WO2021143701A1 - 嘧啶-4(3h)-酮类杂环化合物、其制备方法及其在医药学上的应用 - Google Patents

嘧啶-4(3h)-酮类杂环化合物、其制备方法及其在医药学上的应用 Download PDF

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WO2021143701A1
WO2021143701A1 PCT/CN2021/071375 CN2021071375W WO2021143701A1 WO 2021143701 A1 WO2021143701 A1 WO 2021143701A1 CN 2021071375 W CN2021071375 W CN 2021071375W WO 2021143701 A1 WO2021143701 A1 WO 2021143701A1
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alkyl
ring
cycloalkyl
cyano
group
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PCT/CN2021/071375
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English (en)
French (fr)
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陈向阳
高英祥
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北京诺诚健华医药科技有限公司
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Priority to AU2021208025A priority Critical patent/AU2021208025A1/en
Priority to JP2022543700A priority patent/JP2023511337A/ja
Priority to MX2022008881A priority patent/MX2022008881A/es
Priority to US17/793,571 priority patent/US20230219946A1/en
Priority to CN202180009655.3A priority patent/CN114981268A/zh
Priority to KR1020227028172A priority patent/KR20220130168A/ko
Priority to CA3168452A priority patent/CA3168452A1/en
Priority to EP21741216.2A priority patent/EP4092024A4/en
Publication of WO2021143701A1 publication Critical patent/WO2021143701A1/zh

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    • 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
    • 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
    • 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
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/10—Spiro-condensed systems
    • C07D491/107—Spiro-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00

Definitions

  • the present invention relates to a novel pyrimidine-4(3H)-one heterocyclic compound or a pharmaceutically acceptable salt thereof that regulates or inhibits the activity of SHP2, a pharmaceutical composition containing the compound or a pharmaceutically acceptable salt thereof, and
  • the method for preparing the compound or its pharmaceutically acceptable salt and the compound or its pharmaceutically acceptable salt or a pharmaceutical composition containing the compound or its pharmaceutically acceptable salt are prepared for the treatment and/or prevention of SHP2
  • SHP2 (Src Homology 2 domain-containing Phosphatase 2) is a non-receptor protein tyrosine phosphatase encoded by the PTPN11 (Protein Tyrosine Phosphatase Nonreceptor type 11) gene.
  • SHP2 contains two SH2 domains (Src Homology domain), a PTP domain (Protein Tyrosine Phosphatase domain) and a C-terminal tail. Under normal conditions, SHP2 is in a self-inhibitory conformation, and its N-SH2 binds to PTP, blocking the substrate channel at the catalytic site of PTP, thereby inhibiting the activity of PTP.
  • SH2 binds to bisphosphotyrosine peptides (such as IRS-1)
  • the autoinhibitory interaction of SH2-PTP is released, PTP catalytic sites are exposed, SHP2 is activated, and tyrosine dephosphorylation is catalyzed reaction.
  • SHP2 is widely expressed. As an oncogene, it mediates the activation of a variety of cancer-causing signal transduction channels, such as the RAS-ERK, PI3K-AKT and JAK-STAT pathways, and promotes the survival and proliferation of cancer cells. SHP2 can bind to and dephosphorylate RAS, increase RAS-RAF association, and activate downstream cell proliferation signals. SHP2 also mediates the compensatory activation pathway after MEK and other kinases are inhibited, thereby promoting tumor resistance (Ruess DA, et al., Nat. Med. 2018, 24, 954-960). Therefore, the activation of SHP2 is closely related to the occurrence of many diseases, such as leukemia, melanoma, breast cancer, lung cancer, colon cancer, neuroblastoma, hepatocellular carcinoma and so on.
  • diseases such as leukemia, melanoma, breast cancer, lung cancer, colon cancer, neuroblastoma, hepatocellular carcinoma and so on.
  • SHP2 also plays an important role in the immune checkpoint channel of PD-1 and BTLA (B-and T-Lymphocyte Attenuator), not only inhibiting T-cell activation but also promoting T-cell disability (Li J, et al., Cancer Res. 2015, 75, 508-518).
  • SHP2 As an anti-tumor target, SHP2 has attracted a lot of attention.
  • the SHP2 catalytic site inhibitor Due to the high protein sequence homology of the PTP catalytic site and the high hydrophilicity of the PTP catalytic pocket, the SHP2 catalytic site inhibitor has poor selectivity, poor cell permeability, and low bioavailability.
  • the discovery of Novartis allosteric inhibitor SHP099 (Chen Y, et al., Nature 2016, 535, 148-52) provides a new channel for the development of highly specific oral SHP2 inhibitors.
  • many companies have published some patent applications for SHP2 inhibitors, including WO2015107495, WO2016203405, WO2018057884, WO2018013597, WO2017211303, etc.
  • the present invention designs a compound having a structure represented by the general formula (I), and finds that the compound having such a structure exhibits an excellent SHP2 activity inhibitory effect.
  • the present invention provides a compound represented by general formula (I) or its prodrugs, stable isotope derivatives, pharmaceutically acceptable salts, isomers and mixtures thereof as SHP2 inhibitors:
  • Ring A is a benzene ring or a 6-membered heteroaromatic ring
  • ring B is a 5-membered heteroaromatic ring fused with ring A.
  • the benzene ring and heteroaromatic ring are optionally selected from D, halogen, and cyano.
  • R 1 is H, D, cyano, C 1-2 alkyl, cyclopropyl, -OR a , -NR a R b or -C(O)NR a R b , the alkyl and cyclopropyl
  • One or more hydrogens are optionally substituted by one or more D or fluorine;
  • R 2 is H, C 1-2 alkyl or cyclopropyl, one or more hydrogens of said alkyl and cyclopropyl are optionally substituted by one or more selected from D, fluorine or hydroxyl;
  • R 4a and R 4b are each independently selected from H, D, halo, cyano, -OR a, -NR a R b , -C (O) R a, -C (O) NR a R b, C 1- 6 alkyl group, C 3-6 cycloalkyl group, 4-7 membered heterocyclic group or 5-6 membered heteroaryl group, but R 4a and R 4b cannot be cyano, -OR a or -NR a R b at the same time,
  • the alkyl group, cycloalkyl group, heterocyclic group and heteroaryl group are optionally selected by one or more selected from D, halogen, cyano, oxo, C 1-6 alkyl, C 3-6 cycloalkyl, 4-7 membered heterocyclic group, 5-6 membered heteroaryl group, -OR a , -NR a R b , -C(O)R a or -C(
  • R 5a , R 5b , R 6a and R 6b are each independently selected from H, D, fluorine or methyl;
  • R a and R b are each independently selected from H, C 1-6 alkyl, C 3-6 cycloalkyl or 4-7 membered heterocyclic group, said alkyl, cycloalkyl and heterocyclic group are optionally selected by One or more substituents selected from D, fluorine, cyano, oxo, hydroxyl, -OCH 3 or -NH 2 are substituted.
  • One embodiment of the present invention relates to the compound represented by the above general formula (I) or its pharmaceutically acceptable salts, prodrugs, stable isotope derivatives, isomers and mixtures thereof, which are described in the general formula (II)
  • Ring A is a benzene ring or a 6-membered heteroaromatic ring
  • ring B is a 5-membered heteroaromatic ring fused with ring A.
  • the benzene ring and heteroaromatic ring are optionally selected from D, halogen, and cyano.
  • R 1 is H, D, cyano, C 1-2 alkyl, -OR a or -NR a R b , and one or more hydrogens of the alkyl group are optionally substituted by one or more D or fluorine;
  • R 2 is H or C 1-2 alkyl, one or more hydrogens of the alkyl are optionally substituted by one or more D or fluorine;
  • R 4a and R 4b are each independently selected from H, D, halogen, cyano, -OR a , -NR a R b , -C(O)NR a R b , C 1-6 alkyl, C 3-6 Cycloalkyl, 4-7 membered heterocyclic group or 5-6 membered heteroaryl, but R 4a and R 4b cannot be cyano, -OR a or -NR a R b at the same time, the alkyl, cycloalkyl , Heterocyclyl and heteroaryl are optionally selected by one or more selected from D, halogen, cyano, oxo, C 1-6 alkyl, C 3-6 cycloalkyl, 4-7 membered heterocyclyl, 5-6 membered heteroaryl, -OR a , -NR a R b , -C(O)R a or -C(O)NR a R b substituents; R 4a and
  • R a and R b are each independently selected from H, C 1-6 alkyl, C 3-6 cycloalkyl or 4-7 membered heterocyclic group, said alkyl, cycloalkyl and heterocyclic group are optionally selected by One or more substituents selected from D, fluorine, cyano, oxo, hydroxyl, -OCH 3 or -NH 2 are substituted.
  • Another embodiment of the present invention relates to the compound represented by the above general formula (I) or its pharmaceutically acceptable salts, prodrugs, stable isotope derivatives, isomers and mixtures thereof, which are represented by the general formula (III)
  • Ring A is a benzene ring or a 6-membered heteroaromatic ring
  • ring B is a 5-membered heteroaromatic ring fused with ring A.
  • the benzene ring and heteroaromatic ring are optionally selected from D, halogen, and cyano.
  • X is -O- or -CR 8a R 8b -;
  • R 1 is H, D, cyano, C 1-2 alkyl or -NR a R b , and one or more hydrogens of the alkyl group are optionally substituted by one or more D or fluorine;
  • R 2 is H or C 1-2 alkyl, one or more hydrogens of the alkyl are optionally substituted by one or more D or fluorine;
  • R 7a , R 7b , R 8a , R 8b , R 9a and R 9b are each independently selected from H, D, halogen, cyano, C 1-2 alkyl or -OR c , one or more of the alkyl groups Each hydrogen is optionally substituted by one or more selected from D, fluorine or hydroxyl, but R 7a and R 7a , R 8a and R 8b , R 9a and R 9b optionally cannot be -OR c at the same time;
  • R a and R b are each independently selected from H, C 1-6 alkyl, C 3-6 cycloalkyl or 4-7 membered heterocyclic group, said alkyl, cycloalkyl and heterocyclic group are optionally selected by One or more substituents selected from D, fluorine, cyano, oxo, hydroxyl, -OCH 3 or -NH 2 ;
  • R c is H or C 1-2 alkyl, and one or more hydrogens of the alkyl are optionally substituted by one or more D or fluorine.
  • Another embodiment of the present invention relates to the compound represented by the above general formula (I) or its pharmaceutically acceptable salts, prodrugs, stable isotope derivatives, isomers and mixtures thereof, which are represented by the general formula (IV)
  • Ring A is a benzene ring or a 6-membered heteroaromatic ring
  • ring B is a 5-membered heteroaromatic ring fused with ring A.
  • the benzene ring and heteroaromatic ring are optionally selected from D, halogen, and cyano. , Oxo, C 1-2 alkyl, -OR a or -NR a R b substituents, wherein one or more hydrogens of the alkyl group are optionally substituted by one or more D or fluorine;
  • X is -O- or -CH 2 -;
  • R 1 is independently selected from H, D, C 1-2 alkyl or -NR a R b , one or more hydrogens of the alkyl group are optionally substituted by one or more D or fluorine;
  • R 2 is H or a C 1-2 alkyl group, one or more hydrogens of the alkyl group are optionally substituted by one or more D or fluorine;
  • R 7 is H, D or C 1-2 alkyl, one or more hydrogens of said alkyl are optionally substituted by one or more D or fluorine;
  • R a and R b are each independently H or C 1-2 alkyl, and one or more hydrogens of the alkyl group are optionally substituted with one or more D or fluorine.
  • the present invention further relates to the compound represented by the above general formula (I), wherein the compound is selected from:
  • the compound of the present invention can effectively inhibit the activity of SHP2, and preferably has an IC 50 of less than 50 nM.
  • the compound of the present invention has a significant inhibitory effect on the proliferation of NCI-H358 cells, and preferably its IC 50 is less than 1000 nM.
  • Another aspect of the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a compound represented by general formula (I) or a pharmaceutically acceptable salt, prodrug, stable isotope derivative, isomer and mixture thereof Forms and pharmaceutically acceptable carriers and excipients.
  • the present invention further relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a compound represented by the general formula (I) or a pharmaceutically acceptable salt, prodrug, stable isotope derivative, isomer and mixture thereof, and at least An additional drug, wherein the at least one additional drug includes, but is not limited to, chemotherapeutic drugs, targeted drugs, DNA synthesis inhibitors, antibody drugs, antibody drug conjugates, anti-tumor drugs, immunosuppressants, and the like.
  • Another aspect of the present invention also relates to the compound represented by the general formula (I) or its pharmaceutically acceptable salts, prodrugs, stable isotope derivatives, isomers and mixtures thereof, or the pharmaceutical composition in the preparation of medicines
  • the drug is used to treat or prevent SHP2-mediated related diseases, wherein the diseases include but are not limited to leukemia, Noonan syndrome, leopard spot syndrome, neuroblastoma, lung cancer, breast cancer, colon cancer , Esophageal cancer, stomach cancer, head and neck cancer, etc.
  • the drug can be in any pharmaceutical dosage form, including but not limited to tablets, capsules, solutions, freeze-dried preparations, injections and the like.
  • the pharmaceutical preparation of the present invention can be administered in the form of a dosage unit containing a predetermined amount of active ingredient per dosage unit.
  • a dosage unit may contain, for example, 0.5 mg to 1 g, preferably 1 mg to 700 mg, and particularly preferably 5 mg to 500 mg of the compound of the present invention according to the condition to be treated, the method of administration, and the age, weight and condition of the patient.
  • methods known in the pharmaceutical field can be used to prepare this type of pharmaceutical preparations, such as mixing the active ingredient with one or more excipients and/or adjuvants.
  • compositions of the present invention may be suitable for administration by any desired suitable method, such as oral (including oral or sublingual), rectal, nasal, topical (including oral, sublingual or transdermal), vaginal or parenteral (Including subcutaneous, intramuscular, intravenous or intradermal) method of administration.
  • oral including oral or sublingual
  • rectal including oral or sublingual
  • nasal including oral, sublingual or transdermal
  • vaginal or parenteral including subcutaneous, intramuscular, intravenous or intradermal
  • C xy means the range of carbon atoms, where x and y are both integers, for example, C 3-8 cycloalkyl means a cycloalkyl group with 3-8 carbon atoms, that is, with 3, 4, 5, 6, Cycloalkyl groups of 7 or 8 carbon atoms. It should also be understood that “C 3-8 "also includes any sub-range therein, such as C 3-7 , C 3 6 , C 4-7 , C 4-6 , C 5-6 and the like.
  • Alkyl refers to a saturated linear or branched hydrocarbon group containing 1 to 20 carbon atoms, for example, 1 to 8 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms.
  • alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl Group, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl -2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-Dimethylbutyl, 2-ethylbutyl, etc.
  • Cycloalkyl or carbocyclic refers to a saturated cyclic hydrocarbyl substituent containing 3 to 14 carbon ring atoms.
  • the cycloalkyl group may be a single carbon ring, and usually contains 3 to 8, 3 to 7, or 3 to 6 carbon ring atoms.
  • Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and the like.
  • Cycloalkyl groups can also be fused, bridged or spiro-bonded bi- or tricyclic rings, such as decahydronaphthyl, bicyclo[2.2.2]octane, spiro[3.3]heptane and the like.
  • Heterocyclic group or heterocyclic ring refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic group, which includes 3 to 20 ring atoms, for example, 3 to 14, 3 to 12, 3 to 10 1, 3 to 8, 3 to 6, or 5 to 6 ring atoms, of which one or more ring atoms are selected from nitrogen, oxygen or S(O) m (where m 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.
  • It preferably includes 3 to 12 ring atoms, more preferably 3 to 10 ring atoms, more preferably 4 to 7 ring atoms, more preferably 4 to 6 ring atoms, most preferably 5 or 6 ring atoms, of which 1 to 4 It is a heteroatom, more preferably 1 to 3 are heteroatoms, and most preferably 1 to 2 are heteroatoms.
  • Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, oxbutanyl, piperidinyl, piperazinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, thiomorphyl Linyl, homopiperazinyl, azetidine, etc.
  • Polycyclic heterocyclic groups include fused, bridged or spiro heterocyclic groups, such as octahydrocyclopenta[c]pyrrole, octahydropyrrolo[1,2-a]pyrazine, 3,8- Diazabicyclo[3.2.1]octane, 5-azaspiro[2.4]heptane, 2-oxa-7-azaspiro[3.5]nonane, etc.
  • Aryl or aromatic ring refers to an aromatic monocyclic or condensed polycyclic group containing 6 to 14 carbon atoms, preferably 6 to 10 members, such as phenyl and naphthyl, and most preferably phenyl.
  • the aryl ring may be fused to a heteroaryl, heterocyclic or cycloalkyl ring, wherein the ring connected to the parent structure is an aryl ring.
  • Non-limiting examples include:
  • Heteroaryl or heteroaromatic ring refers to a heteroaromatic system containing 5 to 14 ring atoms, where 1 to 4 ring atoms are selected from heteroatoms including oxygen, sulfur, and nitrogen. Heteroaryl is preferably 5 to 10 members, more preferably heteroaryl is 5 or 6 members, such as furyl, thienyl, pyridyl, pyrrolyl, pyrimidinyl, pyrazinyl, pyrazolyl, imidazolyl, tetrakis Azolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolinyl, isoquinolinyl, indolyl, isoindolyl and the like.
  • the heteroaryl ring may be fused to an aryl, heterocyclic or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring. Non-limiting examples
  • Halogen refers to fluorine, chlorine, bromine or iodine.
  • Cyano refers to -CN.
  • heterocyclic group optionally substituted by an alkyl group means that an alkyl group may but need not be present, and the expression includes the case where the heterocyclic group is substituted by an alkyl group and the case where the heterocyclic group is not substituted by an alkyl group.
  • substitution means that one or more hydrogen atoms in the group, preferably 5, more preferably 1 to 3 hydrogen atoms are independently substituted with a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without too much effort. For example, an amino group or a hydroxyl group having free hydrogen may be unstable when combined with a carbon atom having an unsaturated (e.g., olefinic) bond.
  • the substituents include, but are not limited to, halogen, cyano, nitro, oxo, -SF 5 , C 1-4 alkyl, C 3-7 cycloalkyl, 4-7 membered heterocyclic group, phenyl, 5 -6-membered heteroaryl and so on.
  • “Isomers” refer to compounds that have the same molecular formula but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are called “stereoisomers”. Stereoisomers include optical isomers, geometric isomers and conformational isomers.
  • the compounds of the present invention may exist in the form of optical isomers.
  • Optical isomers include enantiomers and diastereomers.
  • Enantiomers means that two stereoisomers are mirror images of each other but not superimposable with each other.
  • a racemic mixture or racemate refers to a mixture of equal numbers of left and right enantiomers of a chiral molecule.
  • Diastereoisomers means that two stereoisomers are not mirror images of each other and cannot be superimposed.
  • optical isomer When the optical isomer is a single isomer and its absolute configuration is determined, it is the absolute configuration of "R” or “S” according to the configuration of the substituent on the chiral carbon atom; when the absolute configuration of the optical isomer The configuration is not determined, and it is (+) or (-) according to the measured optical rotation value.
  • Methods of preparing and separating optical isomers are known in the art.
  • the compounds of the present invention may also exist in geometric isomers.
  • the present invention considers various geometric isomers and mixtures thereof resulting from the distribution of substituents around carbon-carbon double bonds, carbon-nitrogen double bonds, cycloalkyl or heterocyclic groups. Substituents around a carbon-carbon double bond or carbon-nitrogen bond are designated as Z or E configuration, and substituents around a cycloalkyl or heterocyclic ring are designated as cis or trans configuration.
  • the compounds of the present invention may also exhibit tautomerism, such as keto-enol tautomerism.
  • the present invention includes any tautomeric or stereoisomeric forms and mixtures thereof, and is not limited to any one of the tautomeric or stereoisomeric forms used in the naming or chemical structural formula of the compound.
  • isotopes refer to all isotopes of atoms occurring in the compounds of the present invention. Isotopes include those atoms that have the same atomic number but different mass numbers. Examples of isotopes suitable for incorporation into the compounds of the present invention are hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as but not limited to 2 H(D), 3 H, 13 C, 14 C, 15 N, respectively , 17 O, 18 O, 31 P, 32 P, 35 S, 18 F and 36 Cl.
  • the isotope-labeled compounds of the present invention can generally be prepared by conventional techniques known to those skilled in the art or by methods similar to those described in the appended examples using appropriate isotope-labeled reagents instead of non-isotopically-labeled reagents. Such compounds have various potential uses, for example as standards and reagents in the determination of biological activity. In the case of stable isotopes, such compounds have the potential to advantageously alter biological, pharmacological or pharmacokinetic properties.
  • Deuterium (D) is a preferred isotope of the present invention. For example, hydrogen in methyl, methylene or methine can be replaced by deuterium.
  • the compounds of the present invention can be administered in the form of prodrugs.
  • Prodrug refers to a derivative of the biologically active compound of the invention that is converted into a derivative of the biologically active compound of the present invention under physiological conditions in the living body, for example, by oxidation, reduction, hydrolysis, etc. (each of them utilizes enzymes or is performed without the participation of enzymes).
  • prodrugs are compounds in which the amino group in the compound of the present invention is acylated, alkylated or phosphorylated, such as eicosanoylamino, alanylamino, pivaloyloxymethylamino, or
  • the hydroxyl group is acylated, alkylated, phosphorylated or converted into borate, such as acetoxy, palmitoyloxy, pivaloyloxy, succinyloxy, fumaryloxy, alanyloxy Or wherein the carboxyl group is esterified or amidated, or wherein the sulfhydryl group forms a disulfide bridge with a carrier molecule, such as a peptide, that selectively delivers the drug to the target and/or to the cytosol of the cell.
  • a carrier molecule such as a peptide
  • “Pharmaceutically acceptable salt” or “pharmaceutically acceptable salt” refers to salts made from pharmaceutically acceptable bases or acids, including inorganic bases or acids and organic bases or acids.
  • the present invention also includes their corresponding pharmaceutically acceptable salts. Therefore, the compounds of the invention containing acidic groups can exist in the form of salts and can be used in accordance with the invention, for example as alkali metal salts, alkaline earth metal salts or as ammonium salts. More specific examples of such salts include sodium, potassium, calcium, magnesium, or salts with ammonia or organic amines, such as ethylamine, ethanolamine, triethanolamine, or amino acids.
