WO2024083086A1 - Bicyclic derivative integrin inhibitor - Google Patents

Bicyclic derivative integrin inhibitor Download PDF

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Publication number
WO2024083086A1
WO2024083086A1 PCT/CN2023/124799 CN2023124799W WO2024083086A1 WO 2024083086 A1 WO2024083086 A1 WO 2024083086A1 CN 2023124799 W CN2023124799 W CN 2023124799W WO 2024083086 A1 WO2024083086 A1 WO 2024083086A1
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substituted
unsubstituted
ring
membered
heteroalkane
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PCT/CN2023/124799
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French (fr)
Chinese (zh)
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郝宇
黄逸安
裴成奎
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上海如凌生物医药有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/438—The ring being spiro-condensed with carbocyclic or heterocyclic ring systems
    • A—HUMAN NECESSITIES
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • 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
    • A—HUMAN NECESSITIES
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/538—1,4-Oxazines, e.g. morpholine ortho- or peri-condensed with carbocyclic ring systems
    • A—HUMAN NECESSITIES
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    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
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    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
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    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • 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
    • C07D471/10—Spiro-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D498/04—Ortho-condensed systems
    • 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 the field of medicinal chemistry, and in particular to a class of bicyclic derivatives as inhibitors of ⁇ v ⁇ 1, ⁇ v ⁇ 6 and ⁇ v ⁇ 8 integrins, pharmaceutical compositions comprising such compounds, and their use in the treatment and/or prevention of conditions requiring inhibitors of ⁇ v ⁇ 1, ⁇ v ⁇ 6 and ⁇ v ⁇ 8 integrins.
  • Integrins also known as integrins and junction proteins, are a large family of cell surface receptors. They are heterodimeric transmembrane glycoproteins formed by the combination of ⁇ subunits and ⁇ subunits. In mammals, 18 ⁇ and 8 ⁇ subunits have been found. These subunits are non-covalently bound to form at least 24 different integrins, which are differentially expressed by various cells and recognize a variety of ligands, mediating information transmission between cells and the extracellular matrix and between adjacent cells. Integrins are a type of cell adhesion molecules, consisting of three parts: the extracellular region, the transmembrane region, and the intracellular region.
  • the extracellular N-terminal domain binds to specific ligands, and the intracellular region is connected to the cytoskeleton protein through ⁇ -actinin, talin, and focal adhesion proteins to form a ligand-integrin-cytoskeleton transmembrane system.
  • integrin can transmit signals bidirectionally through the cell membrane. One is outside-in signal transmission. Integrins bind to ligands in the ECM, causing conformational changes in integrins, which aggregate on the cell membrane to form focal adhesions, activate multiple intracellular signaling pathways, and transduce signals from the extracellular to the intracellular, thereby regulating cell adhesion, diffusion, migration, proliferation, differentiation, and remodeling. The other is inside-out signaling.
  • Intracellular signals induce conformational changes in talin, causing talin to bind to the intracellular domain of the integrin ⁇ subunit. This binding unties the link between the ⁇ subunit tail and the ⁇ subunit tail in the cytoplasm, and the conformation of the integrin head changes, increasing its affinity for binding to extracellular ligands and enhancing the cell's adhesion ability.
  • integrins can be divided into three types: laminin-binding integrins, collagen-binding integrins, and arginine-glycine-aspartic acid sequence (Arg-Gly-Asp, RGD) integrins.
  • integrin ⁇ v ⁇ 1 is expressed on activated fibroblasts and mesangial cells; integrin ⁇ v ⁇ 6 can bind to the RGD sequence of TGF- ⁇ 1, achieve cell-cell contact and activate TGF- ⁇ 1; ⁇ v ⁇ 8 can bind to the RGD sequence in TGF- ⁇ 3LAP, present the LAP complex to matrix metalloproteinases on the cell surface, and activate TGF- ⁇ 1.
  • av ⁇ 6 and av ⁇ 1 play a major role in renal and pulmonary tissue fibrosis, while av ⁇ 1 plays a dominant role in liver fibrosis.
  • Integrin av ⁇ 1 is a low-affinity fibronectin receptor that is highly expressed in basal epithelial cells and has the function of promoting the migration of keratinocytes on the underlying fibronectin EDA. Blocking the interaction between integrin av ⁇ 1 and TGF- ⁇ 1 helps inhibit TGF- ⁇ 1 activity and block the process of fibrosis. It has been reported that the expression of integrin ⁇ v ⁇ 6 is very low in normal lung tissue, but when inflammation and fibrosis occur in lung injury, av ⁇ 6 is rapidly expressed (Hatley et al, Angewandte Chemie International Edition, 2018, 57(13): 3298.).
