)和水(0.500mL)中，氮气置换3次。在90℃的氮气氛围下反应12小时。反应体系加入水(2.00mL)，再用乙酸乙酯(5.00mL*3)萃取3次。合并有机相，用饱和氯化钠水溶液(10.0mL*3)洗涤3次，无水硫酸钠干燥，过滤，浓缩滤液得到化合物4-2。LCMS:MS(ESI)m/z＝330.1[M+H]+。
第2步：
在0℃下，化合物4-2(85.0mg,241umol,1eq)溶于乙腈(0.500mL)中，缓慢滴加SO2Cl2(97.6mg,723umol,72.3uL,3.00eq)，在0℃下搅拌1小时。在0℃下，将反应体系缓慢加入NaHCO3(aq,2.00mL)中，用乙酸乙酯(2.00mL*3)萃取3次。合并有机相，饱和氯化钠水溶液(5.00mL*3)洗涤3次，无水硫酸钠干燥，过滤，浓缩滤液得到化合物4-3。LCMS:m/z＝341.0(M+H)+
第3步：
室温下，将化合物4-3(50.0mg,146umol,1.00eq)，化合物4-4(27.0mg,176umol,1.20eq),DIEA(56.8mg,440umol,76.6uL,3.00eq)溶于乙腈(0.500mL)中，80℃下反应12小时。反应体系加入水(1.00mL)，再用乙酸乙酯(2.00mL*3)萃取3次。合并有机相，用饱和氯化钠水溶液(4.00mL*3)洗涤3次，无水硫酸钠干燥，过滤，浓缩滤液得到粗产品，粗产品经制备-高 效液相色谱法(FA condition)纯化后得到化合物4。LCMS:m/z＝422.2[M+H]+。1H NMR:(400MHz,DMSO-d6)δ8.81-8.72(m,1H),8.44-8.32(m,1H),7.93-7.81(m,1H),7.77-7.65(m,1H),5.02-4.96(m,1H),4.07-3.93(m,1H),3.82(br dd,J＝4.8,10.7Hz,2H),3.58-3.47(m,1H),3.04(dt,J＝4.4,10.4Hz,1H),2.33(s,3H),2.07(br d,J＝3.6Hz,6H),1.98-1.83(m,1H),1.72(br d,J＝5.2Hz,2H),1.61-1.20(m,2H)。
体外活性测试
实验例一：体外CDK4/CyclinD1酶活性测试
实验材料：
CDK4/CyclinD1购自Invitrogen；Ulight-4E-BP1多肽，1X检测缓冲液，Eu-anti-phospho-tyrosine抗体购自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：
激酶缓冲液包含：50mM HEPES，1mM EDTA，10mM MgCl2，0.01％Brij-35，pH7.4；
配制激酶缓冲液：200mL缓冲液中加入2.38g HEPES，58mg EDTA，406mg MgCl2，20mg Brij-35，调整pH到7.4。
配置终止液：使用100μL 1M EDTA原液加上0.625uL的1X检测缓冲液与1725uL蒸馏水混合。
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8μM稀释至0.102nM，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，5μL CDK4/CyclinD1酶(0.5ng)，化合物与酶25℃孵育1小时。向微孔板中加入2.5μL底物和ATP的混合物(4mM ATP，100nM Ulight-4E-BP1多肽)，此时化合物终浓度梯度为2μM稀释至0.0256nM，ATP和底物终浓度为1mM和25nM。反应体系置于25℃反应180分钟。反应结束后，每孔加入5μL终止液，25℃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℃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)。
实验例二：体外CDK6/CyclinD1酶活性测试
实验材料：
CDK6/CyclinD1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：
激酶缓冲液包含：50mM HEPES，1mM EDTA，10mM MgCl2，0.01％Brij-35，pH7.4；
配制激酶缓冲液：200mL缓冲液中加入2.38g HEPES，58mg EDTA，406mg MgCl2，20mg Brij-35，调整pH到7.4。
配置终止液：使用100μL 1M EDTA原液加上0.625uL的1X检测缓冲液与1725uL蒸馏水混合。
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8μM稀释至0.102nM，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，5μL CDK6/CyclinD1酶(2ng)，2.5μL底物和ATP的混合物(4mMATP，50nM Ulight-4E-BP1多肽)，此时化合物终浓度梯度为2μM稀释至0.0256nM，ATP和底物终浓度为1mM和12.5nM。反应体系置于25℃反应60分钟。反应结束后，每孔加入5μL终止液，25℃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℃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)。
实验例三：体外MCF-7细胞活性测试
实验材料：
EMEM培养基自维森特；胎牛血清自Biosera；盘尼西林/链霉素抗生素购自源培；CellTiter-Glo(细胞活率化学发光检测试剂)试剂购自Promega；MCF-7细胞系购自普诺赛；Envision多标记分析仪(PerkinElmer)。
实验方法：