  • the compounds of the present invention containing basic groups can exist in the form of salts and can be used in the form of their addition salts with inorganic or organic acids according to the present invention.
  • suitable acids include hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, propylene Acid, pivalic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, Adipic acid and other acids known to those skilled in the art.
  • the present invention also includes internal salts or betaines in addition to the salt forms mentioned.
  • Each salt can be obtained by conventional methods known to those skilled in the art, for example, by contacting these with organic or inorganic acids or bases in a solvent or dispersant or by anion exchange or cation exchange with other salts.
  • “Pharmaceutical composition” refers to containing one or more of the compounds described herein or their pharmaceutically acceptable salts, prodrugs, stable isotope derivatives, isomers and mixtures thereof, and other components such as pharmaceutically acceptable carriers And excipients.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredient and thus the biological activity.
  • terapéuticaally effective amount refers to the amount of the compound of the present invention that can effectively inhibit the function of SHP2 and/or treat or prevent the disease.
  • patient refers to mammals, especially humans.
  • the present invention also provides a method for preparing the compound.
  • the preparation of the compound of the general formula (I) of the present invention can be accomplished by the following exemplary methods and examples, but these methods and examples should not be considered as limiting the scope of the present invention in any way.
  • the compounds of the present invention can also be synthesized by synthetic techniques known to those skilled in the art, or a combination of methods known in the art and the methods of the present invention can be used.
  • the product obtained in each step of the reaction is obtained by separation techniques known in the art, including but not limited to extraction, filtration, distillation, crystallization, chromatographic separation and the like.
  • the starting materials and chemical reagents required for synthesis can be conventionally synthesized or purchased according to literature (available on SciFinder).
  • the heterocyclic compound of the general formula (I) of the present invention can be synthesized according to the route described in Method A: 1) A1 is substituted with NH in piperidine compound A2 under base catalysis or condensation reaction with a condensing agent to obtain an intermediate A3; 2) A3 then reacts with the sulfhydryl group on the 5,6-fused heteroaromatic ring through Buchwald coupling reaction to produce the product A4.
  • the type of heterocyclic compounds described by the general formula (I) of the present invention can be synthesized according to the route described in Method B: 1) A1 and A2a undergo substitution reaction under base catalysis or condensation reaction with a condensing agent to obtain intermediate A3a; 2) The ketone group in A3a and (R)-2-methylpropane-2-sulfinamide undergo reductive amination to form A5a; 3) A5a and the sulfhydryl group on the 5,6-fused heteroaromatic ring are formed by Buchwald coupling reaction A6a; 4) A6a is deprotected in an acidic environment to obtain product A4a.
  • the starting materials of the present invention can be synthesized by or according to methods known in the art, or can be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc., Beijing Coupling Chemicals And other chemical companies.
  • the structure of the compound is determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS).
  • NMR nuclear magnetic resonance
  • MS mass spectrometry
  • DMSO-d 6 deuterated dimethyl sulfoxide
  • CDC1 3 deuterated chloroform
  • CD 3 OD deuterated methanol
  • the internal standard is Tetramethylsilane (TMS)
  • TMS Tetramethylsilane
  • ESI Agilent SQD mass spectrometer
  • HPLC uses Agilent 1260DAD high pressure liquid chromatograph (Poroshell120EC-C18, 50 ⁇ 3.0mm, 2.7 ⁇ m column) or Waters Arc high pressure liquid chromatograph (Sunfire C18, 150 ⁇ 4.6mm, 5 ⁇ m column).
  • reaction temperature is room temperature (20-30°C).
  • the reactions are all carried out under an argon atmosphere or a nitrogen atmosphere.
  • the argon atmosphere or nitrogen atmosphere means that the reaction flask is connected to an argon or nitrogen balloon with a volume of about 1L.
  • the hydrogen atmosphere means that the reaction flask is evacuated and then filled with hydrogen (repeated 3 times), and then a hydrogen balloon with a volume of about 1L is connected.
  • the microwave reaction uses a CEM Discover-SP type microwave reactor.
  • the monitoring of the reaction process in the examples uses Agilent liquid mass spectrometry (1260/6120), or thin layer chromatography (TLC), and the thickness of the silica gel plate used is 0.15-0.2 mm (Qingdao Ocean GF254).
  • the compound is purified by column chromatography or thin layer chromatography, wherein the column chromatography uses Qingdao Ocean’s 200-300 mesh silica gel, and the thin layer chromatography uses Qingdao Ocean’s GF254 silica gel plate with a thickness of 0.4 to 0.5 mm.
  • Column chromatography or thin layer chromatography developing solvent system usually has a) dichloromethane and methanol system, b) petroleum ether and ethyl acetate system, or as shown in the examples.
  • the volume ratio of the solvent is adjusted according to the polarity of the compound, and a small amount of triethylamine or other acidic or alkaline reagents can also be added for further adjustment.
  • the purification of the compound also used Waters mass spectrometry guided automatic preparation system (mass detector: SQD2), according to the polarity of the compound, an appropriate acetonitrile/water (containing 0.1% trifluoroacetic acid or formic acid, or 0.05% ammonia) gradient was used in 20mL/ The flow rate of min was used to elute the reversed-phase high pressure column (XBridge-C18, 19 ⁇ 150mm, 5 ⁇ m).
  • 1N diluted hydrochloric acid can be added after purification using an automatic preparation system, and then the solvent can be removed under reduced pressure to obtain hydrochloride.
  • DMF refers to N,N-dimethylformamide
  • DIPEA N,N-diisopropylethylamine
  • DBU refers to 1,8-diazabicyclo[5.4.0]undec-7-ene.
  • NBS N-bromosuccinimide
  • NIS refers to N-iodosuccinimide.
  • XantPhos refers to 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene.
  • the abbreviation Pd 2 (dba) 3 refers to tris(dibenzylideneacetone)dipalladium.
  • Carter condensing agent refers to benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate.
  • 2,4-Dichloro-5-iodopyrimidine 1a (8.0g, 29.1mmol) was dissolved in tetrahydrofuran (100mL), sodium hydroxide solution (1N, 45mL) was added at 0°C, the reaction mixture was warmed to room temperature and stirred for 16 hours .
  • the pH was adjusted to acidity with citric acid, and extraction was performed with ethyl acetate (60 mL ⁇ 3).
  • the organic phases were combined and washed with saturated brine (30 mL ⁇ 3), dried over anhydrous sodium sulfate, and dissolved under reduced pressure to obtain the target product 1b (6.7 g, solid), yield: 91%.
  • a methanol solution (30%, 158 mg, 0.88 mmol) of sodium methoxide (30%, 158 mg, 0.88 mmol) was added to a methanol (20 mL) solution of 1 g (200 mg, 0.8 mmol), and the mixture was stirred at room temperature for 2 hours. Solubility was removed under reduced pressure to obtain the target product for 1 h (100 mg, solid), with a yield of 73%. The product was used directly in the next reaction without purification.
  • the organic phase was dried with anhydrous sodium sulfate, filtered to remove the desiccant, and the solvent was removed under reduced pressure.
  • Potassium tert-butoxide (204 mg, 1.82 mmol) was added to the solution of 2b (240 mg, 0.91 mmol) in tetrahydrofuran (5 mL), and the mixture was stirred at room temperature for 2 hours. It was acidified to pH 4 with hydrochloric acid (6N), and extracted with ethyl acetate (20 mL). The organic phase was dried with anhydrous sodium sulfate, filtered to remove the desiccant, and desolventized under reduced pressure to obtain the target product 2c (130 mg, solid), yield: 87%.
  • a methanol solution (30%, 443.5 mg, 2.46 mmol) of sodium methoxide (30%, 443.5 mg, 2.46 mmol) was added to a methanol (20 mL) solution of 3b (560 mg, 2.24 mmol), and the mixture was stirred at 30°C for 1 hour. Solubility was removed under reduced pressure to obtain the target product 3c (200 mg, solid), yield: 51%. The product was used directly in the next reaction without purification.
  • a methanol solution (30%, 182 mg, 1.01 mmol) of sodium methoxide (30%, 182 mg, 1.01 mmol) was added to a methanol (20 mL) solution of 4d (230 mg, 0.92 mmol), and the mixture was stirred at 30°C for 4 hours. The solvent was removed under reduced pressure to obtain the target product 4e (150 mg, solid), with a yield of 95%. The product was used directly in the next reaction without purification.
  • a methanol solution (30%, 317 mg, 1.76 mmol) of sodium methoxide (30%, 317 mg, 1.76 mmol) was added to a methanol (20 mL) solution of 5b (400 mg, 1.6 mmol), and the mixture was stirred at room temperature for 2 hours.
  • the reaction mixture was poured into water (100 mL), the pH was adjusted to weakly acidic with citric acid, and extracted with ethyl acetate (30 mL ⁇ 3).
  • the organic phases were combined and washed with saturated brine (20 mL ⁇ 3), dried over anhydrous sodium sulfate, filtered to remove the desiccant, and dissolved under reduced pressure.
  • Potassium tert-butoxide (672 mg, 6.0 mmol) was added to the solution of 6b (500 mg, 2.0 mmol) in tetrahydrofuran (10 mL), and the mixture was stirred at room temperature for 2 hours. It was acidified to pH 4 with hydrochloric acid (6N) and extracted with dichloromethane (20 mL). The organic phase was dried with anhydrous sodium sulfate, filtered to remove the desiccant, and dissolved under reduced pressure to obtain the target product 6c (300 mg, solid), yield: 100%.
  • Trimethyloxonium tetrafluoroborate (733 mg, 4.95 mmol) was added to a solution of 7b (760 mg, 3.3 mmol) in ethyl acetate (15 mL) under ice cooling, and the reaction mixture was stirred at room temperature for 4 hours. Add petroleum ether (30 mL) to dilute, stir for 10 minutes, and filter. The filtrate was added with ethyl acetate (20 mL), and washed with sodium bicarbonate solution (30 mL ⁇ 3) and saturated brine (20 mL ⁇ 3).
  • a methanol solution (30%, 93 mg, 0.52 mmol) of sodium methoxide (30%, 93 mg, 0.52 mmol) was added to a methanol (20 mL) solution of 7d (140 mg, 0.47 mmol), and the mixture was stirred at 30°C for 32 hours. The solvent was removed under reduced pressure to obtain the target product 7e (100 mg, solid), with a yield of 96%. The product was used directly in the next reaction without purification.
  • a methanol solution (30%, 45.5 mg, 0.25 mmol) of sodium methoxide (30%, 45.5 mg, 0.25 mmol) was added to a methanol (3 mL) solution of 8 g (60 mg, 0.21 mmol), and the mixture was stirred at 30°C for 4 hours. Solubility was removed under reduced pressure to obtain the target product for 8h (40 mg, solid), yield: 92%. The product was directly used in the next reaction without purification.
  • a methanol solution (30%, 130 mg, 0.72 mmol) of sodium methoxide (30%, 130 mg, 0.72 mmol) was added to a methanol (20 mL) solution of 9d (195 mg, 0.60 mmol), and the mixture was stirred at 30°C for 16 hours. The solvent was removed under reduced pressure to obtain the target product 9e (220 mg, solid, crude product). The product was used directly in the next reaction without purification.
  • Oxalyl chloride (27.6 g, 218.9 mmol) was added dropwise to dichloromethane (500 mL) at -30°C, the temperature was lowered to -78°C, and dimethyl sulfoxide (21.7 g, 278.6 mmol) was added dropwise. After the reaction mixture was stirred at -78°C for 30 minutes, a solution of 10b (35 g, 199 mmol) in dichloromethane (100 mL) was slowly added and stirred for 1 hour. Triethylamine (100.5g, 995mmol) was added dropwise, stirring was continued at -78°C for 30 minutes, and then stirred at room temperature overnight.
  • a tetrahydrofuran solution (1M, 193 mL, 193 mmol) of tetrabutylammonium fluoride (1M, 193 mL, 193 mmol) was added to a tetrahydrofuran (400 mL) solution of 10e (55 g, 128.5 mmol), and the mixture was stirred at room temperature for 2 hours.
  • Dilute with saturated sodium bicarbonate solution and water (100 mL, 1/2 v/v) add ethyl acetate (200 mL), separate the layers, and extract the aqueous phase with ethyl acetate (200 mL ⁇ 3).
  • the reaction mixture 10g (8.5g, 32.9mmol), Dess-Martin oxidant (27.4g, 64.56mmol) and dichloromethane (200mL) were stirred at 0°C for 2 hours.
  • Saturated sodium bicarbonate solution and saturated sodium thiosulfate solution (100mL, 1/1v/v) were added, stirred vigorously, and separated.
  • the aqueous phase was extracted with dichloromethane (200 mL ⁇ 3).
  • the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure.
  • reaction mixture 10j (60mg, 0.15mmol), (R)-2-methylpropane-2-sulfinamide (36.3mg, 0.3mmol), tetraethyl titanate (137mg, 0.6mmol) and tetrahydrofuran (5mL) Heat to 90°C and stir for 4 hours. Cool to 0°C, add methanol (2 mL) and lithium borohydride in tetrahydrofuran (2.0M, 0.15 mL, 0.3 mmol), and stir for 1 hour. Water (5 mL) and dichloromethane (20 mL) were added, and the layers were separated. The aqueous phase was extracted with dichloromethane (10 mL ⁇ 3).
  • the aqueous phase was extracted with dichloromethane (10 mL ⁇ 3), and the organic phases were combined and dried over anhydrous sodium sulfate, filtered, and dissolved under reduced pressure.
  • a tetrahydrofuran solution (1M, 303 mL, 303 mmol) of tetrabutylammonium fluoride (1M, 303 mL, 303 mmol) was added to a solution of 11e (90 g, 223 mmol) in tetrahydrofuran (1300 mL), and the mixture was stirred at room temperature overnight.
  • Dilute with saturated sodium bicarbonate solution and water (800 mL, 1/2 v/v) add ethyl acetate (800 mL), separate the layers, and extract the aqueous phase with ethyl acetate (500 mL ⁇ 3).
  • Dissolve 12a (86 mg, 0.166 mmol) in methanol (5 mL), add a dioxane solution of hydrogen chloride (4.0 M, 1 mL), and stir at room temperature for 1 hour. Concentrated under reduced pressure, and the residue was purified by reverse-phase high performance liquid chromatography to obtain the target product 12 (16.8 mg, solid), yield: 19%.
  • reaction mixture 1-benzyl-4-methylpiperidine-4-carbonitrile 16a (2g, 9.3mmol), di-tert-butyl dicarbonate (6.1g, 27.9mmol), nickel chloride hexahydrate (2.2g, 9.3 mmol) and methanol (50 mL) were stirred at room temperature for 15 minutes. Cool to 0°C, add sodium borohydride (1.77 g, 46.5 mmol), warm to room temperature, and stir for 8 hours. The solvent was removed under reduced pressure, and the residue was suspended in methylene chloride and filtered.
  • reaction mixture 17c (180mg, 0.35mmol), 2c (70mg, 0.42mmol), DIPEA (135mg, 1.05mmol), Pd 2 (dba) 3 (32mg, 0.035mmol), XantPhos (20mg, 0.035mmol) and dioxygen
  • Potassium tert-butoxide (224 mg, 2 mmol) was added to a solution of 1 g (250 mg, 1 mmol) in tetrahydrofuran (5 mL), and the mixture was stirred at room temperature for 2 hours. It was quenched with water and washed with ethyl acetate. The aqueous phase was acidified to pH 4 with hydrochloric acid (6N) and extracted with ethyl acetate. The organic phase was dried with anhydrous sodium sulfate, filtered to remove the desiccant, and dissolved under reduced pressure to obtain the target product 18a (100 mg, solid), yield: 67%.
  • reaction mixture 17c (180mg, 0.35mmol), 18a (63mg, 0.42mmol), DIPEA (135mg, 1.05mmol), Pd 2 (dba) 3 (32mg, 0.035mmol), XantPhos (20mg, 0.035mmol) and dioxy
  • Recombinant human SHP2 full-length protein was expressed and purified by Tsinghua University's protein purification and identification platform, and the double phosphorylated polypeptide (H2N-LN(pY)IDLDLV-(dPEG8)LST(pY)ASINFQK-amide) was synthesized by GenScript Biotechnology Co., Ltd.
  • the alternative substrate DiFMUP was purchased from Thermo Fisher Scientific (Cat. No. D6567).
  • the reaction buffer contains the following components: 60mM HEPES (pH 7.2), 75mM NaCl, 75mM KCl, 1mM EDTA, 0.05% TWEEN 20, and 5mM DTT.
  • the compound was dissolved and diluted to 100 ⁇ M in DMSO (Sigma, product number D5879), and then serially diluted with DMSO to the lowest concentration of 6.1 nM by 4 times, and then diluted 25 times with reaction buffer at each concentration point.
  • DMSO Sigma, product number D5879
  • the fluorescence signal value is positively correlated with the degree of dephosphorylation of the substrate, which reflects the catalytic activity of SHP2 phosphatase.
  • the experiment not added as 100% inhibition proteome group, but did not increase protein plus compound group group as 0% inhibition, compound using XLfit software drawing inhibition curves and calculate their IC 50 inhibition, the experimental results shown in Table.
  • the example compounds of the present invention have an inhibitory effect on the activity of SHP2, and preferably IC 50 is less than 50 nM.
  • the luminescent cell viability test was used to evaluate the effects of the compounds of the present invention on the proliferation of NCI-H358 human non-small cell lung cancer cells.
  • the compound was dissolved and diluted to 5mM in DMSO (Sigma, catalog number D5879), and then a 4-fold serial dilution was performed with DMSO to the lowest concentration of 0.31 ⁇ M, and RPMI 1640 medium (Thermo Fisher Scientific, catalog number) was used for each concentration point. 11995073) diluted 50 times. If the compound IC 50 value is low, the initial concentration of the compound can be reduced.
  • NCI-H358 ATCC, catalog number CRL-5807 cells are cultured in RPMI 1640 complete medium [RPMI 1640 medium contains 10% FBS (GBICO, catalog number 10099-141) and 100 units/mL penicillin streptomycin mixture (Thermo Fisher Scientific, catalog number 15140122)].
  • the compounds of the examples of the present invention have an inhibitory effect on cell proliferation, and preferably IC 50 is less than 1000 nM.
  • the effect of the compounds of the present invention on possible arrhythmias was evaluated by measuring the blocking effect on the hERG potassium ion channel.
  • Compound solution Dissolve the test compound in DMSO and make a 10 mM stock solution, then dilute it to 3 mM with DMSO, and then dilute it to a 3 ⁇ M solution with extracellular fluid for subsequent use.
  • HEK293 cell line (Creacell, product number A-0320) stably overexpressing hERG potassium channels contains 10% fetal bovine serum (Gibco, product number 1428478) and 0.8 mg/mL G418 (Amresco, product number E859-5G).
  • DMEM medium Gibco, catalog number 1009-141
  • PBS Gibco, catalog number 1009-141
  • TrypLE TM Express solution (Gibco, catalog number 12604021), and incubate at 37°C for 30 seconds.
  • Whole-cell patch clamp voltage stimulation scheme for recording the whole-cell hERG potassium current when the whole-cell seal is formed, the cell membrane voltage is clamped to -80mV.
  • the clamping voltage is depolarized from -80mV to -50mV and maintained for 0.5 seconds (as a leakage current detection), then stepped to 30mV and maintained for 2.5 seconds, and then quickly restored to -50mV and maintained for 4 seconds to excite the tail current of the hERG channel ( Peak tail current), the hERG potassium current is recorded every 10 seconds.
  • the experimental data is collected by EPC-10 amplifier (HEKA) and stored in PatchMaster (HEKA v2x73) software.
  • Sutter Instruments is used to draw a capillary glass tube (Sutter Instruments) into a recording electrode.
  • the coverslip with the cells is removed from the 24-well plate placed in the incubator, and then placed under an inverted microscope.
  • the whole-cell recording mode perform slow capacitance compensation and record the membrane capacitance and series resistance, during which no leakage compensation is given.