  • integrin av ⁇ 6 In patients with primary biliary cirrhosis (PBC), alcoholic fatty liver disease, hepatitis B, hepatitis C and other diseases, the mRNA expression of integrin av ⁇ 6 is increased. In chronic inflammatory and fibrotic diseases related to kidney disease, the expression of integrin av ⁇ 6 is significantly increased compared with normal kidney tissue. In addition, integrin av ⁇ 6 is significantly highly expressed in biopsy samples of patients with diabetes, Goodpasture's syndrome, Alport syndrome, lupus nephritis, etc. (Koivisto et al, The international journal of biochemistry & cell biology, 2018, 99: 186). Integrin ⁇ v ⁇ 8 is extremely important for the regulation of TGF ⁇ .
  • L-TGF ⁇ Its only binding ligand is L-TGF ⁇ , which is highly expressed in a variety of cancer cells, especially tumor cells with high Tregs. L-TGF ⁇ is mainly expressed in immune cells such as T cells.
  • ⁇ v ⁇ 8 promotes the differentiation of Tregs in the tumor microenvironment, thereby promoting tumor growth. The mechanism of action is that after T cell GARP/L-TGFbeta binds to ⁇ v ⁇ 8, Conformational changes occur, releasing activated TGF ⁇ , which binds to TGF ⁇ R on T cells and promotes the differentiation of T cells into Tregs.
  • conditional deletion of lung fibroblasts ⁇ v ⁇ 8 can inhibit mouse airway fibrosis.
  • Tissue fibrosis can occur in many organs and is a common fibrotic disease, including idiopathic pulmonary fibrosis (IPF), non-alcoholic fatty liver disease (NASH), cirrhosis, renal fibrosis, scleroderma, myocardial fibrosis, etc. Tissue damage and inflammation are important causes of fibrosis. Due to inflammation, organ parenchymal cells undergo necrosis, local immune cells are activated, and a variety of blood cells enter the site of injury. The activated immune cells produce a large number of highly biologically active cytokines and chemokines, leading to local activation of mesenchymal cells.
  • ECM extracellular matrix
  • cytokines cytokines
  • chemokines chemokines
  • angiogenic factors cytokines
  • angiogenic factors cytokines, chemokines, and angiogenic factors.
  • the abnormal increase and excessive deposition of extracellular matrix lead to pathological changes and tissue fibrosis (Ricard-Blumet al, Matrix Biology, 2018, 68: 122.).
  • the main feature of fibrosis is the formation and deposition of excessive fibrous connective tissue. Chronic fibrotic damage can destroy tissue structure, impair organ function, and eventually lead to organ failure.
  • the integrin family has attracted much attention as a key regulatory factor in chronic inflammation, fibrosis, and tumor immunity. There is an urgent need to develop a class of integrin inhibitors with novel structures that are safe and effective.
  • the purpose of the present invention is to provide a class of integrin inhibitors with novel structures of bicyclic derivatives.
  • Such compounds contain a bicyclic structure, have good inhibitory activity and certain drug selectivity against integrins ⁇ v ⁇ 1, ⁇ v ⁇ 6 and ⁇ v ⁇ 8, excellent oral pharmacokinetic properties, and can effectively inhibit the increased expression of fibronectin and collagen in in vitro and in vivo pharmacodynamic models of fibrotic diseases.