将MCF-7细胞种于白色96孔板中，80μL细胞悬液每孔，其中包含2000个MCF-7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0.0256nM，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20μL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0.128nM，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25μL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25μL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。
实验例四：体外T47D细胞活性测试
实验材料：
1640培养基自Vivacell；胎牛血清自Biosera；盘尼西林/链霉素抗生素购自源培；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0.0256μM，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0.128nM，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％将实验例一～实验例四的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)，结果如表1所示。
表1
从表1可知，实施例1～4制得的化合物对CDK4和CDK6具有不同程度的抑制作用，尤其对CDK4具有高度选择性抑制；能有效抑制MCF7细胞和T47D细胞的增殖。
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 (15)

  1. 一种具有通式(I)结构的2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体：
    其中：
    R1分别独立地选自卤素、氰基、三卤甲基、硝基、羧基或酰基，n1选自1或2；
    R2、R4、R5分别独立地选自H、卤素、C1-C4烷基、C1-C4烷氧基，n2选自1或2，n4选自1～4的整数，n5选自1～3的整数；
    R3选自H、C1-C4烷基、C1-C4烷氧基、(二烷氨基)甲基、酰基、羟基取代的烷基；
    X表示O或NR6；
    R6选自烷基磺酰基；
    Y表示N或CR7；
    R7选自卤素、氰基、三卤甲基、硝基、羧基、酰基。
  2. 如权利要求1所述的2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体，其特征在于，所述通式(I)为式(I-1)结构：
  3. 如权利要求1或2所述的2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体，其特征在于，通式(I)具备如下(1)～(2)至少一个特征：
    (1)R1选自卤素、氰基；
    (2)R3选自C1-C4烷基、(二烷氨基)甲基、酰基、羟基取代的烷基。
  4. 如权利要求3所述的2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体，其特征在于，通式(I)具备如下(1)～(2)至少一个特征：
    (1)R1选自Cl、氰基；
    (2)R3选自甲基、(二甲氨基)甲基、乙酰基、2-羟基异丙基。
  5. 如权利要求1或2所述的2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体，其特征在于，通式(I)具备如下(1)～(3)至少一个特征：
    (1)R2为H；
    (2)R4为H；
    (3)R5为H。
  6. 如权利要求1或2所述的2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体，其特征在于，X表示O或NR6；R6为甲基磺酰基。
  7. 如权利要求1或2所述的2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体，其特征在于，Y表示N或CR7；R7选自卤素、氰基。
  8. 如权利要求7所述的2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体，其特征在于，R7选自F、氰基。
  9. 如权利要求1或2所述的2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体，其特征在于，所述通式(I)选自式(II-1)～(II-9)中任一结构：
  10. 如权利要求1～9任一项所述的2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体在制备蛋白激酶抑制剂中的应用，所述蛋白激酶抑制剂包括CDK4抑制剂和CDK6抑制剂。
  11. 如权利要求10所述的应用，所述蛋白激酶抑制剂为CDK4抑制剂。
  12. 如权利要求1～9任一项所述的2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体在制备治疗或预防蛋白激酶介导疾病药物中的应用，所述蛋白激酶包括CDK4和CDK6。
  13. 如权利要求12所述的应用，所述蛋白激酶为CDK4。
  14. 如权利要求12所述的应用，所述蛋白激酶介导疾病为癌症，所述癌症包括乳腺癌、非小细胞肺癌、结肠癌、前列腺癌、甲状腺癌、恶性黑色素瘤、神经母细胞瘤和乳腺样分泌癌中的至少一种。
  15. 一种药用组合物，其特征在于，包括：
    活性成分和药学上可接受的载体，所述活性成分包括权利要求1～9任一项所述的2-氨基嘧啶类化合物、或其药学上可接受的盐、或其立体异构体。
PCT/CN2023/117270 2022-09-30 2023-09-06 2-氨基嘧啶类化合物及其应用、药用组合物 Ceased WO2024066986A1 (zh)

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