Abstract

本发明涉及适用于抑制或调控SHP2的嘧啶-4(3H)-酮类杂环化合物、其制备方法及其在医药学上的应用。具体而言，本发明涉及一种通式(I)所示的化合物及其可药用的盐、含有所述化合物或其可药用的盐的药物组合物、应用所述化合物或其可药用的盐治疗和/或预防由SHP2介导的相关病症、特别是癌症的方法以及所述化合物或其可药用的盐的制备方法。本发明还涉及所述化合物或其可药用的盐或含有所述化合物或其可药用的盐的药物组合物在制备用于治疗和/或预防SHP2介导的相关病症的药物中的用途。其中通式(I)的各取代基与说明书中的定义相同。

Description

嘧啶-4(3H)-酮类杂环化合物、其制备方法及其在医药学上的应用 技术领域
本发明涉及一种新的调控或抑制SHP2活性的嘧啶-4(3H)-酮类杂环化合物或其可药用的盐、含有所述化合物或其可药用的盐的药物组合物、所述化合物或其可药用的盐的制备方法以及所述化合物或其可药用的盐或含有所述化合物或其可药用的盐的药物组合物在制备用于治疗和/或预防由SHP2介导的相关性病症、特别是癌症的药物中的用途和其使用方法。
背景技术
SHP2(Src Homology 2 domain-containing Phosphatase 2)是由PTPN11(Protein Tyrosine Phosphatase Nonreceptor type 11)基因编码的非受体蛋白酪氨酸磷酸酶。SHP2包含两个SH2结构域(Src Homology domain)、一个PTP结构域(Protein Tyrosine Phosphatase domain)和一个C端尾部。在通常状态下，SHP2处于自抑制构象，其N-SH2与PTP结合，阻断PTP催化位点的底物通道，从而抑制了PTP的活性。当SH2与双磷酸酪氨酸肽(如IRS-1)结合时，解除了SH2-PTP的自抑制相互作用，暴露出PTP催化位点，使SHP2处于激活状态，催化酪氨酸的去磷酸化反应。
SHP2广泛表达，作为致癌基因，介导多种致癌细胞信号转导通道的激活，如RAS-ERK、PI3K-AKT和JAK-STAT通路，促进癌细胞的存活和增殖。SHP2可结合并去磷酸化RAS，增加RAS-RAF关联，激活下游细胞增殖信号。SHP2还介导MEK等激酶被抑制之后的代偿性激活途径，从而促进肿瘤的耐药(Ruess DA,et al.,Nat.Med.2018,24,954-960)。因此，SHP2的激活与多种疾病的发生密切相关，如白血病、黑色素瘤、乳腺癌、肺癌、结肠癌、神经母细胞瘤、肝细胞癌等。
SHP2还在PD-1和BTLA(B-and T-Lymphocyte Attenuator)的免疫检查点通道中起重要作用，不仅抑制T-细胞活化而且促进T-细胞的失能(Li J,et al.,Cancer Res.2015,75,508-518)。
因此，SHP2作为一个抗肿瘤的靶点，吸引了众多的关注。但由于PTP催化位点的高度蛋白序列同源性以及PTP催化口袋的高亲水性，导致SHP2催化位点抑制剂的选择性差、细胞通透性差以及生物利用度底。诺华变构抑制剂SHP099的发现(Chen Y,et al.,Nature 2016,535,148-52)，为开发高特异性口服SHP2抑制剂提供了新的渠道。目前有多个公司公开了一些SHP2抑制剂的专利申请，其中包括WO2015107495、WO2016203405、WO2018057884、WO2018013597、 WO2017211303等。本发明设计具有通式(I)所示结构的化合物，并发现具有此类结构的化合物表现出优异的SHP2活性抑制效果。
发明内容
本发明提供作为SHP2抑制剂的一种通式(I)所示的化合物或其前药、稳定同位素衍生物、可药用的盐、异构体及其混合物形式：
Figure PCTCN2021071375-appb-000001
其中：
环A为苯环或6元杂芳环，环B为与环A稠合的5元杂芳环，所述苯环和杂芳环任选被一个或多个选自D、卤素、氰基、氧代、C 1-6烷基、C 3-6环烷基、4-7元杂环基、5-6元杂芳基、-OR a、-NR aR b、-C(O)R a、-C(O)NR aR b、-S(O) 2R a、-S(O) 2NR aR b、-NR aS(O) 2R b或-P(O)(CH 3) 2的取代基所取代，其中所述烷基、环烷基、杂环基和杂芳基任选被一个或多个选自D、卤素、氰基、氧代、C 1-6烷基、C 3-6环烷基、4-7元杂环基、-OR a、-NR aR b、-C(O)R a或-C(O)NR aR b的取代基所取代；
R 1为H、D、氰基、C 1-2烷基、环丙基、-OR a、-NR aR b或-C(O)NR aR b，所述烷基和环丙基的一个或多个氢任选被一个或多个D或氟所取代；
R 2为H、C 1-2烷基或环丙基，所述烷基和环丙基的一个或多个氢任选被一个或多个选自D、氟或羟基所取代；
R 4a和R 4b各自独立地选自H、D、卤素、氰基、-OR a、-NR aR b、-C(O)R a、-C(O)NR aR b、C 1-6烷基、C 3-6环烷基、4-7元杂环基或5-6元杂芳基，但R 4a和R 4b不能同时为氰基、-OR a或-NR aR b，所述烷基、环烷基、杂环基和杂芳基任选被一个或多个选自D、卤素、氰基、氧代、C 1-6烷基、C 3-6环烷基、4-7元杂环基、5-6元杂芳基、-OR a、-NR aR b、-C(O)R a或-C(O)NR aR b的取代基所取代；R 4a和R 4b任选与其所联接的碳原子共同组成一个C 3-7碳环或4-8元杂环；
R 5a、R 5b、R 6a和R 6b各自独立地选自H、D、氟或甲基；
R a和R b各自独立地选自H、C 1-6烷基、C 3-6环烷基或4-7元杂环基，所述烷基、环烷基和杂环基任选被一个或多个选自D、氟、氰基、氧代、羟基、-OCH 3或-NH 2的取代基所取代。
本发明的一个实施方案涉及上述通式(I)所示的化合物或其可药用的盐、前药、稳定同位素衍生物、异构体及其混合物形式，其为通式(II)所述的化合物或其可药用的盐、前药、稳定同位素衍生物、异构体及其混合物形式：
Figure PCTCN2021071375-appb-000002
其中：
环A为苯环或6元杂芳环，环B为与环A稠合的5元杂芳环，所述苯环和杂芳环任选被一个或多个选自D、卤素、氰基、氧代、C 1-6烷基、C 3-6环烷基、4-7元杂环基、5-6元杂芳基、-OR a、-NR aR b、-C(O)R a、-C(O)NR aR b、-S(O) 2R a、-S(O) 2NR aR b或-NR aS(O) 2R b的取代基所取代，其中所述烷基、环烷基、杂环基和杂芳基任选被一个或多个选自D、卤素、氰基、氧代、C 1-6烷基、C 3-6环烷基、4-7元杂环基、-OR a、-NR aR b、-C(O)R a或-C(O)NR aR b的取代基所取代；
R 1为H、D、氰基、C 1-2烷基、-OR a或-NR aR b，所述烷基的一个或多个氢任选被一个或多个D或氟所取代；
R 2为H或C 1-2烷基，所述烷基的一个或多个氢任选被一个或多个D或氟所取代；
R 4a和R 4b各自独立地选自H、D、卤素、氰基、-OR a、-NR aR b、-C(O)NR aR b、C 1-6烷基、C 3-6环烷基、4-7元杂环基或5-6元杂芳基，但R 4a和R 4b不能同时为氰基、-OR a或-NR aR b，所述烷基、环烷基、杂环基和杂芳基任选被一个或多个选自D、卤素、氰基、氧代、C 1-6烷基、C 3-6环烷基、4-7元杂环基、5-6元杂芳基、-OR a、-NR aR b、-C(O)R a或-C(O)NR aR b的取代基所取代；R 4a和R 4b任选与其所联接的碳原子共同组成一个C 3-7碳环或4-7元杂环；
R a和R b各自独立地选自H、C 1-6烷基、C 3-6环烷基或4-7元杂环基，所述烷基、环烷基和杂环基任选被一个或多个选自D、氟、氰基、氧代、羟基、-OCH 3或-NH 2的取代基所取代。
本发明的另一个实施方案涉及上述通式(I)所示的化合物或其可药用的盐、前药、稳定同位素衍生物、异构体及其混合物形式，其为通式(III)所述的化合物或其可药用的盐、前药、稳定同位素衍生物、异构体及其混合物形式：
Figure PCTCN2021071375-appb-000003
其中：
环A为苯环或6元杂芳环，环B为与环A稠合的5元杂芳环，所述苯环和杂芳环任选被一个或多个选自D、卤素、氰基、氧代、C 1-6烷基、C 3-6环烷基、4-7元杂环基、5-6元杂芳基、-OR a、-NR aR b、-C(O)R a或-C(O)NR aR b的取代基所取代，其中所述烷基、环烷基、杂环基和杂芳基任选被一个或多个选自D、卤素、氰基、氧代、C 1-2烷基、-OR a或-NR aR b的取代基所取代；
X为-O-或-CR 8aR 8b-；
R 1为H、D、氰基、C 1-2烷基或-NR aR b，所述烷基的一个或多个氢任选被一个或多个D或氟所取代；
R 2为H或C 1-2烷基，所述烷基的一个或多个氢任选被一个或多个D或氟所取代；
R 7a、R 7b、R 8a、R 8b、R 9a和R 9b各自独立地选自H、D、卤素、氰基、C 1-2烷基或-OR c，所述烷基的一个或多个氢任选被一个或多个选自D、氟或羟基所取代，但R 7a和R 7a、R 8a和R 8b、R 9a和R 9b任选不能同时为-OR c；
R a和R b各自独立地选自H、C 1-6烷基、C 3-6环烷基或4-7元杂环基，所述烷基、环烷基和杂环基任选被一个或多个选自D、氟、氰基、氧代、羟基、-OCH 3或-NH 2的取代基所取代；
R c为H或C 1-2烷基，所述烷基的一个或多个氢任选被一个或多个D或氟所取代。
本发明的另一个实施方案涉及上述通式(I)所示的化合物或其可药用的盐、前药、稳定同位素衍生物、异构体及其混合物形式，其为通式(IV)所述的化合物或其可药用的盐、前药、稳定同位素衍生物、异构体及其混合物形式：
Figure PCTCN2021071375-appb-000004
其中：
环A为苯环或6元杂芳环，环B为与环A稠合的5元杂芳环，所述苯环和杂芳环任选被一个或多个选自D、卤素、氰基、氧代、C 1-2烷基、-OR a或-NR aR b的取代基所取代，其中所述烷基的一个或多个氢任选被一个或多个D或氟所取代；
X为-O-或-CH 2-；
R 1独立地选自H、D、C 1-2烷基或-NR aR b，所述烷基的一个或多个氢任选被一个或多个D或氟所取代；
R 2为H或C 1-2烷基，所述烷基的一个或多个氢任选被一个或多个D 或氟所取代；
R 7为H、D或C 1-2烷基，所述烷基的一个或多个氢任选被一个或多个D或氟所取代；
R a和R b各自独立地为H或C 1-2烷基，所述烷基的一个或多个氢任选被一个或多个D或氟所取代。
本发明进一步涉及上述通式(I)所示的化合物，其中所述化合物选自：
Figure PCTCN2021071375-appb-000005
Figure PCTCN2021071375-appb-000006
Figure PCTCN2021071375-appb-000007
Figure PCTCN2021071375-appb-000008
或其可药用的盐、前药、稳定同位素衍生物、异构体及其混合物形式。
本发明化合物能够有效抑制SHP2的活性，优选其IC 50小于50nM。本发明化合物对NCI-H358细胞增殖具有显著抑制效应，优选其IC 50小于1000nM。
本发明的另一方面涉及一种药物组合物，所述药物组合物包含通式(I)所示的化合物或其可药用的盐、前药、稳定同位素衍生物、异构体及其混合物形式和药学上可接受的载体和赋形剂。
本发明进一步涉及一种药物组合物，所述药物组合物包含通式(I)所示的化合物或其可药用的盐、前药、稳定同位素衍生物、异构体及其混合物形式和至少一种额外的药物，其中所述至少一种额外的药物包括但不限于化疗药物、靶向药物、DNA合成抑制剂、抗体药物、抗体药物偶联物、抗肿瘤药物、免疫抑制剂等。
本发明的另一方面还涉及通式(I)所示的化合物或其可药用的盐、前药、稳定同位素衍生物、异构体及其混合物形式或所述药物组合物在制备药物中的用途，所述药物用于治疗或者预防SHP2介导的相关疾病，其中所述疾病包括但不限于白血病、努南综合征、豹斑综合征、神经母细胞瘤、肺癌、乳腺癌、结肠癌、食道癌、胃癌、头颈癌等。
根据本发明，所述药物可以是任何药物剂型，包括但不限于片剂、胶囊剂、溶液剂、冻干制剂、注射剂等。
本发明的药物制剂可以以每剂量单位包含预定量的活性成分的剂量单位形式给药。这种单位可根据治疗的病症、给药方法和患者的年龄、体重和状况包含例如0.5毫克至1克，优选1毫克至700毫克，特别优选5毫克至500毫克的本发明的化合物。此外，可以使用制药领域中公知的方法制备这种类型的药物制剂，比如把活性成分与一种或多种辅料和/或佐剂混合。
本发明药物制剂可适于通过任何所需的合适方法给药，例如通过经口(包括口腔或舌下)、直肠、经鼻、局部(包括口腔、舌下或经皮)、***或肠道外(包括皮下、肌内、静脉内或皮内)方法给药。
具体实施方式
除非另有相反陈述，否则下列用在说明书和权利要求书中的术语具有下述含义。
“C x-y”表示碳原子数的范围，其中x和y均为整数，例如C 3-8环烷基表示具有3-8个碳原子的环烷基，即具有3、4、5、6、7或8个碳原子的环烷基。还应理解，“C 3-8”还包含其中的任意亚范围，例如C 3-7、C 3- 6、C 4-7、C 4-6、C 5-6等。
“烷基”指含有1至20个碳原子，例如1至8个碳原子、1至6个碳原子或1至4个碳原子的饱和的直链或支链的烃基基团。烷基的非限制性实例包括甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、仲丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、1-乙基-2-甲基丙基、1,1,2-三甲基丙基、1,1-二甲基丁基、1,2-二甲基丁基、2,2-二甲基丁基、1,3-二甲基丁基、2-乙基丁基等。
“环烷基或碳环”指含有3至14个碳环原子的饱和环状烃基取代基。环烷基可以是单碳环，通常含有3至8个、3至7个或3至6个碳环原子。单环环烷基的非限制性实例包括环丙基、环丁基、环戊基、环己基、环庚基等。环烷基还可以是稠合、桥接或螺合到一起的双或三环，如十氢萘基、二环[2.2.2]辛烷、螺[3.3]庚烷等。
“杂环基或杂环”指饱和或部分不饱和的单环或多环环状基团，其包括3至20个环原子，例如可以是3至14个、3至12个、3至10个、3至8个、3至6个或5至6个环原子，其中一个或多个环原子选自氮、氧或S(O) m(其中m是整数0至2)，但不包括-O-O-、-O-S-或-S-S-的环部分，其余环原子为碳。优选包括3至12个环原子，更优选3至10个环原子，更优选4至7个环原子，更优选4至6个环原子，最优选5或6个环原子，其中1～4个是杂原子，更优选1～3个是杂原子，最优选1～2个是杂原子。单环杂环基的非限制性实例包含吡咯烷基、噁丁环基、 哌啶基、哌嗪基、四氢呋喃基、四氢吡喃基、四氢噻喃基、吗啉基、硫代吗啉基、高哌嗪基、吖丁啶基等。多环杂环基包括稠合、桥接或螺合多环杂环基，如八氢环戊二烯并[c]吡咯、八氢吡咯并[1,2-a]吡嗪、3,8-二氮杂二环[3.2.1]辛烷、5-氮杂螺[2.4]庚烷、2-氧杂-7-氮杂螺[3.5]壬烷等。
“芳基或芳环”指含有6至14个碳原子的芳香族单环或稠合多环基团，优选为6至10元，例如苯基和萘基，最优选苯基。所述芳基环可以稠合于杂芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为芳基环，非限制性实例包括：
Figure PCTCN2021071375-appb-000009
Figure PCTCN2021071375-appb-000010
等。
“杂芳基或杂芳环”指包含5至14个环原子的杂芳族体系，其中1至4个环原子选自包括氧、硫和氮的杂原子。杂芳基优选为5至10元，更优选杂芳基是5元或6元，例如呋喃基、噻吩基、吡啶基、吡咯基、嘧啶基、吡嗪基、吡唑基、咪唑基、四唑基、噁唑基、异噁唑基、噻唑基、异噻唑基、喹啉基、异喹啉基、吲哚基、异吲哚基等。所述杂芳基环可以稠合于芳基、杂环基或环烷基环上，其中与母体结构连接在一起的环为杂芳基环，非限制性实例包括：
Figure PCTCN2021071375-appb-000011
Figure PCTCN2021071375-appb-000012
等。
“卤素”指氟、氯、溴或碘。
“氰基”指-CN。
“氧代”指＝O。
“任选”意味着随后所描述的事件或环境可以但不必发生，该表述包括该事件或环境发生或不发生的情形。例如，“任选被烷基取代的杂环基团”意味着烷基可以但不必须存在，该表述包括杂环基团被烷基取代的情形和杂环基团不被烷基取代的情形。
“取代”指基团中的一个或多个氢原子，优选为5个，更优选为1～3个氢原子彼此独立地被相应数目的取代基取代。不言而喻，取代基仅处在它们的可能的化学位置，本领域技术人员能够在不付出过多努力的情况下确定(通过实验或理论)可能或不可能的取代。例如，具有游离氢的氨基或羟基与具有不饱和(如烯属)键的碳原子结合时可能是不稳定的。所述取代基包括但不限于卤素、氰基、硝基、氧代、-SF 5、C 1-4烷基、C 3-7环烷基、4-7元杂环基、苯基、5-6元杂芳基等。
“异构体”指具有相同分子式但其原子结合的性质或顺序或其原子的空间排列不同的化合物。原子空间排列不同的异构体称为“立体异构体”。立体异构体包括旋光异构体、几何异构体和构象异构体。
本发明的化合物可以以旋光异构体形式存在。旋光异构体包括对映异构体和非对映异构体。对映异构体是指两个立体异构体彼此镜像但相互不可重叠。外消旋混合物或外消旋体是指手性分子的左右手对映异构体数量相等的混合物。非对映异构体是指两个立体异构体不是彼此的镜像且不可重叠。当旋光异构体为单一异构体且其绝对构型确定，根据手性碳原子上取代基的构型，其为“R”或“S”的绝对构型；当旋光异构体的绝对构型未确定，依据所测的旋光值，其为(+)或(-)。制备和分离光学异构体的方法是本领域中已知的。
本发明的化合物也可以存在几何异构体。本发明考虑由碳-碳双键、碳-氮双键、环烷基或杂环基团周围的取代基的分布所产生的各种几何异构体和其混合物。碳-碳双键或碳-氮键周围的取代基指定为Z或E构型，环烷基或杂环周围的取代基指定为顺式或反式构型。
本发明的化合物还可能显示互变异构现象，例如酮-烯醇互变异构。
应该理解，本发明包括任何互变异构或立体异构形式和其混合物，并且不仅仅限于化合物的命名或化学结构式中所使用的任何一个互变异构或立体异构形式。
“同位素”是指在本发明化合物中出现的原子的所有同位素。同位素包括具有相同原子序数但不同质量数的那些原子。适合并入本发明化合物中的同位素的实例是氢、碳、氮、氧、磷、硫、氟和氯，分别例如但不限于 2H(D)、 3H、 13C、 14C、 15N、 17O、 18O、 31P、 32P、 35S、 18F和 36Cl。本发明的同位素标记化合物通常可通过本领域技术人员已知的传统技术或通过与所附实施例中描述的那些类似的方法使用适当的同位素标记的试剂代替非同位素标记的试剂来制备。这样的化合物具有各种潜在用途，例如作为测定生物活性中的标样和试剂。在稳定同位素的情况下，这样的化合物具有有利地改变生物、药理学或药代动力学性质的潜力。氘(D)为本发明的优选同位素，比如甲基、亚甲基或次甲基中的氢可被氘取代。
本发明的化合物可以以前药的形式给予。“前药”是指在活体内的生理条件下例如通过氧化、还原、水解等(它们各自利用酶或在没有酶参与下进行)转化成本发明的生物活性化合物的衍生物。前药的实例是下述化合物：其中本发明的化合物中的氨基被酰化、烷基化或磷酸化，例如二十烷酰基氨基、丙氨酰氨基、新戊酰氧基甲基氨基，或其中羟基被酰化、烷基化、磷酸化或转化成硼酸盐，例如乙酰氧基、棕榈酰氧基、新戊酰氧基、琥珀酰氧基、富马酰氧基、丙氨酰氧基，或其中羧基被酯化或酰胺化，或其中巯基与选择性地向靶和/或向细胞的胞质溶胶递送药物的载体分子，例如肽形成二硫桥键。这些化合物可以由本发明的化 合物根据公知方法制备。
“可药用的盐”或者“药学上可接受的盐”是指由可药用的碱或酸，包括无机碱或酸和有机碱或酸制成的盐。在本发明的化合物含有一个或多个酸性或碱性基团的情况下，本发明还包含它们相应的可药用盐。因此，含有酸性基团的本发明的化合物可以以盐形式存在并可根据本发明使用，例如作为碱金属盐、碱土金属盐或作为铵盐。这样的盐的更确切实例包括钠盐、钾盐、钙盐、镁盐或与氨或有机胺，例如乙胺、乙醇胺、三乙醇胺或氨基酸的盐。含有碱性基团的本发明的化合物可以以盐形式存在并可根据本发明以它们与无机或有机酸的加成盐的形式使用。合适的酸的实例包括盐酸、氢溴酸、磷酸、硫酸、硝酸、甲磺酸、对甲苯磺酸、萘二磺酸、草酸、乙酸、酒石酸、乳酸、水杨酸、苯甲酸、甲酸、丙酸、特戊酸、丙二酸、琥珀酸、庚二酸、富马酸、马来酸、苹果酸、氨基磺酸、苯基丙酸、葡糖酸、抗坏血酸、异烟酸、柠檬酸、己二酸和本领域技术人员已知的其它酸。如果本发明的化合物在分子中同时含有酸性和碱性基团，本发明除所提到的盐形式外还包括内盐或内铵盐。各盐可通过本领域技术人员已知的常规方法获得，例如通过在溶剂或分散剂中使这些与有机或无机酸或碱接触或通过与其它盐阴离子交换或阳离子交换。
“药物组合物”指含有一种或多种本文所述的化合物或其可药用的盐、前药、稳定同位素衍生物、异构体及其混合物形式以及其他组分例如可药用的载体和赋形剂的组合物。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
因此，在本申请中当提及“化合物”、“本发明化合物”或“本发明所述化合物”时，包括所有所述化合物形式，例如其可药用的盐、前药、稳定同位素衍生物、异构体及其混合物。