  • Y1 is C1 ⁇ C6 alkylene, -O-, -(C1 ⁇ C6 alkylene)-O-, -NH-, -(C1 ⁇ C6 alkylene)-NH-;
  • Y2 is C1 ⁇ C6 alkylene, -O-, -(C1 ⁇ C6 alkylene)-O-, -C(O)-(C1 ⁇ C6 alkylene)-NH-, -NH-, -NH-(C1 ⁇ C6 alkylene)-, -(C1 ⁇ C6 alkylene)-NH-;
  • R1 is a substituted or unsubstituted 6-10 membered aromatic ring, a substituted or unsubstituted 5-8 membered heteroaromatic ring, a substituted or unsubstituted wherein the C ring and the D ring are each independently a substituted or unsubstituted C6-C10 aromatic ring, a substituted or unsubstituted 5-8 membered heteroaromatic ring, a substituted or unsubstituted 5-8 membered cycloalkane ring, or a substituted or unsubstituted 5-8 membered heteroalkane ring;
  • R2 is a hydrogen atom, a substituted or unsubstituted C6-C10 aromatic ring, a substituted or unsubstituted 5-8 membered heteroaromatic ring, a substituted or unsubstituted C8-C16 condensed ring, or -L1-L2;
  • -L1- is selected from none, -(substituted or unsubstituted C1-C6 alkylene)-, -(substituted or unsubstituted C1-C6 alkyleneoxy) -(substituted or unsubstituted C1-C6 alkylenethio)-, -(substituted or unsubstituted C3-C8 cycloalkyl)-, -(substituted or unsubstituted C3-C8 heterocycloalkyl)-, -(substituted or unsubstituted C6-C10 aryl)-, -(substituted or unsubstituted C5-C8 heteroaryl)-,
  • L2 is selected from none, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C1-C6 alkylthio, substituted or unsubstituted C3-C8 cycloalkyl, -(C1-C3 alkyl)-substituted or unsubstituted C3-C8 cycloalkyl, -(C1-C3 alkoxy)-substituted or unsubstituted C3-C8 cycloalkyl, -O-substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C3-C8 heteroalkylcyclyl, -(C1-C3 alkyl)-substituted or unsubstituted C3-C8 heteroalkylcyclyl, -(C1-C3
  • X is an oxygen atom or a nitrogen atom
  • R 3a when X is an oxygen atom, R 3a is a hydrogen atom, a C1-C6 alkyl group, a substituted or unsubstituted C6-C10 aromatic ring, and R 3b is absent; when X is a nitrogen atom, R 3a is a hydrogen atom, a hydroxyl group, a C1-C6 alkyl group, a substituted or unsubstituted C6-C10 aromatic ring, and R 3b is a hydrogen atom;
  • A is a ring selected from the group consisting of a substituted or unsubstituted four-membered cycloalkane ring, a substituted or unsubstituted five-membered cycloalkane ring, a substituted or unsubstituted six-membered cycloalkane ring, a substituted or unsubstituted seven-membered cycloalkane ring, a substituted or unsubstituted eight-membered cycloalkane ring, a substituted or unsubstituted four-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring, a substituted or unsubstituted six-membered heteroalkane ring, a substituted or unsubstituted seven-membered heteroalkane ring, a substituted or unsubstituted eight-membered heteroalkane ring,
  • B is a ring selected from the group consisting of a substituted or unsubstituted four-membered cycloalkane ring, a substituted or unsubstituted five-membered cycloalkane ring, a substituted or unsubstituted six-membered cycloalkane ring, a substituted or unsubstituted seven-membered cycloalkane ring, a substituted or unsubstituted eight-membered cycloalkane ring, a substituted or unsubstituted four-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring, a substituted or unsubstituted six-membered heteroalkane ring, a substituted or unsubstituted seven-membered heteroalkane ring, a substituted or unsubstituted eight-membered heteroalkane ring,
  • A is a ring selected from the group consisting of a substituted or unsubstituted four-membered cycloalkane ring, a substituted or unsubstituted five-membered cycloalkane ring, a substituted or unsubstituted six-membered cycloalkane ring, a substituted or unsubstituted seven-membered cycloalkane ring, a substituted or unsubstituted eight-membered cycloalkane ring, a substituted or unsubstituted four-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring, a substituted or unsubstituted six-membered heteroalkane ring, a substituted or unsubstituted a substituted seven-membered heteroalkane ring, a substituted or unsubstituted eight-membered heteroalkane
  • B is a ring selected from the group consisting of a substituted or unsubstituted four-membered cycloalkane ring, a substituted or unsubstituted five-membered cycloalkane ring, a substituted or unsubstituted six-membered cycloalkane ring, a substituted or unsubstituted seven-membered cycloalkane ring, a substituted or unsubstituted eight-membered cycloalkane ring, a substituted or unsubstituted C6-C10 aromatic ring, a substituted or unsubstituted four-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring, a substituted or unsubstituted six-membered heteroalkane ring, a substituted or unsubstituted seven-membered heteroalkane ring, a substituted
  • n 0, 1, 2 or 3;
  • heteroaromatic ring, heteroalkyl ring or heteroaryl group each independently has 1-3 (preferably 1, 2 or 3) heteroatoms selected from N, O and S.
  • Y1 is none.
  • Y1 is C1 ⁇ C3 alkylene, -(C1 ⁇ C3 alkylene)-NH-.
  • Y 1 is C1-C3 alkylene.
  • Y1 is a methylene group.
  • Y2 is C1-C3 alkylene, -O-, -(C1-C3 alkylene)-O-, -C(O)-(C1-C3 alkylene)-NH-, -NH-, -NH-(C1-C3 alkylene)-, -(C1-C3 alkylene)-NH-.
  • Y2 is C1-C3 alkylene, -C(O)-(C1-C3 alkylene)-NH-, -(C1-C3 alkylene)-NH-.