在本文中，术语“治疗有效量”是指包括可有效抑制SHP2的功能和/或治疗或防止所述疾病的本发明化合物的量。
在本文中，术语“患者”是指哺乳动物，尤其是人类。
合成方法
本发明还提供制备所述化合物的方法。本发明通式(I)所述化合物的制备可通过以下示例性方法和实施例完成，但这些方法和实施例不应以任何方式被认为是对本发明范围的限制。也可通过本领域技术人员所知的合成技术合成本发明所述的化合物，或者综合使用本领域已知方法和本发明所述方法。每步反应所得的产物用本领域已知的分离技术得到，包括但不限于萃取、过滤、蒸馏、结晶、色谱分离等。合成所需的起始物料和化学试剂可以根据文献(可从SciFinder上查询)常规合成或购买。
本发明通式(I)所述杂环化合物可按照方法A所述路线合成：1) A1与哌啶类化合物A2中的NH在碱催化下进行取代反应或用缩合剂进行缩合反应得到中间体A3；2)A3接着与5,6-稠合杂芳环上的巯基通过Buchwald耦合反应生成产物A4。
方法A：
Figure PCTCN2021071375-appb-000013
本发明通式(I)所述的一类杂环化合物可按照方法B所述路线合成：1)A1与A2a在碱催化下进行取代反应或用缩合剂进行缩合反应得到中间体A3a；2)A3a中的酮基和(R)-2-甲基丙烷-2-亚磺酰胺发生还原胺化生成A5a；3)A5a再与5,6-稠合杂芳环上的巯基通过Buchwald耦合反应生成A6a；4)A6a在酸性环境下脱保护得到产物A4a。
方法B：
Figure PCTCN2021071375-appb-000014
实施例
本发明的起始原料可以采用或按照本领域已知的方法来合成，或可购买自ABCR GmbH&Co.KG、Acros Organics、Aldrich Chemical Company、韶远化学科技(Accela ChemBio Inc.)、北京偶合化学品等化学品公司。
化合物的结构是通过核磁共振(NMR)或质谱(MS)来确定的。NMR的测定是用Bruker ASCEND-400核磁仪，测定溶剂为氘代二甲基亚砜(DMSO-d 6)、氘代氯仿(CDC1 3)、氘代甲醇(CD 3OD)等，内标为四甲基甲硅烷(TMS)，化学位移是以10 -6(ppm)作为单位给出。MS的测定使用Agilent SQD(ESI)质谱仪(安捷伦6120)。
HPLC使用安捷伦1260DAD高压液相色谱仪(Poroshell120EC-C18， 50×3.0mm，2.7μm色谱柱)或Waters Arc高压液相色谱仪(Sunfire C18，150×4.6mm，5μm色谱柱)。
实施例中如无特殊说明，反应温度为室温(20～30℃)。
实施例中如无特殊说明，反应均在氩气气氛或氮气气氛下进行。氩气气氛或氮气气氛是指反应瓶连接一个约1L容积的氩气或氮气气球。
氢气气氛是指反应瓶在抽真空再充入氢气(反复3次)后，连接一个约1L容积的氢气气球。
微波反应使用CEM Discover-SP型微波反应器。
实施例中的反应进程的监测使用安捷伦的液质联用色谱仪(1260/6120)，也可采用薄层层析(TLC)，所用硅胶板的厚度为0.15～0.2mm(青岛海洋GF254)。
化合物的纯化采用柱层析或薄层层析，其中柱层析使用青岛海洋的200～300目硅胶，薄层层析使用青岛海洋的厚度为0.4～0.5mm的GF254硅胶板。
柱层析或薄层层析展开溶剂体系通常有a)二氯甲烷和甲醇体系，b)石油醚和乙酸乙酯体系，或如实施例中所示。溶剂的体积比根据化合物的极性不同而进行调节，也可以加入少量的三乙胺、或其它酸性或碱性试剂进一步调节。
化合物的纯化还采用Waters的质谱导向自动制备***(质谱检测器：SQD2)，根据化合物的极性用适当的乙腈/水(含0.1％三氟乙酸或甲酸，或0.05％氨水)梯度于20mL/min的流速洗脱反相高压柱(XBridge-C18，19×150mm，5μm)。部分实施例可在使用自动制备***纯化后加入1N稀盐酸，然后减压除去溶剂，得到盐酸盐。
缩写DMF是指N,N-二甲基甲酰胺。
缩写DIPEA是指N,N-二异丙基乙胺。
缩写DBU是指1,8-二氮杂双环[5.4.0]十一碳-7-烯。
缩写NBS是指N-溴代琥珀酰亚胺。
缩写NIS是指N-碘代琥珀酰亚胺。
缩写XantPhos是指4,5-双(二苯基膦)-9,9-二甲基氧杂蒽。
缩写Pd 2(dba) 3是指三(二亚苄基丙酮)二钯。
卡特缩合剂是指苯并三氮唑-1-基氧基三(二甲基氨基)磷鎓六氟磷酸盐。
实施例1
(R)-5-((1H-吡咯并[2,3-b]吡啶-4-基)硫代)-2-(1-氨基-8-氮杂螺[4.5]癸烷-8-基)-3-甲基嘧啶-4(3H)-酮盐酸盐
Figure PCTCN2021071375-appb-000015
第一步
2-氯-5-碘嘧啶-4(3H)-酮(1b)
将2,4-二氯-5-碘嘧啶1a(8.0g，29.1mmol)溶于四氢呋喃(100mL)，0℃下加入氢氧化钠溶液(1N，45mL)，反应混合物升至室温，搅拌16小时。用柠檬酸调节pH值至酸性，用乙酸乙酯(60mL×3)萃取。有机相合并后用饱和食盐水(30mL×3)洗涤，经无水硫酸钠干燥，减压脱溶，得到目标产物1b(6.7g，固体)，产率：91％。
MS m/z(ESI):257[M+1]
第二步
2-氯-5-碘-3-甲基嘧啶-4(3H)-酮(1c)
将1b(5.6g，21.7mmol)溶于四氢呋喃(100mL)，加入碘甲烷(3.7g，26mmol)和DIPEA(8.4g，65.1mmol)，加热至60℃，搅拌16小时。倒入水(120mL)中，用乙酸乙酯(80mL×3)萃取。有机相合并后用饱和食盐水(30mL×3)洗涤，经无水硫酸钠干燥，过滤，减压浓缩。残余物用硅胶柱层析纯化(乙酸乙酯/石油醚＝3/7)得到目标产物1c(2.2g，固体)，产率：37％。
MS m/z(ESI):271[M+1]
第三步
8-(5-碘-1-甲基-6-羰基-1,6-二氢嘧啶-2-基)-8-氮杂螺[4.5]癸烷-1-酮(1d)
向1c(1.0g，3.65mmol)的乙腈(25mL)溶液中加入8-氮杂螺[4.5]癸烷-1-酮(盐酸盐，586mg，3.1mmol)和碳酸钾(1.51g，11mmol)，反应混合物加热至80℃，搅拌16小时。降至室温，将反应混合物倒入 水(50mL)中，用乙酸乙酯(30mL×3)萃取。有机相合并后用饱和食盐水(20mL×3)洗涤，经无水硫酸钠干燥，过滤，减压浓缩。残余物用硅胶柱层析纯化(乙酸乙酯/石油醚＝7/3)得到目标产物1d(1.2g，固体)，产率：85％。
MS m/z(ESI):388[M+1]
第四步
(R)-N-((R)-8-(5-碘-1-甲基-6-羰基-1,6-二氢嘧啶-2-基)-8-氮杂螺[4.5]癸烷-1-基)-2-甲基丙烷-2-亚磺酰胺(1e)
向1d(1.2g，3.1mmol)的四氢呋喃(30mL)溶液中加入(R)-2-甲基丙烷-2-亚磺酰胺(750mg，6.2mmol)和钛酸四乙酯(2.82g，12.4mmol)，加热至90℃，搅拌16小时。冷却至0℃，加入甲醇(10mL)和硼氢化锂的四氢呋喃溶液(2.0M，1.5mL，3mmol)，搅拌1小时。加入氯化铵溶液淬灭，过滤。滤液用乙酸乙酯(25mL×3)萃取，有机相合并后用饱和食盐水(20mL×3)洗涤，经无水硫酸钠干燥，过滤，减压浓缩。残余物用硅胶柱层析纯化(乙酸乙酯/石油醚＝2/1)得到目标产物1e(1.4g，固体)，产率：93％。
MS m/z(ESI):493[M+1]
第五步
3-((1H-吡咯并[2,3-b]吡啶-4-基)硫代)丙酸乙酯(1g)
向4-溴-1H-吡咯并[2,3-b]吡啶1f(300mg，1.52mmol)的二氧六环(20mL)溶液中加入3-巯基丙酸乙酯(408mg，3.04mmol)、DIPEA(588mg，4.56mmol)、Pd 2(dba) 3(140mg，0.152mmol)和XantPhos(132mg，0.228mmol)。反应混合物在氮气下加热至100℃，反应2小时。降至室温，减压脱溶，残余物用硅胶柱层析纯化(乙酸乙酯/石油醚＝7/3)得到目标产物1g(200mg，油状物)，产率：53％。
MS m/z(ESI):251[M+1]
第六步
1H-吡咯并[2,3-b]吡啶-4-硫醇酸钠(1h)
向1g(200mg,0.8mmol)的甲醇(20mL)溶液中加入甲醇钠的甲醇溶液(30％，158mg，0.88mmol)，室温下搅拌2小时。减压脱溶，得到目标产物1h(100mg，固体)，产率：73％。产物不经纯化直接用于下一步反应。
MS m/z(ESI):151[M+1]
第七步
(R)-N-((R)-8-(5-((1H-吡咯并[2,3-b]吡啶-4-基)硫代)-1-甲基-6-羰基-1,6-二氢嘧啶-2-基)-8-氮杂螺[4.5]癸烷-1-基)-2-甲基丙烷-2-亚磺酰胺(1i)
将1h(55mg，0.32mmol)溶于二氧六环(6mL)，加入1e(82mg，0.16mmol)、DIPEA(128mg，0.99mmol)、Pd 2(dba) 3(30mg，0.033mmol)和XantPhos(29mg，0.049mmol)。反应混合物在氮气保护下 加热至80℃，反应2小时。降至室温，减压脱溶，残余物用硅胶柱层析纯化(甲醇/二氯甲烷＝1/9)得到目标产物1i(30mg，固体)，产率：37％。
MS m/z(ESI):515[M+1]
第八步
(R)-5-((1H-吡咯并[2,3-b]吡啶-4-基)硫代)-2-(1-氨基-8-氮杂螺[4.5]癸烷-8-基)-3-甲基嘧啶-4(3H)-酮盐酸盐(1)
将1i(30mg，0.058mmol)溶于甲醇(5mL)，加入氯化氢的二氧六环溶液(4.0M，5mL)，室温搅拌2小时。减压浓缩，残余物用反相高效液相制备色谱纯化，得到目标产物1(7.1mg，固体)，产率：31％。
MS m/z(ESI):411[M+1]
1H NMR(400MHz,DMSO-d 6)δ12.68(s,1H),8.24–8.14(m,5H),7.68(s,1H),6.91–6.87(m,1H),6.74(s,1H),3.75–3.64(m,2H),3.43(s,3H),3.19–3.08(m,3H),2.08–2.05(m,1H),1.92–1.64(m,7H),1.54–1.51(m,1H),1.44–1.41(m,1H).
实施例2
(R)-2-(1-氨基-8-氮杂螺[4.5]癸烷-8-基)-3-甲基-5-((1-甲基-1H-吡咯并[2,3-b]吡啶-4-基)硫代)嘧啶-4(3H)-酮甲酸盐
Figure PCTCN2021071375-appb-000016
第一步
3-((1-甲基-1H-吡咯并[2,3-b]吡啶-4-基)硫代)丙酸乙酯(2b)
向4-溴-1-甲基-1H-吡咯并[2,3-b]吡啶2a(200mg，0.95mmol)、3-巯基丙酸乙酯(255mg，1.9mmol)、DIPEA(368mg，2.85mmol)、XantPhos(55mg，0.095mmol)和二氧六环(10mL)的混合物中加入Pd 2(dba) 3(87mg，0.095mmol)，氮气保护下加热至100℃，反应2小时。降至室温，加水(10mL)，用乙酸乙酯(20mL×3)萃取。有机相用无水硫酸钠干燥，过滤除去干燥剂，减压脱溶，残余物用硅胶柱层析纯化(乙酸乙酯/石油醚＝3/7)得到目标产物2b(240mg，固体)，产率：96％。
MS m/z(ESI):265[M+1]
第二步
1-甲基-1H-吡咯并[2,3-b]吡啶-4-硫醇(2c)
向2b(240mg，0.91mmol)的四氢呋喃(5mL)溶液中加入叔丁醇钾(204mg，1.82mmol)，室温下搅拌2小时。用盐酸(6N)酸化至pH为4，用乙酸乙酯(20mL)萃取。有机相用无水硫酸钠干燥，过滤除去干燥剂，减压脱溶，得到目标产物2c(130mg，固体)，产率：87％。
MS m/z(ESI):165[M+1]
第三步
(R)-2-甲基-N-((R)-8-(1-甲基-5-((1-甲基-1H-吡咯并[2,3-b]吡啶-4-基)硫代)-6-羰基-1,6-二氢嘧啶-2-基)-8-氮杂螺[4.5]癸烷-1-基)丙烷-2-亚磺酰胺(2d)
向2c(50mg，0.24mmol)、1e(110mg，0.22mmol)、DIPEA(85mg，0.66mmol)、XantPhos(13mg，0.022mmol)和二氧六环(5mL)的混合物中加入Pd 2(dba) 3(20mg，0.022mmol)，氮气保护下加热至100℃，反应2小时。降至室温，减压脱溶，残余物用硅胶柱层析纯化(甲醇/二氯甲烷＝1/9)得到目标产物2d(80mg，固体)，产率：68％。
MS m/z(ESI):529[M+1]
第四步
(R)-2-(1-氨基-8-氮杂螺[4.5]癸烷-8-基)-3-甲基-5-((1-甲基-1H-吡咯并[2,3-b]吡啶-4-基)硫代)嘧啶-4(3H)-酮甲酸盐(2)
将2d(80mg，0.14mmol)溶于甲醇(8mL)，加入氯化氢的二氧六环溶液(4.0M，2mL)，室温搅拌2小时。减压浓缩，残余物用反相高效液相制备色谱纯化，得到目标产物2(21.7mg，固体)，产率：34％。
MS m/z(ESI):425[M+1]
1H NMR(400MHz,DMSO-d 6)δ8.34(s,1H),8.14(s,1H),8.03(d,J＝5.1Hz,1H),7.51(d,J＝3.5Hz,1H),6.58(d,J＝5.1Hz,1H),6.44(d,J＝3.5Hz,1H),3.80(s,3H),3.66–3.58(m,2H),3.41(s,3H),3.07(t,J＝11.5Hz,2H),2.99–2.96(m,1H),2.0–1.92(m,1H),1.80–1.68(m,4H),1.60–1.53(m,3H),1.40(d,J＝12.9Hz,1H),1.33(d,J＝13.1Hz,1H).
实施例3
(R)-5-((1H-吲唑-4-基)硫代)-2-(1-氨基-8-氮杂螺[4.5]癸烷-8-基)-3-甲基嘧啶-4(3H)-酮盐酸盐
Figure PCTCN2021071375-appb-000017
第一步
3-((1H-吲唑-4-基)硫代)丙酸乙酯(3b)
向4-溴-1H-吲唑3a(500mg，2.53mmol)的二氧六环(20mL)溶液中加入3-巯基丙酸乙酯(680mg，5.06mmol)、DIPEA(979mg，7.59mmol)、Pd 2(dba) 3(231mg，0.253mmol)和XantPhos(219mg，0.397mmol)。反应混合物在氮气下加热至100℃，反应2小时。降至室温，减压脱溶，残余物用硅胶柱层析纯化(乙酸乙酯/石油醚＝2.6/1)得到目标产物3b(560mg，固体)，产率：89％。
MS m/z(ESI):251[M+1]
第二步
1H-吲唑-4-硫醇酸钠(3c)
向3b(560mg，2.24mmol)的甲醇(20mL)溶液中加入甲醇钠的甲醇溶液(30％，443.5mg，2.46mmol)，30℃下搅拌1小时。减压脱溶，得到目标产物3c(200mg，固体)，产率：51％。产物不经纯化直接用于下一步反应。
MS m/z(ESI):151[M+1]
第三步
(R)-N-((R)-8-(5-((1H-吲唑-4-基)硫代)-1-甲基-6-羰基-1,6-二氢嘧啶-2-基)-8-氮杂螺[4.5]癸烷-1-基)-2-甲基丙烷-2-亚磺酰胺(3d)
向3c(70mg，0.41mmol)的二氧六环(6mL)溶液中加入1e(50mg，0.10mmol)、DIPEA(37.2mg，0.30mmol)、Pd 2(dba) 3(9.1mg，0.01mmol)和XantPhos(8.7mg，0.015mmol)。反应混合物在氮气保护下加热至80℃，反应4小时。降至室温，减压脱溶，残余物用硅胶柱层析纯化(甲醇/二氯甲烷＝1/9)得到目标产物3d(50mg，固体)，产率：96％。
MS m/z(ESI):515[M+1]
第四步
(R)-5-((1H-吲唑-4-基)硫代)-2-(1-氨基-8-氮杂螺[4.5]癸烷-8-基)-3-甲基嘧啶-4(3H)-酮盐酸盐(3)
将3d(50mg，0.097mmol)溶于甲醇(5mL)，加入氯化氢的二氧六环溶液(4.0M，5mL)，室温搅拌2小时。减压浓缩，残余物用反相 高效液相制备色谱纯化，得到目标产物3(16mg，固体)，产率：41％。
MS m/z(ESI):411[M+1]
1H NMR(400MHz,DMSO-d 6)δ8.14(brs,3H),8.06(s,1H),7.97(s,1H),7.40(d,J＝8.4Hz,1H),7.24(dd,J＝8.3,7.2Hz,1H),6.83(d,J＝6.8Hz,1H),3.60(d,J＝13.5Hz,1H),3.52(d,J＝13.3Hz,1H),3.40(s,3H),3.17–3.12(m,1H),3.05–2.98(m,2H),2.07–2.04(m,1H),1.77–1.62(m,7H),1.49(d,J＝12.8Hz,1H),1.39(d,J＝13.1Hz,1H).
实施例4
(R)-2-(1-氨基-8-氮杂螺[4.5]癸烷-8-基)-3-甲基-5-(吡唑并[1,5-a]吡啶-4-基硫代)嘧啶-4(3H)-酮甲酸盐
Figure PCTCN2021071375-appb-000018
第一步
4-溴吡唑并[1,5-a]吡啶-3-羧酸乙酯(4b)
向3-溴吡啶4a(5.0g，32.0mmol)的乙腈(25mL)溶液中加入O-(2,4-二硝基苯基)羟胺(6.37g，32.0mmol)，加热至40℃，搅拌16小时。减压脱溶，残余物用***打浆，干燥后得到黄色固体(3.8g)。
将上述产物(2.0g，5.6mmol)溶于DMF(20mL)，加入碳酸钾(12.1g，88.1mmol)和丙炔酸乙酯(7.1g，52.8mmol)，反应混合物在室温下搅拌20小时。倒入水(50mL)中，用乙酸乙酯(30mL×3)萃取。有机相合并后用饱和食盐水(20mL×3)洗涤，经无水硫酸钠干燥，减压脱溶。残余物用硅胶柱层析纯化(乙酸乙酯/石油醚＝3/7)得到目标产物4b(350mg，固体)，产率：8％。
MS m/z(ESI):269[M+1]
第二步
4-溴吡唑并[1,5-a]吡啶(4c)
将4b(350mg，1.30mmol)溶于氢溴酸溶液(12mL)，加热至100℃，搅拌6小时。降至室温，用氢氧化钠溶液(2N)中和，用乙酸乙酯(20mL×3)萃取。有机相合并后用饱和食盐水(15mL×3)洗涤，经无水硫酸钠干燥，减压脱溶。残余物用硅胶柱层析纯化(乙酸乙酯/石油醚＝1/4)得到目标产物4c(200mg，固体)，产率：79％。
MS m/z(ESI):197[M+1]
第三步
3-(吡唑并[1,5-a]吡啶-4-基硫代)丙酸乙酯(4d)
向4c(200mg，1.02mmol)的二氧六环(20mL)溶液中加入3-巯基丙酸乙酯(680mg，5.06mmol)、DIPEA(394mg，3.06mmol)、Pd 2(dba) 3(93.3mg，0.102mmol)和XantPhos(88.4mg，0.153mmol)。反应混合物在氮气下加热至100℃，反应2小时。降至室温，减压脱溶，残余物用硅胶柱层析纯化(乙酸乙酯/石油醚＝3/7)得到目标产物4d(240mg，固体)，产率：94％。
MS m/z(ESI):251[M+1]
第四步
吡唑并[1,5-a]吡啶-4-硫醇酸钠(4e)
向4d(230mg，0.92mmol)的甲醇(20mL)溶液中加入甲醇钠的甲醇溶液(30％，182mg，1.01mmol)，30℃下搅拌4小时。减压脱溶，得到目标产物4e(150mg，固体)，产率：95％。产物不经纯化直接用于下一步反应。
MS m/z(ESI):151[M+1]
第五步
(R)-2-甲基-N-((R)-8-(1-甲基-6-羰基-5-(吡唑并[1,5-a]吡啶-4-基硫代)-1,6-二氢嘧啶-2-基)-8-氮杂螺[4.5]癸烷-1-基)丙烷-2-亚磺酰胺(4f)
向4e(85.3mg，0.495mmol)的二氧六环(6mL)溶液中加入1e(70mg，0.142mmol)、DIPEA(54.9mg，0.426mmol)、Pd 2(dba) 3(12.9mg，0.0142mmol)和XantPhos(12.3mg，0.0213mmol)。反应混合物在氮气保护下加热至80℃，反应4小时。降至室温，减压脱溶，残余物用硅胶柱层析纯化(甲醇/二氯甲烷＝1/13)得到目标产物4f(40mg，固体)，产率：55％。
MS m/z(ESI):515[M+1]
第六步
(R)-2-(1-氨基-8-氮杂螺[4.5]癸烷-8-基)-3-甲基-5-(吡唑并[1,5-a]吡啶-4-基硫代)嘧啶-4(3H)-酮甲酸盐(4)
将4f(40mg，0.078mmol)溶于甲醇(5mL)，加入氯化氢的二氧六环溶液(4.0M，5mL)，室温搅拌2小时。减压浓缩，残余物用反相高效液相制备色谱纯化，得到目标产物4(16mg，固体)，产率：51％。
MS m/z(ESI):411[M+1]
1H NMR(400MHz,DMSO-d 6)δ8.56(d,J＝6.9Hz,1H),8.39(s,1H),8.09(s,1H),8.03(d,J＝2.2Hz,1H),6.89(d,J＝7.0Hz,1H),6.80(t,J＝7.0Hz,1H),6.63(d,J＝1.7Hz,1H),3.64–3.53(m,2H),3.39(s,3H),3.05–2.97(m,3H),1.98–1.95(m,1H),1.80–1.67(m,4H),1.64–1.53(m,3H),1.40(d,J＝13.0Hz,1H),1.32(d,J＝13.3Hz,1H).
实施例5
(R)-2-(1-氨基-8-氮杂螺[4.5]癸烷-8-基)-3-甲基-5-(吡唑并[1,5-a]吡啶 -5-基硫代)嘧啶-4(3H)-酮盐酸盐
Figure PCTCN2021071375-appb-000019
第一步
3-(吡唑并[1,5-a]吡啶-5-基硫代)丙酸乙酯(5b)
向5-溴吡唑并[1,5-a]吡啶5a(320mg，1.62mmol)的二氧六环(20mL)溶液中加入3-巯基丙酸乙酯(326mg，2.43mmol)、DIPEA(627mg，4.86mmol)、Pd 2(dba) 3(148mg，0.162mmol)和XantPhos(140mg，0.243mmol)。反应混合物在氮气下加热至100℃，反应6小时。降至室温，减压脱溶，残余物用硅胶柱层析纯化(乙酸乙酯/石油醚＝3/1)得到目标产物5b(400mg，油状物)，产率：97％。
MS m/z(ESI):251[M+1]
第二步
吡唑并[1,5-a]吡啶-5-硫醇(5c)
向5b(400mg，1.6mmol)的甲醇(20mL)溶液中加入甲醇钠的甲醇溶液(30％，317mg，1.76mmol)，室温下搅拌2小时。将反应混合物倒入水(100mL)中，用柠檬酸调节pH至弱酸性，用乙酸乙酯(30mL×3)萃取。有机相合并后用饱和食盐水(20mL×3)洗涤，经无水硫酸钠干燥，过滤除去干燥剂，减压脱溶。残余物用硅胶柱层析纯化(乙酸乙酯/石油醚＝2/3)得到目标产物5c(100mg，油状物)，产率：42％。
MS m/z(ESI):151[M+1]
第三步
(R)-2-甲基-N-((R)-8-(1-甲基-6-羰基-5-(吡唑并[1,5-a]吡啶-5-基硫代)-1,6-二氢嘧啶-2-基)-8-氮杂螺[4.5]癸烷-1-基)丙烷-2-亚磺酰胺(5d)
将5c(30mg，0.20mmol)溶于二氧六环(6mL)，加入1e(50mg，0.10mmol)、DIPEA(38.7mg，0.30mmol)、Pd 2(dba) 3(9.0mg，0.010mmol)和XantPhos(8.5mg，0.015mmol)。反应混合物在氮气保护下加热至80℃，反应2小时。降至室温，减压脱溶，残余物用硅胶柱层析纯化(甲醇/二氯甲烷＝1/9)得到目标产物5d(60mg，固体，粗品)。
MS m/z(ESI):515[M+1]
第四步
(R)-2-(1-氨基-8-氮杂螺[4.5]癸烷-8-基)-3-甲基-5-(吡唑并[1,5-a]吡啶-5-基硫代)嘧啶-4(3H)-酮盐酸盐(5)
将5d(60mg，粗品)溶于甲醇(5mL)，加入氯化氢的二氧六环溶液(4.0M，5mL)，室温搅拌2小时。减压浓缩，残余物用反相高效液 相制备色谱纯化，得到目标产物5(14.1mg，固体)，两步产率：34％。
MS m/z(ESI):411[M+1]
1H NMR(400MHz,DMSO-d 6)δ8.56(d,J＝7.3Hz,1H),8.13(s,1H),8.06(brs,3H),7.95(s,1H),7.94(d,J＝2.2Hz,2H),7.33(d,J＝1.4Hz,1H),6.68(dd,J＝7.3,2.1Hz,1H),6.44(dd,J＝2.2,0.7Hz,1H),3.66(d,J＝13.4Hz,1H),3.58(d,J＝13.3Hz,1H),3.41(s,3H),3.21–3.14(m,1H),3.10–3.02(m,2H),2.07–2.03(m,1H),1.87–1.58(m,7H),1.50(d,J＝12.8Hz,1H),1.41(d,J＝12.9Hz,1H).
实施例6
(R)-5-((1H-吲唑-5-基)硫代)-2-(1-氨基-8-氮杂螺[4.5]癸烷-8-基)-3-甲基嘧啶-4(3H)-酮盐酸盐
Figure PCTCN2021071375-appb-000020
第一步
3-((1H-吲唑-5-基)硫代)丙酸乙酯(6b)
向5-溴-1H-吲唑6a(520mg，2.65mmol)、3-巯基丙酸乙酯(533mg，3.98mmol)、DIPEA(1.02g，7.95mmol)、XantPhos(153mg，0.265mmol)和二氧六环(10mL)的混合物中加入Pd 2(dba) 3(243mg，0.265mmol)，氮气保护下加热至100℃，反应2小时。降至室温，减压脱溶，残余物用硅胶柱层析纯化(乙酸乙酯/石油醚＝1/1)得到目标产物6b(630mg，固体)，产率：95％。
MS m/z(ESI):251[M+1]
第二步
1H-吲唑-5-硫醇(6c)
向6b(500mg，2.0mmol)的四氢呋喃(10mL)溶液中加入叔丁醇钾(672mg，6.0mmol)，室温下搅拌2小时。用盐酸(6N)酸化至pH为4，用二氯甲烷(20mL)萃取。有机相用无水硫酸钠干燥，过滤除去干燥剂，减压脱溶，得到目标产物6c(300mg，固体)，产率：100％。
MS m/z(ESI):151[M+1]
第三步
(R)-N-((R)-8-(5-((1H-吲唑-5-基)硫代)-1-甲基-6-羰基-1,6-二氢嘧啶-2-基)-8-氮杂螺[4.5]癸烷-1-基)-2-甲基丙烷-2-亚磺酰胺(6d)
向6c(60mg，0.4mmol)、1e(100mg，0.2mmol)、DIPEA(77mg，0.6mmol)、XantPhos(12mg，0.02mmol)和二氧六环(5mL)的混 合物中加入Pd 2(dba) 3(18mg，0.02mmol)，氮气保护下加热至100℃，反应2小时。降至室温，减压脱溶，残余物用硅胶柱层析纯化(乙酸乙酯/石油醚＝1/1)得到目标产物6d(70mg，固体)，产率：67％。
MS m/z(ESI):515[M+1]
第四步
(R)-5-((1H-吲唑-5-基)硫代)-2-(1-氨基-8-氮杂螺[4.5]癸烷-8-基)-3-甲基嘧啶-4(3H)-酮盐酸盐(6)
将6d(70mg，0.14mmol)溶于甲醇(5mL)，加入氯化氢的二氧六环溶液(4.0M，2mL)，室温搅拌2小时。减压浓缩，残余物用反相高效液相制备色谱纯化，得到目标产物6(30mg，固体)，产率：54％。
MS m/z(ESI):411[M+1]
1H NMR(400MHz,DMSO-d 6)δ8.28(s,3H),8.05(s,1H),7.79(s,1H),7.69(s,1H),7.55(d,J＝8.7Hz,1H),7.34(dd,J＝8.7,1.6Hz,1H),3.56(d,J＝13.2Hz,1H),3.49(d,J＝13.5Hz,1H),3.39(s,3H),3.17–3.13(m,1H),3.06–2.96(m,2H),2.07–2.01(m,1H),1.90–1.61(m,7H),1.52(d,J＝13.1Hz,1H),1.39(d,J＝13.0Hz,1H).
实施例7
(R)-2-(1-氨基-8-氮杂螺[4.5]癸烷-8-基)-5-((4-氯-2-甲基-2H-吲唑-5-基)硫代)-3-甲基嘧啶-4(3H)-酮盐酸盐
Figure PCTCN2021071375-appb-000021
第一步
5-溴-4-氯-2H-吲唑(7b)
在冰浴冷却下向4-溴-3-氯-2-甲基苯胺7a(1.0g，4.58mmol)的醋酸(20mL)溶液中加入亚硝酸钠(396mg，5.73mmol)的水(4mL)溶液，室温下搅拌1小时。旋蒸除去大部分溶剂，残余物悬浮于水(30mL)中，过滤。滤饼用水(25mL×3)洗涤，空气中干燥后得到目标产物7b(780mg，固体)，产率：74％。产物不经纯化直接用于下一步反应。
MS m/z(ESI):231[M+1]
第二步
5-溴-4-氯-2-甲基-2H-吲唑(7c)
在冰浴冷却下向7b(760mg，3.3mmol)的乙酸乙酯(15mL)溶液中加入三甲基氧鎓四氟硼酸盐(733mg，4.95mmol)，反应混合物在室温下搅拌4小时。加入石油醚(30mL)稀释，搅拌10分钟，过滤。滤液加入乙酸乙酯(20mL)，用碳酸氢钠溶液(30mL×3)和饱和食盐水(20mL×3)洗涤。有机相经无水硫酸钠干燥，减压浓缩，残余物用硅胶柱层析纯化(乙酸乙酯/石油醚＝1.4/1)得到目标产物7c(460mg，固体)，产率：57％。
MS m/z(ESI):245[M+1]
第三步
3-((4-氯-2-甲基-2H-吲唑-5-基)硫代)丙酸乙酯(7d)
向7c(440mg，1.8mmol)的二氧六环(20mL)溶液中加入3-巯基丙酸乙酯(483mg，3.6mmol)、DIPEA(697mg，5.4mmol)、Pd 2(dba) 3(165mg，0.18mmol)和XantPhos(156mg，0.27mmol)。反应混合物在氮气下加热至100℃，反应16小时。降至室温，减压脱溶，残余物用硅胶柱层析纯化(乙酸乙酯/石油醚＝1.7/1)得到目标产物7d(300mg，固体)，产率：59％。
MS m/z(ESI):299[M+1]
第四步
4-氯-2-甲基-2H-吲唑-5-硫醇钠(7e)
向7d(140mg，0.47mmol)的甲醇(20mL)溶液中加入甲醇钠的甲醇溶液(30％，93mg，0.52mmol)，30℃下搅拌32小时。减压脱溶，得到目标产物7e(100mg，固体)，产率：96％。产物不经纯化直接用于下一步反应。
MS m/z(ESI):199[M+1]
第五步
(R)-N-((R)-8-(5-((4-氯-2-甲基-2H-吲唑-5-基)硫代)-1-甲基-6-羰基-1,6-二氢嘧啶-2-基)-8-氮杂螺[4.5]癸烷-1-基)-2-甲基丙烷-2-亚磺酰胺(7f)
将7e(80mg，0.36mmol)溶于二氧六环(8mL)，加入1e(100mg，0.20mmol)、DIPEA(77mg，0.60mmol)、Pd 2(dba) 3(18mg，0.020mmol)和XantPhos(18mg，0.030mmol)。反应混合物在氮气保护下加热至90℃，反应2小时。降至室温，将反应混合物倒入乙酸乙酯(15mL)和石油醚(15mL)的混合溶剂中，过滤，滤饼干燥，得到目标产物7f(61mg，固体)，产率：54％。
MS m/z(ESI):563[M+1]
第六步
(R)-2-(1-氨基-8-氮杂螺[4.5]癸烷-8-基)-5-((4-氯-2-甲基-2H-吲唑-5-基)硫代)-3-甲基嘧啶-4(3H)-酮盐酸盐(7)
将7f(61mg，0.11mmol)溶于甲醇(5mL)，加入氯化氢的二氧六环溶液(4.0M，5mL)，室温搅拌2小时。减压浓缩，残余物用反相高效液相制备色谱纯化，得到目标产物7(16mg，固体)，产率：31％。
MS m/z(ESI):459[M+1]
1H NMR(400MHz,DMSO-d 6)δ8.43(s,1H),8.12(brs,3H),7.90(s,1H),7.51(dd,J＝9.0,0.7Hz,1H),6.99(d,J＝9.0Hz,1H),4.17(s,3H),3.60(d,J＝13.3Hz,1H),3.53(d,J＝13.3Hz,1H),3.40(s,3H),3.16–3.13(m,1H),3.06–2.98(m,2H),2.07–2.04(m,1H),1.87–1.60(m,7H),1.49(d,J＝13.0Hz,1H),1.39(d,J＝13.1Hz,1H).
实施例8
(R)-2-(1-氨基-8-氮杂螺[4.5]癸烷-8-基)-5-((4-氯吡唑并[1,5-a]吡啶-5-基)硫代)-3-甲基嘧啶-4(3H)-酮甲酸盐
Figure PCTCN2021071375-appb-000022
第一步
[[(2,4,6-三甲苯基)磺酰基]氧基]氨基甲酸叔丁酯(8b)
将2,4,6-三甲基苯磺酰氯8a(2.0g，9.15mmol)和羟基氨基甲酸叔丁酯(1.22g，9.15mmol)溶于叔丁基甲基醚(30mL)，降至0℃，逐滴加入三乙胺(1.4mL，10.1mmol)，然后升至室温，搅拌4小时。加水洗涤，有机相用无水硫酸钠干燥，减压脱溶。残余物用硅胶柱层析纯化(乙酸乙酯/石油醚＝1/5)得到目标产物8b(2.5g，固体)，产率：87％。
MS m/z(ESI):338[M+23],216[M+1-100]
第二步
O-(2,4,6-三甲基苯磺酰基)羟胺(8c)
0℃下向三氟醋酸(20mL)中分批加入8b(2.5g，7.93mmol)，反应混合物在0℃下搅拌2小时。缓慢加入冰水，搅拌15分钟。过滤，用水洗涤滤饼直到滤液呈中性。滤饼干燥后得到目标产物8c(1.5g，固体)，产率：88％。
MS m/z(ESI):216[M+1]
第三步
5-溴-4-氯吡唑并[1,5-a]吡啶-3-羧酸乙酯(8e)
将4-溴-3-氯吡啶8d(1.4g，7.28mmol)溶于乙腈(25mL)，加入8c(1.6g，7.28mmol)，反应混合物在40℃下搅拌反应过夜。过滤，滤饼干燥后得到黄色固体产物(1.7g)。
将上述产物(1.6g，3.92mmol)和丙炔酸乙酯(0.42g，4.32mmol)溶于N,N-二甲基甲酰胺(15mL)，加入碳酸钾(1.1g，7.84mmol)，室温搅拌3小时。加水淬灭，用乙酸乙酯(100mL×3)萃取。有机相合并后用饱和食盐水洗涤，经无水硫酸钠干燥，减压脱溶。残余物用硅胶柱层析纯化(乙酸乙酯/石油醚＝1/2)得到目标产物8e(120mg，固体)，产率：5.8％。
MS m/z(ESI):303,305[M+1]
第四步
5-溴-4-氯吡唑并[1,5-a]吡啶(8f)
将8e(110mg，0.36mmol)溶于氢溴酸溶液(3mL)，加热至100℃，搅拌3小时。降至室温，加入氢氧化钠溶液(2M)中和至中性，用二氯甲烷(30mL×3)萃取。有机相合并后用饱和食盐水洗涤，经无水硫酸钠干燥，减压脱溶。残余物用硅胶柱层析纯化(乙酸乙酯/石油醚＝3/7)得到目标产物8f(65mg，固体)，产率：77％。
MS m/z(ESI):231,233[M+1]
第五步
3-((4-氯吡唑并[1,5-a]吡啶-5-基)硫代)丙酸乙酯(8g)
向8f(60mg，0.26mmol)的二氧六环(5mL)溶液中加入3-巯基丙酸乙酯(38mg，0.29mmol)、DIPEA(67mg，0.52mmol)、Pd 2(dba) 3(24mg，0.026mmol)和XantPhos(30mg，0.052mmol)。反应混合物在氮气下加热至100℃，反应3小时。降至室温，减压脱溶，残余物用硅胶柱层析纯化(甲醇/二氯甲烷＝1/28)得到目标产物8g(60mg，固体)，产率：81％。
MS m/z(ESI):285[M+1]
第六步
4-氯吡唑并[1,5-a]吡啶-5-硫醇酸钠(8h)
向8g(60mg，0.21mmol)的甲醇(3mL)溶液中加入甲醇钠的甲醇溶液(30％，45.5mg，0.25mmol)，30℃下搅拌4小时。减压脱溶，得到目标产物8h(40mg，固体)，产率：92％。产物不经纯化直接用于 下一步反应。
MS m/z(ESI):185[M+1]
第七步
(R)-N-((R)-8-(5-((4-氯吡唑并[1,5-a]吡啶-5-基)硫代)-1-甲基-6-羰基-1,6-二氢嘧啶-2-基)-8-氮杂螺[4.5]癸烷-1-基)-2-甲基丙烷-2-亚磺酰胺(8i)
将8h(40mg，0.19mmol)溶于二氧六环(5mL)，加入1e(95mg，0.19mmol)、DIPEA(49mg，0.38mmol)、Pd 2(dba) 3(17mg，0.02mmol)和XantPhos(22mg，0.04mmol)。反应混合物在氮气保护下加热至100℃，反应4小时。降至室温，减压脱溶，残余物用硅胶柱层析纯化(甲醇/二氯甲烷＝1/28)得到目标产物8i(32mg，油状物)，产率：30％。
MS m/z(ESI):549[M+1]
第八步
(R)-2-(1-氨基-8-氮杂螺[4.5]癸烷-8-基)-5-((4-氯吡唑并[1,5-a]吡啶-5-基)硫代)-3-甲基嘧啶-4(3H)-酮甲酸盐(8)
将8i(32mg，0.06mmol)溶于甲醇(2mL)，加入氯化氢的二氧六环溶液(4.0M，1mL)，室温搅拌1小时。减压浓缩，残余物用反相高效液相制备色谱纯化，得到目标产物8(13mg，固体)，产率：47％。
MS m/z(ESI):445[M+1]
1H NMR(400MHz,DMSO-d 6)δ8.54(d,J＝7.3Hz,1H),8.35(s,1H),8.19(s,1H),8.06(d,J＝2.2Hz,1H),6.60(d,J＝1.8Hz,1H),6.48(d,J＝7.3Hz,1H),3.69–3.60(m,2H),3.41(s,3H),3.09(t,J＝11.7Hz,2H),3.02–2.99(m,1H),2.00–1.91(m,1H),1.76–1.50(m,7H),1.42(d,J＝13.2Hz,1H),1.35(d,J＝12.9Hz,1H).
实施例9
(R)-5-((2-(1-氨基-8-氮杂螺[4.5]癸烷-8-基)-1-甲基-6-羰基-1,6-二氢嘧啶-5-基)硫代)-4-氯-2-甲基-2H-吲唑-3-甲腈
Figure PCTCN2021071375-appb-000023
第一步
5-溴-4-氯-2-甲基-2H-吲唑-3-甲醛(9a)
将7c(800mg，3.28mmol)溶于四氢呋喃(20mL)，降温至-78℃，加入二异丙基氨基锂的四氢呋喃溶液(2M，2.95mL，5.9mmol)，搅拌90分钟，升温至0℃，继续搅拌30分钟。再次冷却至-78℃，逐滴加入DMF(0.76mL)，搅拌0.5小时，升至室温，继续搅拌2小时。加入饱和氯化铵溶液淬灭，用乙酸乙酯萃取。有机相经无水硫酸钠干燥，过滤除去干燥剂，减压脱溶。残余物用硅胶柱层析纯化(乙酸乙酯/石油醚＝3/7)得到目标产物9a(520mg，固体)，产率：60％。
MS m/z(ESI):273[M+1]
第二步
5-溴-4-氯-2-甲基-2H-吲唑-3-甲醛肟(9b)
将9a(490mg，1.8mmol)溶于异丙醇(8mL)、甲醇(8mL)和水(8mL)中，加入盐酸羟胺(497mg，7.2mmol)和碳酸钠(763mg，7.2mmol)，加热至50℃，搅拌16小时。倒入水(60mL)中，用乙酸乙酯(50mL×3)萃取。有机相合并后用饱和食盐水(20mL×3)洗涤，经无水硫酸钠干燥，减压脱溶。残余物用硅胶柱层析纯化(乙酸乙酯/石油醚＝3/7)得到目标产物9b(470mg，固体)，产率：92％。
MS m/z(ESI):288[M+1]
第三步
5-溴-4-氯-2-甲基-2H-吲唑-3-甲腈(9c)
将9b(470mg，1.63mmol)溶于乙腈(30mL)，加入醋酸铜(147mg，0.81mmol)，加热至85℃，搅拌过夜。减压脱溶，残余物倒入水(50mL)中，用乙酸乙酯(30mL×3)萃取。有机相合并后用饱和食盐水(25mL×3)洗涤，经无水硫酸钠干燥后减压脱溶，得到目标产物9c(390mg，固体)，产率：89％。
MS m/z(ESI):270[M+1]
第四步
3-((4-氯-3-氰基-2-甲基-2H-吲唑-5-基)硫代)丙酸乙酯(9d)
向9c(380mg，1.4mmol)的二氧六环(5mL)溶液中加入3-巯基丙酸乙酯(385mg，2.8mmol)、DIPEA(542mg，4.2mmol)、Pd 2(dba) 3(128mg，0.14mmol)和XantPhos(121mg，0.21mmol)。反应混合物在氮气保护下加热至100℃，反应16小时。降至室温，减压脱溶，残余物用硅胶柱层析纯化(乙酸乙酯/石油醚＝1/1)得到目标产物9d(350mg，固体)，产率：77％。
MS m/z(ESI):324[M+1]
第五步
4-氯-3-氰基-2-甲基-2H-吲唑-5-硫醇酸钠(9e)
向9d(195mg，0.60mmol)的甲醇(20mL)溶液中加入甲醇钠的甲醇溶液(30％，130mg，0.72mmol)，30℃下搅拌16小时。减压脱 溶，得到目标产物9e(220mg，固体，粗品)。产物不经纯化直接用于下一步反应。
MS m/z(ESI):224[M+1]
第六步
(R)-N-((R)-8-(5-((4-氯-3-氰基-2-甲基-2H-吲唑-5-基)硫代)-1-甲基-6-羰基-1,6-二氢嘧啶-2-基)-8-氮杂螺[4.5]癸烷-1-基)-2-甲基丙烷-2-亚磺酰胺(9f)
将9e(89.2mg，0.36mmol)溶于二氧六环(6mL)，加入1e(120mg，0.24mmol)、DIPEA(92.8mg，0.72mmol)、Pd 2(dba) 3(21.9mg，0.024mmol)和XantPhos(20.8mg，0.036mmol)。反应混合物在氮气保护下加热至80℃，反应16小时。降至室温，减压脱溶，残余物用硅胶柱层析纯化(甲醇/二氯甲烷＝1/12)得到目标产物9f(90mg，固体)，两步产率：63％。
MS m/z(ESI):588[M+1]
第七步
(R)-5-((2-(1-氨基-8-氮杂螺[4.5]癸烷-8-基)-1-甲基-6-羰基-1,6-二氢嘧啶-5-基)硫代)-4-氯-2-甲基-2H-吲唑-3-甲腈甲酸盐(9)
将9f(90mg，0.15mmol)溶于甲醇(10mL)，加入氯化氢的二氧六环溶液(4.0M，10mL)，室温搅拌2小时。减压浓缩，残余物用反相高效液相制备色谱纯化，得到目标产物9(14mg，固体)，产率：19％。
MS m/z(ESI):484[M+1]
1H NMR(400MHz,DMSO-d 6)δ8.14(s,1H),7.79(brs,3H),7.75(d,J＝9.0Hz,1H),7.05(d,J＝9.1Hz,1H),4.34(s,3H),3.69(d,J＝14.1Hz,1H),3.61(d,J＝14Hz,1H),3.41(s,3H),3.24–3.16(m,1H),3.13–3.06(m,2H),2.08–2.04(m,1H),1.80–1.64(m,7H),1.49–1.41(m,2H).
实施例10
(S)-2-(4-氨基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-5-((4-氯-2-甲基-2H-吲唑-5-基)硫代)-3-甲基嘧啶-4(3H)-酮
Figure PCTCN2021071375-appb-000024
第一步
2-((叔丁基二甲基甲硅烷基)氧代)乙烷-1-醇(10b)
0℃下向乙二醇10a(124g，2mol)、咪唑(34g，500mmol)和二氯甲烷(500mL)的混合物中逐滴加入叔丁基二甲基氯硅烷(50g，334mmol)，然后反应混合物在室温下搅拌16小时。加水(300mL)和二氯甲烷(600mL)稀释，分液，水相用二氯甲烷(300mL×3)萃取。有机相合并后经无水硫酸钠干燥，过滤，减压浓缩。残余物用硅胶柱层析纯化(乙酸乙酯/石油醚＝1/5)得到目标产物10b(35g，油状物)，产率：10％。
MS m/z(ESI):177[M+1]
第二步
2-((叔丁基二甲基甲硅烷基)氧代)乙醛(10c)
-30℃下向二氯甲烷(500mL)中逐滴加入草酰氯(27.6g，218.9mmol)，降温至-78℃，逐滴加入二甲基亚砜(21.7g，278.6mmol)。反应混合物在-78℃下搅拌30分钟后，缓慢加入10b(35g，199mmol)的二氯甲烷(100mL)溶液，搅拌1小时。逐滴加入三乙胺(100.5g，995mmol)，-78℃下继续搅拌30分钟，然后室温搅拌过夜。先后用水(300mL)、稀盐酸(1N，400mL×2)、饱和碳酸氢钠溶液(400mL)和饱和食盐水(400mL)洗涤。有机相经无水硫酸钠干燥，过滤，减压浓缩，得到目标产物10c(34g，油状物)，产率：98％。
1H NMR(400MHz,CDCl 3)δ9.60(s,1H),4.11(s,2H),0.82(s,9H),0.00(s,6H).
第三步
1-(叔丁基)4-乙基4-(2-((叔丁基二甲基甲硅烷基)氧代)-1-羟基乙基)哌啶-1,4-二羧酸酯(10d)
-10℃下向二异丙基氨基锂的四氢呋喃溶液(2M，148mL，296mmol)和四氢呋喃(400mL)中逐滴加入1-(叔丁基)4-乙基哌啶-1,4-二羧酸酯(50g，197mmol)的四氢呋喃(100mL)溶液，然后反应混合物在0℃下搅拌30分钟。加入10c(34g，197mmol)，0℃下搅拌1小时，室温搅拌1小时。加入饱和碳酸氢钠和水的混合溶液(400mL，1/4v/v)，加入乙酸乙酯(200mL)，分液。水相用乙酸乙酯(200mL×3)萃取。有机相合并后经无水硫酸钠干燥，过滤，减压浓缩。残余物用硅胶柱层析纯化(乙酸乙酯/石油醚＝2/3)得到目标产物10d(75g，油状物)，产率：88％。
MS m/z(ESI):332[M+1-100]
第四步
4-(2-((叔丁基二甲基甲硅烷基)氧代)-1-羟基乙基)-4-(羟甲基)哌啶-1-羧酸叔丁酯(10e)
向10d(75g，174mmol)的四氢呋喃(600mL)溶液中加入硼氢化锂的四氢呋喃溶液(2M，130mL，261mmol)，室温搅拌2小时。降温至0℃，用饱和碳酸氢钠溶液和水(150mL，1/2v/v)淬灭，用乙酸乙酯(200mL)稀释，过滤。滤液分液，水相用乙酸乙酯(200mL×3)萃取。有机相合并后用饱和食盐水洗涤，经无水硫酸钠干燥，过滤，减压浓缩，得到目标产物10e(55g，油状物)，产率：81％。
MS m/z(ESI):290[M+1-100]
第五步
4-(1,2-二羟基乙基)-4-(羟甲基)哌啶-1-羧酸叔丁酯(10f)
向10e(55g，128.5mmol)的四氢呋喃(400mL)溶液中加入四丁基氟化铵的四氢呋喃溶液(1M，193mL，193mmol)，室温搅拌2小时。用饱和碳酸氢钠溶液和水(100mL，1/2v/v)稀释，加入乙酸乙酯(200mL)，分液，水相用乙酸乙酯(200mL×3)萃取。有机相合并后经无水硫酸钠干燥，过滤，减压浓缩。残余物用硅胶柱层析纯化(乙酸乙酯/石油醚＝1/4到1/0)得到目标产物10f(23g，油状物)，产率：65％。
MS m/z(ESI):176[M+1-100]
第六步
4-羟基-2-氧杂-8-氮杂螺[4.5]癸烷-8-羧酸叔丁酯(10g)
将氢化钠(60％，11.7g，292.6mmol)悬浮于四氢呋喃(100mL)中，降至0℃，逐滴加入10f(23g，83.6mmol)的四氢呋喃(100mL)溶液，然后加入4-甲苯磺酰氯(16g，83.6mmol)的四氢呋喃(200mL)溶液。反应混合物在0℃下搅拌2小时。缓慢加入饱和氯化铵溶液(50mL)，剧烈搅拌直至无气体产生。加入饱和氯化铵溶液(100mL)和饱和食盐水(100mL)，用乙酸乙酯(200mL×3)萃取。有机相合并后经无水硫酸钠干燥，过滤，减压浓缩。残余物用硅胶柱层析纯化(乙酸乙 酯/石油醚＝0/1到1/0)得到目标产物10g(8.5g，油状物)，产率：39％。
MS m/z(ESI):158[M+1-100]
第七步
4-羰基-2-氧杂-8-氮杂螺[4.5]癸烷-8-羧酸叔丁酯(10h)
反应混合物10g(8.5g，32.9mmol)、戴斯-马丁氧化剂(27.4g，64.56mmol)和二氯甲烷(200mL)在0℃下搅拌2小时。加入饱和碳酸氢钠溶液和饱和硫代硫酸钠溶液(100mL，1/1v/v)，剧烈搅拌，分液。水相用二氯甲烷(200mL×3)萃取。有机相合并后经无水硫酸钠干燥，过滤，减压浓缩。残余物用硅胶柱层析纯化(乙酸乙酯/石油醚＝2/3)得到目标产物10h(6.2g，油状物)，产率：74％。
MS m/z(ESI):156[M+1-100]
第八步
2-氧杂-8-氮杂螺[4.5]癸烷-4-酮(10i)
向10h(500mg，1.96mmol)的二氯甲烷(8mL)溶液中加入氯化氢的二氧六环溶液(4M，2mL)，室温下搅拌1小时。减压脱溶，得到目标产物10i(盐酸盐，370mg，固体)，产率：99％。产物不经纯化直接用于下一步反应。
MS m/z(ESI):156[M+1]
第九步
8-(5-碘-1-甲基-6-羰基-1,6-二氢嘧啶-2-基)-2-氧杂-8-氮杂螺[4.5]癸烷-4-酮(10j)
向10i(盐酸盐，106mg，0.555mmol)、碳酸钾(153mg，1.11mmol)和乙腈(4mL)的混合物中加入1c(100mg，0.37mmol)，加热至80℃，搅拌16h。降至室温，加入水(5mL)和乙酸乙酯(20mL)，分液。水相用乙酸乙酯(10mL×3)萃取。有机相合并后经无水硫酸钠干燥，过滤，减压浓缩。残余物用硅胶柱层析纯化(乙酸乙酯/石油醚＝1/4)得到目标产物10j(70mg，固体)，产率：49％。
MS m/z(ESI):390[M+1]
第十步
(R)-N-((S)-8-(5-碘-1-甲基-6-羰基-1,6-二氢嘧啶-2-基)-2-氧杂-8-氮杂螺[4.5]癸烷-4-基)-2-甲基丙烷-2-亚磺酰胺(10k)
将反应混合物10j(60mg，0.15mmol)、(R)-2-甲基丙烷-2-亚磺酰胺(36.3mg，0.3mmol)、钛酸四乙酯(137mg，0.6mmol)和四氢呋喃(5mL)加热至90℃，搅拌4小时。冷却至0℃，加入甲醇(2mL)和硼氢化锂的四氢呋喃溶液(2.0M，0.15mL，0.3mmol)，搅拌1小时。加入水(5mL)和二氯甲烷(20mL)，分液。水相用二氯甲烷(10mL×3)萃取。有机相合并后经无水硫酸钠干燥，过滤，减压浓缩。残余物用硅胶柱层析纯化(甲醇/二氯甲烷＝1/9)得到目标产物10k(70mg，固体)，产率：92％。
MS m/z(ESI):495[M+1]
第十一步
(R)-N-((S)-8-(5-((4-氯-2-甲基-2H-吲唑-5-基)硫代)-1-甲基-6-羰基-1,6-二氢嘧啶-2-基)-2-氧杂-8-氮杂螺[4.5]癸烷-4-基)-2-甲基丙烷-2-亚磺酰胺(10l)
向10k(70mg，0.14mmol)、7e(37.4mg，0.17mmol)、DIPEA(54mg，0.42mmol)、XantPhos(8.1mg，0.014mmol)和二氧六环(4mL)的混合物中加入Pd 2(dba) 3(12.8mg，0.014mmol)，在氮气保护下加热至100℃，反应2小时。降至室温，加入水(5mL)和二氯甲烷(20mL)，分液。水相用二氯甲烷(10mL×3)萃取，有机相合并后经无水硫酸钠干燥，过滤，减压脱溶。残余物用硅胶柱层析纯化(甲醇/二氯甲烷＝1/9)得到目标产物10l(50mg，固体)，产率：63％。
MS m/z(ESI):565[M+1]
第十二步
(S)-2-(4-氨基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-5-((4-氯-2-甲基-2H-吲唑-5-基)硫代)-3-甲基嘧啶-4(3H)-酮(10)
将10l(50mg，0.09mmol)溶于甲醇(8mL)，加入氯化氢的二氧六环溶液(4.0M，2mL)，室温搅拌2小时。减压浓缩，残余物用反相高效液相制备色谱纯化，得到目标产物10(5.8mg，固体)，产率：14％。
MS m/z(ESI):461[M+1]
1H NMR(400MHz,DMSO-d 6)δ8.43(s,1H),7.91(s,1H),7.51(d,J＝9.0Hz,1H),6.99(d,J＝9.0Hz,1H),4.18(s,3H),4.02–3.95(m,1H),3.69(d,J＝8.5Hz,1H),3.63(d,J＝8.5Hz,1H),3.39(s,3H),3.15–3.13(m,1H),3.08–3.00(m,2H),1.84–1.76(m,1H),1.72–1.65(m,1H),1.54–1.43(m,2H).
实施例11
2-((3S,4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-5-((4-氯-2-甲基-2H-吲唑-5-基)硫代)-3-甲基嘧啶-4(3H)-酮甲酸盐
Figure PCTCN2021071375-appb-000025
第一步
(S)-2-((叔丁基二甲基甲硅烷基)氧代)丙酸甲酯(11b)
0℃下向咪唑(68.5g，1008mmol)的二氯甲烷(800mL)溶液中逐滴加入(S)-2-羟基丙酸甲酯11a(70g，672mmol)，然后缓慢加入叔丁基二甲基氯硅烷(120g，807mmol)，反应混合物在室温下搅拌16小时。加水(600mL)稀释，用二氯甲烷(500mL×2)萃取。有机相合并后用稀盐酸(1N，300mL×3)洗涤，经无水硫酸钠干燥，过滤，减压浓缩，得到目标产物11b(150g，油状物)，产率：99％。
1H NMR(400MHz,CDCl 3)δ4.32(q,J＝6.7Hz,1H),3.70(s,3H),1.38(d,J＝6.8Hz,3H),0.88(s,9H),0.08(s,3H),0.05(s,3H).
第二步
(S)-2-((叔丁基二甲基甲硅烷基)氧代)丙醛(11c)
将11b(151.2g，693mmol)的二氯甲烷(1200mL)溶液冷却至-78℃，逐滴加入二异丁基氢化铝的正己烷溶液(1M，832mL，832mmol)，升温至-40℃，搅拌1小时。将反应混合物倒入酒石酸钾钠的饱和溶液(140mL)中，加入***(100mL)，室温下搅拌2小时。分液，水相用***萃取。有机相合并后用饱和食盐水洗涤，经无水硫酸钠干燥，过滤，减压浓缩。残余物用硅胶柱层析纯化(乙酸乙酯/石油醚＝4/1)得到目标产物11c(62.3g，油状物)，产率：48％。
1H NMR(400MHz,CDCl 3)δ9.60(s,1H),4.09(q,J＝6.8Hz,1H),1.27(d,J＝6.9Hz,3H),0.91(s,9H),0.10(s,3H),0.08(s,3H).
第三步
1-(叔丁基)4-乙基4-((2S)-2-((叔丁基二甲基甲硅烷基)氧代)-1-羟基丙基)哌啶-1,4-二羧酸酯(11d)
-50℃下向二异丙基氨基锂的四氢呋喃溶液(1M，425mL，425mmol) 和四氢呋喃(800mL)中逐滴加入1-(叔丁基)4-乙基哌啶-1,4-二羧酸酯(72.8g，283.5mmol)的四氢呋喃(50mL)溶液，然后反应混合物在-10℃下搅拌1小时。加入11c(53.3g，283.5mmol)，-10℃下搅拌1小时。升至0℃，继续搅拌1小时，然后室温搅拌1小时。加入饱和碳酸氢钠和水的混合溶液(400mL，1/4v/v)，加入乙酸乙酯(500mL)，分液。水相用乙酸乙酯(500mL×3)萃取。有机相合并后经无水硫酸钠干燥，过滤，减压浓缩，得到目标产物11d(120g，油状物，粗品)。产物不经纯化直接用于下一步反应。
MS m/z(ESI):346[M+1-100]
第四步
4-((2S)-2-((叔丁基二甲基甲硅烷基)氧代)-1-羟基丙基)-4-(羟甲基)哌啶-1-羧酸叔丁酯(11e)
向11d(120g，269.6mmol)的四氢呋喃(1200mL)溶液中加入硼氢化锂的四氢呋喃溶液(2M，270mL，539.2mmol)，室温搅拌16小时。降至0℃，用饱和碳酸氢钠溶液和水(600mL，1/2v/v)淬灭，用乙酸乙酯(800mL)稀释，过滤。滤液分液，水相用乙酸乙酯(500mL×3)萃取。有机相合并后用饱和食盐水(300mL×3)洗涤，经无水硫酸钠干燥，过滤，减压浓缩，得到目标产物11e(90g，油状物，粗品)。产物不经纯化直接用于下一步反应。
MS m/z(ESI):404[M+1]
第五步
4-((2S)-1,2-二羟基丙基)-4-(羟甲基)哌啶-1-羧酸叔丁酯(11f)
向11e(90g，223mmol)的四氢呋喃(1300mL)溶液中加入四丁基氟化铵的四氢呋喃溶液(1M，303mL，303mmol)，室温搅拌过夜。用饱和碳酸氢钠溶液和水(800mL，1/2v/v)稀释，加入乙酸乙酯(800mL)，分液，水相用乙酸乙酯(500mL×3)萃取。有机相合并后用饱和食盐水(300mL×3)洗涤，经无水硫酸钠干燥，过滤，减压浓缩。残余物用硅胶柱层析纯化(乙酸乙酯/石油醚＝49/1)得到目标产物11f(24.5g，油状物)，三步产率：26％。
MS m/z(ESI):290[M+1]
第六步
(3S)-4-羟基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-羧酸叔丁酯(11g)
将氢化钠(60％，10.8g，270.0mmol)悬浮于四氢呋喃(600mL)中，降温至0℃，逐滴加入11f(22.3g，77.1mmol)的四氢呋喃溶液(40mL)，然后加入4-甲苯磺酰氯(14.6g，77.1mmol)的四氢呋喃(50mL)溶液。反应混合物在0℃下搅拌6小时。降至-20℃，缓慢加入饱和氯化铵溶液(10mL)，剧烈搅拌直至无气体产生。用乙酸乙酯(300mL×3)萃取。有机相合并后用饱和食盐水(150mL×3)洗涤，经无水硫酸钠干燥，过滤，减压浓缩。残余物用硅胶柱层析纯化(乙酸 乙酯/石油醚＝1/1)得到目标产物11g(12.1g，油状物)，产率：58％。
MS m/z(ESI):172[M+1-100]
第七步
(S)-3-甲基-4-羰基-2-氧杂-8-氮杂螺[4.5]癸烷-8-羧酸叔丁酯(11h)
0℃下向11g(12.1g，44.6mmol)的二氯甲烷(140mL)溶液中缓慢加入戴斯-马丁氧化剂(37.8g，89.2mmol)，然后室温下搅拌16小时。加入饱和碳酸氢钠溶液至溶液呈中性，用二氯甲烷(150mL×3)萃取。有机相合并后用饱和食盐水(80mL×3)洗涤，经无水硫酸钠干燥，过滤，减压浓缩。残余物用硅胶柱层析纯化(乙酸乙酯/石油醚＝3/7)得到目标产物11h(8.36g，油状物)，产率：70％。
MS m/z(ESI):170[M+1-100]
第八步
(S)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-酮(11i)
将11h(1.2g，4.46mmol)溶于氯化氢的二氧六环溶液(4M，20mL)，室温下搅拌2小时。减压脱溶，得到目标产物11i(盐酸盐，800mg，固体)，产率：87％。
MS m/z(ESI):170[M+1]
第九步
(S)-8-(5-碘-1-甲基-6-羰基-1,6-二氢嘧啶-2-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-酮(11j)
向2-氯-5-碘-3-甲基嘧啶-4(3H)-酮1c(130mg，0.481mmol)的乙腈(20mL)溶液中加入11i(盐酸盐，108mg，0.529mmol)和碳酸钾(199mg，1.44mmol)，加热至80℃，搅拌16小时。降至室温，加入水(50mL)，用乙酸乙酯(30mL×3)萃取。有机相合并后用饱和食盐水(20mL×3)洗涤，经无水硫酸钠干燥，过滤，减压浓缩。残余物用硅胶柱层析纯化(乙酸乙酯/石油醚＝1.3/1)得到目标产物11j(180mg，固体)，产率：92％。
MS m/z(ESI):404[M+1]
第十步
(R)-N-((3S,4S)-8-(5-碘-1-甲基-6-羰基-1,6-二氢嘧啶-2-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-基)-2-甲基丙烷-2-亚磺酰胺(11k)
向11j(180mg，0.446mmol)的四氢呋喃(2mL)溶液中加入(R)-2-甲基丙烷-2-亚磺酰胺(108mg，0.892mmol)和钛酸四乙酯(813mg，3.59mmol)，加热至90℃，搅拌16小时。冷却至0℃，加入甲醇(5mL)和硼氢化锂的四氢呋喃溶液(2.0M，0.23mL，0.46mmol)，搅拌1小时。用饱和氯化铵溶液淬灭，过滤。滤液用乙酸乙酯(25mL×3)萃取。有机相合并后用饱和食盐水(20mL×3)洗涤，经无水硫酸钠干燥，过滤，减压浓缩。残余物用硅胶柱层析纯化(甲醇/二氯甲烷＝1/16)得到目标产物11k(90mg，固体)，产率：39％。
MS m/z(ESI):509[M+1]
第十一步
(R)-N-((3S,4S)-8-(5-((4-氯-2-甲基-2H-吲唑-5-基)硫代)-1-甲基-6-羰基-1,6-二氢嘧啶-2-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-基)-2-甲基丙烷-2-亚磺酰胺(11l)
向11k(80mg，0.157mmol)的二氧六环(5mL)溶液中加入7e(60mg，0.27mmol)、DIPEA(60.7mg，0.471mmol)、XantPhos(14.2mg，0.023mmol)和Pd 2(dba) 3(14.4mg，0.0157mmol)，在氮气保护下加热至80℃，反应4小时。降至室温，减压脱溶。残余物用硅胶柱层析纯化(甲醇/二氯甲烷＝1/13)得到目标产物11l(55mg，固体)，产率：60％。
MS m/z(ESI):579[M+1]
第十二步
2-((3S,4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-5-((4-氯-2-甲基-2H-吲唑-5-基)硫代)-3-甲基嘧啶-4(3H)-酮甲酸盐(11)
将11l(55mg，0.095mmol)溶于甲醇(5mL)，加入氯化氢的二氧六环溶液(4.0M，5mL)，室温搅拌2小时。减压浓缩，残余物用反相高效液相制备色谱纯化，得到目标产物11(16.8mg，固体)，产率：37％。
MS m/z(ESI):475[M+1]
1H NMR(400MHz,DMSO-d 6)δ8.43(s,1H),8.21(s,1H),7.91(s,1H),7.51(d,J＝9.1Hz,1H),6.99(d,J＝9.0Hz,1H),4.18(s,3H),4.11–4.05(m,1H),3.68(d,J＝8.6Hz,1H),3.51(d,J＝8.6Hz,1H),3.47–3.43(m,2H),3.41(s,3H),3.20–3.03(m,2H),3.00(d,J＝4.9Hz,1H),1.89–1.82(m,1H),1.77–1.72(m,1H),1.62–1.53(m,2H),1.11(d,J＝6.4Hz,3H).
实施例12
2-((3S,4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-5-((4-氯吡唑并[1,5-a]吡啶-5-基)硫代)-3-甲基嘧啶-4(3H)-酮甲酸盐
Figure PCTCN2021071375-appb-000026
第一步
(R)-N-((3S,4S)-8-(5-((4-氯吡唑并[1,5-a]吡啶-5-基)硫代)-1-甲基-6-羰基-1,6-二氢嘧啶-2-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-基)-2-甲基 丙烷-2-亚磺酰胺(12a)
将11k(140mg，0.28mmol)溶于二氧六环(16mL)，加入8h(60mg，0.29mmol)、DIPEA(71.5mg，0.56mmol)、Pd 2(dba) 3(25.3mg，0.03mmol)和XantPhos(32mg，0.06mmol)。反应混合物在氮气保护下加热至100℃，反应16小时。降至室温，减压脱溶，残余物用硅胶柱层析纯化(甲醇/二氯甲烷＝1/25)得到目标产物12a(100mg，固体)，产率：64％。
MS m/z(ESI):565[M+1]
第二步
2-((3S,4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-5-((4-氯吡唑并[1,5-a]吡啶-5-基)硫代)-3-甲基嘧啶-4(3H)-酮甲酸盐(12)
将12a(86mg，0.166mmol)溶于甲醇(5mL)，加入氯化氢的二氧六环溶液(4.0M，1mL)，室温搅拌1小时。减压浓缩，残余物用反相高效液相制备色谱纯化，得到目标产物12(16.8mg，固体)，产率：19％。
MS m/z(ESI):461[M+1]
1H NMR(400MHz,DMSO-d 6)δ8.51(d,J＝7.3Hz,1H),8.26(s,1H),8.17(s,1H),8.04(d,J＝2.1Hz,1H),6.59(d,J＝1.5Hz,1H),6.48(d,J＝7.3Hz,1H),4.14–4.07(m,1H),3.72(d,J＝8.7Hz,1H),3.57–3.51(m,3H),3.39(s,3H),3.21–3.07(m,2H),3.06(d,J＝4.9Hz,1H),1.88–1.75(m,2H),1.66–1.55(m,2H),1.12(d,J＝6.4Hz,3H).
实施例13
(R)-5-((1H-吲唑-4-基)硫代)-6-氨基-2-(1-氨基-8-氮杂螺[4.5]癸烷-8-基)-3-甲基嘧啶-4(3H)-酮甲酸盐
Figure PCTCN2021071375-appb-000027
第一步
6-氨基-5-溴-3-甲基嘧啶-2,4(1H,3H)-二酮(13b)
将6-氨基-3-甲基嘧啶-2,4(1H,3H)-二酮(1.0g，7.08mmol)溶于DMF(6mL)，加入NBS(1.38g，7.8mmol)，室温下搅拌16小时。加水(20mL)稀释，过滤。滤饼用水(5mL×2)洗涤，真空干燥后得到目标产物13b(1.0g，固体)，产率：64％。
MS m/z(ESI):220[M+1]
第二步
8-(4-氨基-5-溴-1-甲基-6-羰基-1,6-二氢嘧啶-2-基)-8-氮杂螺[4.5]癸烷-1-酮(13c)
将13b(200mg，0.91mmol)溶于DMF(5mL)，加入8-氮杂螺[4.5]癸烷-1-酮(167mg，1.1mmol)、卡特缩合剂(1.2g，2.73mmol)和DBU(692mg，4.55mmol)，室温下搅拌过夜。加入乙酸乙酯(50mL)稀释，用水(10mL×3)和饱和食盐水(10mL)洗涤。有机相减压脱溶，残余物用硅胶柱层析纯化(乙酸乙酯/石油醚＝7/3)得到目标产物13c(170mg，油状物)，产率：53％。
MS m/z(ESI):355[M+1]
第三步
(R)-N-((R)-8-(4-氨基-5-溴-1-甲基-6-羰基-1,6-二氢嘧啶-2-基)-8-氮杂螺[4.5]癸烷-1-基)-2-甲基丙烷-2-亚磺酰胺(13d)
向13c(160mg，0.45mmol)的四氢呋喃(15mL)溶液中加入(R)-2-甲基丙烷-2-亚磺酰胺(110mg，0.9mmol)和钛酸四乙酯(411mg，1.8mmol)，加热至90℃，搅拌过夜。冷却至室温，加入甲醇(5mL)和硼氢化锂的四氢呋喃溶液(2.0M，0.5mL)，搅拌1小时。减压浓缩，残余物用硅胶柱层析纯化(甲醇/二氯甲烷＝1/20)得到目标产物13d(200mg，油状物)，产率：96％。
MS m/z(ESI):460[M+1]
第四步
(R)-N-((R)-8-(5-((1H-吲唑-4-基)硫代)-4-氨基-1-甲基-6-羰基-1,6-二氢嘧啶-2-基)-8-氮杂螺[4.5]癸烷-1-基)-2-甲基丙烷-2-亚磺酰胺(13e)
将13d(100mg，0.22mmol)溶于二氧六环(10mL)，加入1H-吲唑-4-硫醇酸钠3c(75mg，0.44mmol)、DIPEA(84mg，0.66mmol)、Pd 2(dba) 3(40mg，0.044mmol)和XantPhos(51mg，0.088mmol)。反应混合物在氮气保护下加热至100℃，搅拌过夜。降至室温，减压脱溶，残余物用薄层硅胶制备色谱层析纯化得到目标产物13e(30mg，固体)，产率：26％。
MS m/z(ESI):530[M+1]
第五步
(R)-5-((1H-吲唑-4-基)硫代)-6-氨基-2-(1-氨基-8-氮杂螺[4.5]癸烷-8-基)-3-甲基嘧啶-4(3H)-酮甲酸盐(13)
将13e(30mg，0.056mmol)溶于甲醇(2mL)，加入氯化氢的二 氧六环溶液(4.0M，2mL)，室温搅拌1小时。减压浓缩，残余物用反相高效液相制备色谱纯化，得到目标产物13(5mg，固体)，产率：21％。
MS m/z(ESI):426[M+1]
1H NMR(400MHz,CD 3OD)δ8.54(s,1H),8.12(s,1H),7.30(d,J＝8.3Hz,1H),7.20(t,J＝7.7Hz,1H),6.78(d,J＝7.0Hz,1H),3.65–3.57(m,2H),3.42(s,3H),3.27–3.25(m,1H),3.09(t,J＝12.3Hz,2H),2.22–2.17(m,1H),1.85–1.72(m,7H),1.54(t,J＝13.5Hz,2H).
实施例14
6-氨基-2-((3S,4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-5-((4-氯-2-甲基-2H-吲唑-5-基)硫代)-3-甲基嘧啶-4(3H)-酮甲酸盐
Figure PCTCN2021071375-appb-000028
第一步
(S)-8-(4-氨基-1-甲基-6-羰基-1,6-二氢嘧啶-2-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-酮(14a)
将6-氨基-3-甲基嘧啶-2,4(1H,3H)-二酮13a(400mg，2.8mmol)溶于DMF(25mL)，加入11i(576mg，2.8mmol)、卡特缩合剂(6.18g，14mmol)和DBU(1.29g，8.5mmol)，室温下搅拌过夜。加水(60mL)淬灭，用二氯甲烷和甲醇的混合溶剂(10/1v/v，50mL×3)萃取。有机相合并后用饱和食盐水(20mL×3)洗涤，经无水硫酸钠干燥，减压脱溶。残余物用硅胶柱层析纯化(甲醇/二氯甲烷＝1/9)得到目标产物14a(700mg，油状物)，产率：84％。
MS m/z(ESI):293[M+1]
第二步
(R)-N-((3S,4S)-8-(4-氨基-1-甲基-6-羰基-1,6-二氢嘧啶-2-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-基)-2-甲基丙烷-2-亚磺酰胺(14b)
向14a(700mg，2.3mmol)的四氢呋喃(30mL)溶液中加入(R)- 2-甲基丙烷-2-亚磺酰胺(557mg，4.6mmol)和钛酸四乙酯(2.06g，9.2mmol)，加热至90℃，搅拌16小时。冷却至0℃，加入甲醇(10mL)和硼氢化锂的四氢呋喃溶液(2.0M，1.35mL)，搅拌1小时。加入氯化铵溶液淬灭，过滤，滤液用乙酸乙酯(25mL×3)萃取。有机相合并后经无水硫酸钠干燥，过滤，减压浓缩。残余物用硅胶柱层析纯化(甲醇/二氯甲烷＝1/10)得到目标产物14b(80mg，固体)，产率：8.7％。
MS m/z(ESI):398[M+1]
第三步
(R)-N-((3S,4S)-8-(4-氨基-5-碘-1-甲基-6-羰基-1,6-二氢嘧啶-2-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-基)-2-甲基丙烷-2-亚磺酰胺(14c)
将14b(80mg，0.2mmol)溶于乙腈(10mL)，加入NIS(54.2mg，0.24mmol)，室温搅拌1.5小时。减压脱溶，残余物用硅胶柱层析纯化(甲醇/二氯甲烷＝1/12)得到目标产物14c(100mg，固体)，产率：95％。
MS m/z(ESI):524[M+1]
第四步
(R)-N-((3S,4S)-8-(4-氨基-5-((4-氯-2-甲基-2H-吲唑-5-基)硫代)-1-甲基-6-羰基-1,6-二氢嘧啶-2-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-基)-2-甲基丙烷-2-亚磺酰胺(14d)
将14c(50mg，0.095mmol)溶于二氧六环(6mL)，加入7e(22.8mg，0.114mmol)、DIPEA(36.7mg，0.285mmol)、Pd 2(dba) 3(8.6mg，0.0095mmol)和XantPhos(8.2mg，0.014mmol)。反应混合物在氮气保护下加热至80℃，搅拌16小时。降至室温，加入乙酸乙酯(100mL)稀释，用饱和食盐水洗涤。有机相经无水硫酸钠干燥，减压脱溶，残余物用硅胶柱层析纯化(甲醇/二氯甲烷＝1/14)得到目标产物14d(46mg，固体)，产率：82％。
MS m/z(ESI):594[M+1]
第五步
6-氨基-2-((3S,4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-5-((4-氯-2-甲基-2H-吲唑-5-基)硫代)-3-甲基嘧啶-4(3H)-酮甲酸盐(14)
将14d(46mg，0.077mmol)溶于甲醇(5mL)，加入氯化氢的二氧六环溶液(4.0M，5mL)，室温搅拌2小时。减压浓缩，残余物用反相高效液相制备色谱纯化，得到目标产物14(6.5mg，固体)，产率：17％。
MS m/z(ESI):490[M+1]
1H NMR(400MHz,DMSO-d 6)δ8.33(s,1H),8.20(s,1H),7.46(d,J＝9.0Hz,1H),6.76(d,J＝9.0Hz,1H),4.15(s,3H),4.10–4.06(m,1H),3.69(d,J＝8.5Hz,1H),3.52(d,J＝8.5Hz,1H),3.39–3.37(m,2H),3.29(s, 3H),3.11–2.97(m,3H),1.85–1.71(m,2H),1.63–1.54(m,2H),1.11(d,J＝6.4Hz,3H).
实施例15
6-氨基-2-((3S,4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-5-((4-氯吡唑并[1,5-a]吡啶-5-基)硫代)-3-甲基嘧啶-4(3H)-酮甲酸盐
Figure PCTCN2021071375-appb-000029
第一步
(R)-N-((3S,4S)-8-(4-氨基-5-((4-氯吡唑并[1,5-a]吡啶-5-基)硫代)-1-甲基-6-羰基-1,6-二氢嘧啶-2-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-基)-2-甲基丙烷-2-亚磺酰胺(15a)
将14c(40mg，0.076mmol)溶于二氧六环(4mL)，加入8h(31.3mg，0.152mmol)、DIPEA(29.4mg，0.228mmol)、Pd 2(dba) 3(6.9mg，0.0076mmol)和XantPhos(6.5mg，0.0114mmol)。反应混合物在氮气保护下加热至80℃，搅拌16小时。降至室温，加入乙酸乙酯(100mL)稀释，用饱和食盐水洗涤。有机相经无水硫酸钠干燥，减压脱溶，残余物用硅胶柱层析纯化(甲醇/二氯甲烷＝1/17)得到目标产物15a(35mg，固体)，产率：79％。
MS m/z(ESI):580[M+1]
第二步
6-氨基-2-((3S,4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-5-((4-氯吡唑并[1,5-a]吡啶-5-基)硫代)-3-甲基嘧啶-4(3H)-酮甲酸盐(15)
将15a(35mg，0.06mmol)溶于甲醇(5mL)，加入氯化氢的二氧六环溶液(4.0M，5mL)，室温搅拌2小时。减压浓缩，残余物用反相高效液相制备色谱纯化，得到目标产物15(4.2mg，固体)，产率：15％。
MS m/z(ESI):476[M+1]
1H NMR(400MHz,CD 3OD)δ8.52(s,1H),8.28(d,J＝8.0Hz,1H),7.93(d,J＝2.3Hz,1H),6.55(d,J＝1.7Hz,1H),6.45(d,J＝7.3Hz,1H),4.28–4.23(m,1H),3.89(d,J＝8.9Hz,1H),3.77(d,J＝8.9Hz,1H),3.63–3.56(m,2H),3.43(s,3H),3.24–3.02(m,3H),1.97–1.90(m,2H),1.79–1.69(m,2H),1.26(d,J＝6.5Hz,3H).
实施例16
2-(4-(氨基甲基)-4-甲基哌啶-1-基)-5-((4-氯-2-甲基-2H-吲唑-5-基)硫代)-3-甲基嘧啶-4(3H)-酮甲酸盐
Figure PCTCN2021071375-appb-000030
第一步
((1-苄基-4-甲基哌啶-4-基)甲基)氨基甲酸叔丁酯(16b)
反应混合物1-苄基-4-甲基哌啶-4-甲腈16a(2g，9.3mmol)、二碳酸二叔丁酯(6.1g，27.9mmol)、氯化镍六水合物(2.2g，9.3mmol)和甲醇(50mL)在室温下搅拌15分钟。冷却至0℃，加入硼氢化钠(1.77g，46.5mmol)，升至室温，搅拌8小时。减压脱溶，残余物悬浊于二氯甲烷中，过滤。滤液减压脱溶，残余物用硅胶柱层析纯化(乙酸乙酯/石油醚＝1/1)得到目标产物16b(1.2g，油状物)，产率：40％。
MS m/z(ESI):319[M+1]
第二步
((4-甲基哌啶-4-基)甲基)氨基甲酸叔丁酯(16c)
将16b(1.2g，3.77mmol)溶于甲醇(20mL)，加入钯碳(10％，含水量55％，1.2g)，氢气氛围下室温搅拌3小时。过滤，滤饼用甲醇洗涤，滤液浓缩，得到目标产物16c(810mg，油状物)，产率：94％。
MS m/z(ESI):229[M+1]
第三步
((1-(5-碘-1-甲基-6-羰基-1,6-二氢嘧啶-2-基)-4-甲基哌啶-4-基)甲基)氨基甲酸叔丁酯(16d)
将1c(150mg，0.55mmol)加入16c(150mg，0.66mmol)、碳酸钾(228mg，1.65mmol)和乙腈(10mL)的混合物中，加热至90℃，搅拌16小时。减压浓缩，残余物用硅胶柱层析纯化(甲醇/二氯甲烷＝1/9)得到目标产物16d(150mg，固体)，产率：58％。
MS m/z(ESI):463[M+1]
第四步
((1-(5-((4-氯-2-甲基-2H-吲唑-5-基)硫代)-1-甲基-6-羰基-1,6-二氢嘧啶-2-基)-4-甲基哌啶-4-基)甲基)氨基甲酸叔丁酯(16e)
将反应混合物16d(150mg，0.32mmol)、7e(86mg，0.39mmol)、DIPEA(124mg，0.96mmol)、Pd 2(dba) 3(29mg，0.032mmol)、XantPhos(18.5mg，0.032mmol)和二氧六环(4mL)在氮气保护下加热至90℃， 搅拌3小时。减压脱溶，残余物用硅胶柱层析纯化(甲醇/二氯甲烷＝1/9)得到目标产物16e(130mg，固体)，产率：75％。
MS m/z(ESI):533[M+1]
第五步
2-(4-(氨基甲基)-4-甲基哌啶-1-基)-5-((4-氯-2-甲基-2H-吲唑-5-基)硫代)-3-甲基嘧啶-4(3H)-酮甲酸盐(16)
将16e(130mg，0.24mmol)溶于二氯甲烷(5mL)，加入三氟醋酸(1mL)，室温搅拌1小时。减压浓缩，残余物用反相高效液相制备色谱纯化，得到目标产物16(32.4mg，固体)，产率：31％。
MS m/z(ESI):433[M+1]
1H NMR(400MHz,DMSO-d 6)δ8.43(s,1H),8.37(s,1H),7.91(s,1H),7.51(d,J＝9.0Hz,1H),6.99(d,J＝9.0Hz,1H),4.18(s,3H),3.40–3.36(m,5H),3.17(t,J＝10.2Hz,2H),2.65(s,2H),1.63–1.57(m,2H),1.42(d,J＝11.1Hz,2H),1.01(s,3H).
实施例17
2-((3S,4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-3-甲基-5-((1-甲基-1H-吡咯并[2,3-b]吡啶-4-基)硫代)嘧啶-4(3H)-酮甲酸盐
Figure PCTCN2021071375-appb-000031
第一步
(R)-2-甲基-N-((3S,4S)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-基)丙烷-2-亚磺酰胺(17b)
将(3S,4S)-4-(((R)-叔丁基亚硫酰基)氨基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-羧酸叔丁酯17a(300mg，0.8mmol)溶于二氯甲烷(5mL)，加入三氟醋酸(1mL)，室温搅拌1小时。减压浓缩，得到目标产物17b(350mg，粗品)。产物不经纯化直接用于下一步反应。
MS m/z(ESI):275[M+1]
第二步
(R)-N-((3S,4S)-8-(5-碘-1-甲基-6-羰基-1,6-二氢嘧啶-2-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-基)-2-甲基丙烷-2-亚磺酰胺(17c)
将反应混合物1c(216mg，0.8mmol)、17b(粗品，350mg，0.8mmol)、碳酸钾(562mg，4.05mmol)和乙腈(10mL)加热至80℃，搅拌16小时。减压浓缩，残余物用硅胶柱层析纯化(甲醇/二氯甲烷＝ 1/9)得到目标产物17c(360mg，固体)，产率：89％。
MS m/z(ESI):509[M+1]
第三步
(R)-2-甲基-N-((3S,4S)-3-甲基-8-(1-甲基-5-((1-甲基-1H-吡咯并[2,3-b]吡啶-4-基)硫代)-6-羰基-1,6-二氢嘧啶-2-基)-2-氧杂-8-氮杂螺[4.5]癸烷-4-基)丙烷-2-亚磺酰胺(17d)
将反应混合物17c(180mg，0.35mmol)、2c(70mg，0.42mmol)、DIPEA(135mg，1.05mmol)、Pd 2(dba) 3(32mg，0.035mmol)、XantPhos(20mg，0.035mmol)和二氧六环(5mL)在氮气保护下加热至90℃，搅拌2小时。降至室温，减压脱溶，残余物用硅胶柱层析纯化(甲醇/二氯甲烷＝1/9)得到目标产物17d(160mg，固体)，产率：83％。
MS m/z(ESI):545[M+1]
第四步
2-((3S,4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-3-甲基-5-((1-甲基-1H-吡咯并[2,3-b]吡啶-4-基)硫代)嘧啶-4(3H)-酮甲酸盐(17)
将17d(160mg，0.29mmol)溶于甲醇(4mL)，加入氯化氢的二氧六环溶液(4.0M，2mL)，室温搅拌2小时。减压浓缩，残余物用反相高效液相制备色谱纯化，得到目标产物17(36mg，固体)，产率：28％。
MS m/z(ESI):441[M+1]
1H NMR(400MHz,DMSO-d 6)δ8.22(s,1H),8.14(s,1H),8.03(d,J＝5.1Hz,1H),7.51(d,J＝3.5Hz,1H),6.58(d,J＝5.1Hz,1H),6.44(d,J＝3.5Hz,1H),4.12–4.04(m,1H),3.80(s,3H),3.70(d,J＝8.6Hz,1H),3.53–3.48(m,3H),3.40(s,3H),3.19–3.08(m,2H),3.02(d,J＝4.9Hz,1H),1.88–1.84(m,2H),1.64–1.54(m,2H),1.11(d,J＝6.4Hz,3H).
实施例18
5-((1H-吡咯并[2,3-b]吡啶-4-基)硫代)-2-((3S,4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-3-甲基嘧啶-4(3H)-酮甲酸盐
Figure PCTCN2021071375-appb-000032
第一步
1H-吡咯并[2,3-b]吡啶-4-硫醇(18a)
向1g(250mg，1mmol)的四氢呋喃(5mL)溶液中加入叔丁醇钾(224mg，2mmol)，室温下搅拌2小时。加水淬灭，用乙酸乙酯洗涤。水相用盐酸(6N)酸化至pH为4，用乙酸乙酯萃取。有机相用无水硫酸钠干燥，过滤除去干燥剂，减压脱溶，得到目标产物18a(100mg，固体)，产率：67％。
MS m/z(ESI):151[M+1]
第二步
(R)-N-((3S,4S)-8-(5-((1H-吡咯并[2,3-b]吡啶-4-基)硫代)-1-甲基-6-羰基-1,6-二氢嘧啶-2-基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-基)-2-甲基丙烷-2-亚磺酰胺(18b)
将反应混合物17c(180mg，0.35mmol)、18a(63mg，0.42mmol)、DIPEA(135mg，1.05mmol)、Pd 2(dba) 3(32mg，0.035mmol)、XantPhos(20mg，0.035mmol)和二氧六环(5mL)在氮气保护下加热至90℃，搅拌2小时。降至室温，减压脱溶，残余物用硅胶柱层析纯化(甲醇/二氯甲烷＝1/9)得到目标产物18b(150mg，固体)，产率：80％。
MS m/z(ESI):531[M+1]
第三步
5-((1H-吡咯并[2,3-b]吡啶-4-基)硫代)-2-((3S,4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-3-甲基嘧啶-4(3H)-酮甲酸盐(18)
将18b(150mg，0.28mmol)溶于甲醇(4mL)，加入氯化氢的二氧六环溶液(4.0M，2mL)，室温搅拌2小时。减压浓缩，残余物用反相高效液相制备色谱纯化，得到目标产物18(37.9mg，固体)，产率：31％。
MS m/z(ESI):427[M+1]
1H NMR(400MHz,DMSO-d 6)δ11.73(s,1H),8.25(s,1H),8.14(s,1H),7.99(d,J＝5.1Hz,1H),7.47–7.45(m,1H),6.54(d,J＝5.1Hz,1H),6.42(d,J＝2.3Hz,1H),4.15–4.05(m,1H),3.71(d,J＝8.5Hz,1H),3.52–3.48(m,3H),3.41(s,3H),3.24–3.05(m,3H),1.89–1.75(m,2H),1.65–1.55(m,2H),1.12(d,J＝6.2Hz,3H).
实施例19
2-((3S,4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-5-((4-氯-2-甲基-2H-吲唑-5-基)硫代)-3-(甲基-d3)嘧啶-4(3H)-酮
Figure PCTCN2021071375-appb-000033
Figure PCTCN2021071375-appb-000034
第一步
(3S,4S)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-4-胺(19b)
将(3S,4S)-4-(((R)-叔丁基亚硫酰基)氨基)-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-羧酸叔丁酯19a(95.0g，254mmol)和氯化氢的甲醇溶液(4M，1L)在室温下搅拌4小时。减压浓缩，残余物溶于甲醇(500mL)，缓慢加入石油醚和乙酸乙酯的混合溶剂(1/1，4L)，室温搅拌2小时。过滤，滤饼干燥后得到目标产物19b(64.1g，固体，双盐酸盐)，产率：>100％。
MS m/z(ESI):171[M+1]
1H NMR(400MHz,DMSO-d 6)δ9.12(brs,2H),8.33(s,3H),4.25–4.19(m,1H),3.82(d,J＝9.1Hz,1H),3.64(d,J＝9.1Hz,1H),3.47(brs,1H),3.27–3.24(m,1H),3.19–3.16(m,1H),2.94–2.85(m,2H),2.03–1.85(m,2H),1.79–1.67(m,2H),1.23(d,J＝6.5Hz,3H).
第二步
2-氯-5-碘-3-(甲基-d3)嘧啶-4(3H)-酮(19c)
向2-氯-5-碘嘧啶-4(3H)-酮1b(5.0g，19.5mmol)的四氢呋喃(100mL)溶液中加入DBU(3.85g，25.3mmol)，降至0℃，加入氘代碘甲烷(3.39g，23.4mmol)，加热至50℃，搅拌6小时。减压脱溶，残余物加入乙酸乙酯(3.5L)，用稀盐酸(0.5N，1L×)和食盐水(800mL×2)洗涤。有机相经无水硫酸钠干燥，过滤，减压浓缩。残余物用硅胶柱层析纯化(乙酸乙酯/石油醚＝1/19到1/4)得到目标产物19c(3.3g，固体)，产率：62％。
MS m/z(ESI):274[M+1]
第三步
2-((3S,4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-5-碘-3-(甲基-d3)嘧啶-4(3H)-酮(19d)
反应混合物19c(1.8g，6.5mmol)，19b(1.67g，6.9mmol，双盐酸盐)，碳酸铯(9.5g，29.3mmol)和乙腈(60mL)在氮气保护下室温搅拌3小时。减压浓缩，残余物用硅胶柱层析纯化(乙酸乙酯/石油醚＝9/1)得到目标产物19d(2.4g，固体)，产率：91％。
MS m/z(ESI):408[M+1]
第四步
2-((3S,4S)-4-氨基-3-甲基-2-氧杂-8-氮杂螺[4.5]癸烷-8-基)-5-((4-氯-2-甲基-2H-吲唑-5-基)硫代)-3-(甲基-d3)嘧啶-4(3H)-酮(19)
反应混合物7e(1.9g，9.0mmol)，19d(2.3g，5.65mmol)，碘化亚铜(430mg，2.26mmol)，1,10-菲罗啉(814mg，4.52mmol)，磷酸钾(2.3g，11.3mmol)和二氧六环(50mL)在氮气保护下加热至100℃，搅拌过夜。降至室温，减压脱溶。残余物用硅胶柱层析纯化(甲醇/二氯甲烷＝1/9)，得到的粗品产物(2.5g)加入乙腈(25mL)，90℃下搅拌3小时，然后室温下搅拌16小时。过滤，滤饼在40℃下真空干燥4小时，得到目标产物19(1.47g，固体)，产率：54％。
MS m/z(ESI):478[M+1]
1H NMR(400MHz,DMSO-d 6)δ8.42(s,1H),7.91(s,1H),7.50(dd,J＝9.0,0.9Hz,1H),6.98(d,J＝9.0Hz,1H),4.17(s,3H),4.05–4.02(m,1H),3.64(d,J＝8.5Hz,1H),3.47(d,J＝8.5Hz,1H),3.48–3.37(m,2H),3.15–3.05(m,2H),2.90(d,J＝5.1Hz,1H),1.86–1.80(m,1H),1.74–1.68(m,1H),1.59–1.50(m,2H),1.38(brs,2H),1.07(d,J＝6.4Hz,3H).
生物学实验
SHP2的活性抑制测试
通过使用替代底物DiFMUP以快速荧光测定方法评估本发明的化合物对SHP2活性的影响
实验方法概述如下：
重组人SHP2全长蛋白由清华大学蛋白纯化和鉴定平台表达纯化，双磷酸化多肽(H2N-LN(pY)IDLDLV-(dPEG8)LST(pY)ASINFQK-amide)由金斯瑞生物科技有限公司合成，替代底物DiFMUP购自Thermo Fisher Scientific(货号为D6567)，反应缓冲液包含以下组分：60mM HEPES(pH 7.2)、75mM NaCl、75mM KCl、1mM EDTA、0.05％TWEEN 20和5mM DTT。
将化合物在DMSO(Sigma，货号为D5879)中溶解稀释至100μM，然后用DMSO进行4倍的系列稀释至最低浓度为6.1nM，每个浓度点再使用反应缓冲液稀释25倍。
向384孔检测板(Corning，货号为3575)中添加10μL化合物溶液和10μL 0.25nM的SHP2蛋白溶液，混合均匀后室温孵育30分钟；加入10μL 0.5μM双磷酸化多肽(溶解于反应缓冲液)，混合均匀后室温孵育30-60分钟；随后加入10μL 60μM的DiFMUP溶液(溶解于反应缓冲液)，混合均匀后室温放置。30分钟后荧光信号在激发波长340nm/发射波长450nm下用酶标仪(EnSpire，Perkin Elmer)检测。荧光信号值与底物的脱磷酸化程度呈正相关，从而反映SHP2磷酸酶的催化活性。该实验中未加蛋白组作为100％抑制组，加蛋白但是未加化合物组 作为0％抑制组，使用XLfit软件绘制化合物抑制曲线并计算其抑制的IC 50，实验结果见表。
化合物编号 IC 50(nM)
1. 23
2. 20
3. 20
4. 126
5. 801
6. 877
7. 12
8. 8.6
9. 8.8
10. 36
11. 4.0
12. 3.1
13. 8.7
14. 2.5
15. 2.7
16. 56
17. 14
18. 5.5
19. 11
本发明的实施例化合物对SHP2的活性具有抑制效应，优选IC 50小于50nM。
NCI-H358细胞增殖抑制的测定
使用发光细胞活力测试实验评估本发明的化合物对NCI-H358人非小细胞肺癌细胞增殖的影响。
实验方法概述如下：
将化合物在DMSO(Sigma，货号为D5879)中溶解并稀释至5mM，然后用DMSO进行4倍的系列稀释至最低浓度为0.31μM，每个浓度点再用RPMI 1640培养基(Thermo Fisher Scientific，货号为11995073)稀释50倍。如果化合物IC 50值较低，可降低化合物的起始浓度。
NCI-H358(ATCC，货号为CRL-5807)细胞被培养在RPMI 1640完全培养基[RPMI 1640培养基中含有10％FBS(GBICO，货号为10099-141)和100units/mL青链霉素混合液(Thermo Fisher Scientific，货号为15140122)]中。将细胞(15000个细胞/mL)接种于96孔板的90μL完全培养基中，培养过夜后每孔加入10μL化合物溶液，于37℃、5％CO 2的培养箱中培养5天，然后按CellTilter-Glo(CTG)试剂盒(Promega， 货号为G7572)说明书操作后，将细胞培养板取出平衡至室温，加50μL CTG试剂充***解，室温放置10分钟后用酶标仪(EnVision，Perkin Elmer)读取冷光信号。实验中，加10μM阳性对照物RMC-4550组作为阴性对照(100％抑制)，加0.2％DMSO组作为阳性对照(0％抑制)，使用XLfit软件绘制化合物抑制曲线并计算其抑制的IC 50，实验结果下表。
化合物编号 IC 50(nM)
1 310
2 383
3 434
4 2359
7 575
8 309
9 1345
10 1763
11 165
12 264
14 108
15 342
17 540
18 335
19 222
本发明的实施例化合物对细胞增殖具有抑制效应，优选IC 50为小于1000nM。
hERG钾离子通道阻断的测定
通过测定对hERG钾离子通道阻断作用评估本发明的化合物对可能心律失常的影响。
实验方法概述如下：
细胞外液：140mM NaCl、3.5mM KCl、1mM MgCl 2、2mM CaCl 2、10mM D-glucose、10mM HEPES、1.25mM NaH 2PO 4、pH＝7.4。
电极内液：20mM KCl、115mM K-aspartate、1mM MgCl 2、5mM EGTA、10mM HEPES、2mM Na 2-ATP、pH＝7.2。
化合物溶液：将待测化合物在DMSO中溶解并配成10mΜ的储液，再用DMSO将其稀释为3mM，接着用细胞外液稀释成3μΜ的溶液，以备后续使用。
细胞培养：hERG钾通道稳定过表达的HEK293细胞系(Creacell，货号为A-0320)在含有10％胎牛血清(Gibco，货号为1428478)和0.8mg/mL G418(Amresco，货号为E859-5G)的DMEM培养基(Gibco， 货号为11995-065)中培养，培养温度为37℃，二氧化碳浓度为5％。除去旧培养基并用PBS(Gibco，货号为1009-141)洗一次，然后加入1mL TrypLE TM Express溶液(Gibco，货号为12604021)，37℃孵育30秒。当细胞从培养皿底脱离，加入5mL 37℃预热的完全培养基，转移细胞悬液至无菌的离心管中，于1000rpm离心5分钟后收集细胞。将细胞接种于6cm细胞培养皿，每个细胞培养皿接种细胞量为2.5*10 5(最终体积：5mL)。膜片钳检测实验之前将3*10 3个细胞铺到盖玻片上，在24孔板中培养(最终体积：500μL)，18个小时后进行检测。
全细胞膜片钳记录全细胞hERG钾电流的电压刺激方案：当形成全细胞封接后细胞膜电压钳制于-80mV。钳制电压由-80mV除极至-50mV并维持0.5秒(作为漏电流检测)，然后阶跃至30mV并维持2.5秒，再迅速恢复至-50mV并维持4秒以激发出hERG通道的尾电流(Peak tail current)，每隔10秒记录1次hERG钾电流。实验数据由EPC-10放大器(HEKA)进行采集并储存于PatchMaster(HEKA v2x73)软件中。
测定：用微电极拉制仪(Sutter Instruments)将毛细玻璃管(Sutter Instruments)拉制成记录电极。将载有细胞的盖玻片从放置于培养箱的24孔板中取出，然后置于倒置显微镜下。记录电极内灌注电极内液，然后操纵微电极操纵仪(Sutter Instruments)，将记录电极接触到细胞表面，给予负压抽吸，形成GΩ封接；接着进行快速电容补偿；然后继续给予负压抽吸，直至吸破细胞膜，形成全细胞记录模式。在全细胞记录模式下，进行慢速电容的补偿并记录膜电容及串联电阻，期间不给予漏电补偿。当全细胞记录的hERG尾电流稳定3-5分钟后，将8mL不含化合物的细胞外液(空白对照)和8mL 3μM待测化合物溶液利用重力灌流的方法，依次流经记录浴槽从而作用于细胞5分钟(或者至电流稳定)。每一个细胞在不含化合物的细胞外液中检测到的电流作为自己的对照组。独立重复检测2-3个细胞。所有电生理实验在室温下进行。
数据分析：首先将待测化合物作用后的电流和空白对照电流标准化
Figure PCTCN2021071375-appb-000035
然后计算与这个化合物浓度对应的抑制率
Figure PCTCN2021071375-appb-000036
Figure PCTCN2021071375-appb-000037
Figure PCTCN2021071375-appb-000038

Claims (9)

  1. 一种通式(I)所示的化合物：
    Figure PCTCN2021071375-appb-100001
    或其可药用的盐、前药、稳定同位素衍生物、异构体及其混合物形式，其中：
    环A为苯环或6元杂芳环，环B为与环A稠合的5元杂芳环，所述苯环和杂芳环任选被一个或多个选自D、卤素、氰基、氧代、C 1-6烷基、C 3-6环烷基、4-7元杂环基、5-6元杂芳基、-OR a、-NR aR b、-C(O)R a、-C(O)NR aR b、-S(O) 2R a、-S(O) 2NR aR b、-NR aS(O) 2R b或-P(O)(CH 3) 2的取代基所取代，其中所述烷基、环烷基、杂环基和杂芳基任选被一个或多个选自D、卤素、氰基、氧代、C 1-6烷基、C 3-6环烷基、4-7元杂环基、-OR a、-NR aR b、-C(O)R a或-C(O)NR aR b的取代基所取代；
    R 1为H、D、氰基、C 1-2烷基、环丙基、-OR a、-NR aR b或-C(O)NR aR b，所述烷基和环丙基的一个或多个氢任选被一个或多个D或氟所取代；
    R 2为H、C 1-2烷基或环丙基，所述烷基和环丙基的一个或多个氢任选被一个或多个选自D、氟或羟基所取代；
    R 4a和R 4b各自独立地选自H、D、卤素、氰基、-OR a、-NR aR b、-C(O)R a、-C(O)NR aR b、C 1-6烷基、C 3-6环烷基、4-7元杂环基或5-6元杂芳基，但R 4a和R 4b不能同时为氰基、-OR a或-NR aR b，所述烷基、环烷基、杂环基和杂芳基任选被一个或多个选自D、卤素、氰基、氧代、C 1-6烷基、C 3-6环烷基、4-7元杂环基、5-6元杂芳基、-OR a、-NR aR b、-C(O)R a或-C(O)NR aR b的取代基所取代；R 4a和R 4b任选与其所联接的碳原子共同组成一个C 3-7碳环或4-8元杂环；
    R 5a、R 5b、R 6a和R 6b各自独立地选自H、D、氟或甲基；
    R a和R b各自独立地选自H、C 1-6烷基、C 3-6环烷基或4-7元杂环基，所述烷基、环烷基和杂环基任选被一个或多个选自D、氟、氰基、氧代、羟基、-OCH 3或-NH 2的取代基所取代。
  2. 根据权利要求1所述的化合物或其可药用的盐、前药、稳定同位素衍生物、异构体及其混合物形式，其为以下通式(II)的化合物：
    Figure PCTCN2021071375-appb-100002
    其中：
    环A为苯环或6元杂芳环，环B为与环A稠合的5元杂芳环，所述苯环和杂芳环任选被一个或多个选自D、卤素、氰基、氧代、C 1-6烷基、C 3-6环烷基、4-7元杂环基、5-6元杂芳基、-OR a、-NR aR b、-C(O)R a、-C(O)NR aR b、-S(O) 2R a、-S(O) 2NR aR b或-NR aS(O) 2R b的取代基所取代，其中所述烷基、环烷基、杂环基和杂芳基任选被一个或多个选自D、卤素、氰基、氧代、C 1-6烷基、C 3-6环烷基、4-7元杂环基、-OR a、-NR aR b、-C(O)R a或-C(O)NR aR b的取代基所取代；
    R 1为H、D、氰基、C 1-2烷基、-OR a或-NR aR b，所述烷基的一个或多个氢任选被一个或多个D或氟所取代；
    R 2为H或C 1-2烷基，所述烷基的一个或多个氢任选被一个或多个D或氟所取代；
    R 4a和R 4b各自独立地选自H、D、卤素、氰基、-OR a、-NR aR b、-C(O)NR aR b、C 1-6烷基、C 3-6环烷基、4-7元杂环基或5-6元杂芳基，但R 4a和R 4b不能同时为氰基、-OR a或-NR aR b，所述烷基、环烷基、杂环基和杂芳基任选被一个或多个选自D、卤素、氰基、氧代、C 1-6烷基、C 3-6环烷基、4-7元杂环基、5-6元杂芳基、-OR a、-NR aR b、-C(O)R a或-C(O)NR aR b的取代基所取代；R 4a和R 4b任选与其所联接的碳原子共同组成一个C 3-7碳环或4-7元杂环；
    R a和R b各自独立地选自H、C 1-6烷基、C 3-6环烷基或4-7元杂环基，所述烷基、环烷基和杂环基任选被一个或多个选自D、氟、氰基、氧代、羟基、-OCH 3或-NH 2的取代基所取代。
  3. 根据权利要求1或2所述的化合物或其可药用的盐、前药、稳定同位素衍生物、异构体及其混合物形式，其为以下通式(III)的化合物：
    Figure PCTCN2021071375-appb-100003
    其中：
    环A为苯环或6元杂芳环，环B为与环A稠合的5元杂芳环，所 述苯环和杂芳环任选被一个或多个选自D、卤素、氰基、氧代、C 1-6烷基、C 3-6环烷基、4-7元杂环基、5-6元杂芳基、-OR a、-NR aR b、-C(O)R a或-C(O)NR aR b的取代基所取代，其中所述烷基、环烷基、杂环基和杂芳基任选被一个或多个选自D、卤素、氰基、氧代、C 1-2烷基、-OR a或-NR aR b的取代基所取代；
    X为-O-或-CR 8aR 8b-；
    R 1为H、D、氰基、C 1-2烷基或-NR aR b，所述烷基的一个或多个氢任选被一个或多个D或氟所取代；
    R 2为H或C 1-2烷基，所述烷基的一个或多个氢任选被一个或多个D或氟所取代；
    R 7a、R 7b、R 8a、R 8b、R 9a和R 9b各自独立地选自H、D、卤素、氰基、C 1-2烷基或-OR c，所述烷基的一个或多个氢任选被一个或多个选自D、氟或羟基所取代，但R 7a和R 7a、R 8a和R 8b、R 9a和R 9b任选不能同时为-OR c；
    R a和R b各自独立地选自H、C 1-6烷基、C 3-6环烷基或4-7元杂环基，所述烷基、环烷基和杂环基任选被一个或多个选自D、氟、氰基、氧代、羟基、-OCH 3或-NH 2的取代基所取代；
    R c为H或C 1-2烷基，所述烷基的一个或多个氢任选被一个或多个D或氟所取代。
  4. 根据前述权利要求任一项所述的化合物或其可药用的盐、前药、稳定同位素衍生物、异构体及其混合物形式，其为以下通式(IV)的化合物：
    Figure PCTCN2021071375-appb-100004
    其中：
    环A为苯环或6元杂芳环，环B为与环A稠合的5元杂芳环，所述苯环和杂芳环任选被一个或多个选自D、卤素、氰基、氧代、C 1-2烷基、-OR a或-NR aR b的取代基所取代，其中所述烷基的一个或多个氢任选被一个或多个D或氟所取代；
    X为-O-或-CH 2-；
    R 1为H、D、C 1-2烷基或-NR aR b，所述烷基的一个或多个氢任选被一个或多个D或氟所取代；
    R 2为H或C 1-2烷基，所述烷基的一个或多个氢任选被一个或多个D或氟所取代；
    R 7为H、D或C 1-2烷基，所述烷基的一个或多个氢任选被一个或多个D或氟所取代；
    R a和R b各自独立地为H或C 1-2烷基，所述烷基的一个或多个氢任选被一个或多个D或氟所取代。
  5. 根据前述权利要求任一项所述的化合物或其可药用的盐、前药、稳定同位素衍生物、异构体及其混合物形式，所述化合物选自：
    Figure PCTCN2021071375-appb-100005
    Figure PCTCN2021071375-appb-100006
  6. 一种药物组合物，其包含权利要求1-5任一项所述的化合物或其可药用的盐、前药、稳定同位素衍生物、异构体及其混合物形式和一种或多种药学上可接受的载体和赋形剂。
  7. 一种药物组合物，所述药物组合物包含根据权利要求1-5任一项所述的化合物或其可药用的盐、前药、稳定同位素衍生物、异构体及其混合物形式和至少一种额外的药物，其中所述至少一种额外的药物包括但不限于化疗药物、靶向药物、DNA合成抑制剂、抗体药物、抗体药物偶联物、抗肿瘤药物、免疫抑制剂等。
  8. 根据权利要求1-5任一项所述的化合物或其可药用的盐、前药、稳定同位素衍生物、异构体及其混合物形式或根据权利要求6-7所述的药物组合物在制备用于治疗和/或预防与SHP2活性异常相关疾病的药物中的用途，所述疾病包括但不限于癌症，比如白血病、努南综合症、豹斑综合征、神经母细胞瘤、黑色素瘤、肺癌、乳腺癌、食道癌、结肠癌、头颈癌、胃癌等。
  9. 一种治疗和/或预防SHP2相关性疾病的方法，其包含治疗有效量的根据权利要求1-7任一项所述的化合物或其前药、稳定同位素衍生物、可药用的盐、异构体及其混合物形式，或包含所述化合物的药物组合物。
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