  • Y2 is -O-, -(C1-C3 alkylene)-O-.
  • Y2 is C1-C3 alkylene.
  • Y2 is methylene, ethylene or -O-.
  • R 1 is substituted or unsubstituted
  • the C ring is a C6-C10 aromatic ring or a 5-8 membered heteroaromatic ring
  • the D ring is a substituted or unsubstituted 5-8 membered heteroaromatic ring or a substituted or unsubstituted 5-8 membered heteroalkyl ring.
  • R 1 is substituted or unsubstituted
  • the C ring is a C6 aromatic ring or a 5-6-membered nitrogen-containing heteroaromatic ring
  • the D ring is a substituted or unsubstituted 5-6-membered nitrogen-containing heteroaromatic ring or a substituted or unsubstituted 5-6-membered nitrogen-containing heteroalkane ring.
  • the nitrogen-containing heteroaromatic ring or nitrogen-containing heteroalkyl ring of the C ring or the D ring optionally further includes 1 or 2 O atoms.
  • R 1 is substituted or unsubstituted
  • the D ring is a substituted or unsubstituted 5-6 membered nitrogen-containing heteroaromatic ring or a substituted or unsubstituted 5-6 membered nitrogen-containing heteroalkane ring.
  • R 1 is a substituted or unsubstituted 5-8 membered nitrogen-containing heteroaromatic ring.
  • R 1 is a substituted or unsubstituted group selected from the following group:
  • R 1 is a substituted or unsubstituted pyridine ring, a substituted or unsubstituted 1,2,3,4-tetrahydronaphthyridine ring, a substituted or unsubstituted quinoline ring, or a substituted or unsubstituted azaindole ring.
  • R 1 is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • R 2 is a C6-C10 aromatic ring (preferably a C6 aromatic ring) substituted by R c , wherein R c is hydrogen, halogen, C1-C6 alkyl, C3-C8 cycloalkyl, or C1-C6 alkoxy.
  • the C8-C16 fused ring refers to a ring with 8 to 16 carbon atoms in which two or three rings are fused, and each ring in the fused ring is independently a 6-membered aromatic ring, a 5-6-membered heteroaromatic ring, a 5-6-membered cycloalkane ring, or a 5-6-membered heteroalkane ring.
  • R 2 is a C9-C10 fused ring.
  • the C9-C10 fused ring is preferably a 6-membered aromatic ring fused with a 5-6-membered heteroaromatic ring, more preferably a 6-membered aromatic ring fused with a 6-membered heteroaromatic ring.
  • R2 is a hydrogen atom or -L1-L2; wherein -L1- is -(substituted or unsubstituted C6-C10 aryl)-, and L2 is a substituted or unsubstituted 5-8 membered heteroaryl.
  • R2 is -L1-L2.
  • -L1- is selected from -(substituted or unsubstituted C3-C8 cycloalkyl)-, -(substituted or unsubstituted C3-C8 heterocycloalkyl)-, -(substituted or unsubstituted C6-C10 aryl)-, and -(substituted or unsubstituted C5-C8 heteroaryl)-.
  • -L1- is selected from -(substituted or unsubstituted C6-C10 aryl)-, -(substituted or unsubstituted 5-8 membered heteroaryl)-.
  • -L1- is selected from -(substituted or unsubstituted C6 aryl)-, -(substituted or unsubstituted 6-membered heteroaryl)-.
  • L2 is none, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C1-C6 alkylthio, substituted or unsubstituted C3-C8 cycloalkyl, -(C1-C3 alkylene)-substituted or unsubstituted C3-C8 cycloalkyl, -(C1-C3 alkyleneoxy)-substituted or unsubstituted C3-C8 cycloalkyl, -O-substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted 3-8 membered heteroalkyl ring group, -(C1-C3 alkylene)-substituted or unsubstituted 3-8 membered heteroalkyl ring group, -(C1-C3 al
  • L2 is selected from substituted or unsubstituted 3-8 membered cycloalkyl, -(C1-C3 alkylene)-substituted or unsubstituted C3-C8 cycloalkyl, -(C1-C3 alkyleneoxy)-substituted or unsubstituted 3-8 membered cycloalkyl, -O-substituted or unsubstituted 3-8 membered cycloalkyl, substituted or unsubstituted 3-8 membered heteroalkyl ring group, -(C1-C3 alkylene)-substituted or unsubstituted 3-8 membered heteroalkyl ring group, -(C1-C3 alkyleneoxy)-substituted or unsubstituted 3-8 membered heteroalkyl ring group, -O-substituted or unsubstituted 3-8 membered hetero
  • L2 is selected from a substituted or unsubstituted 3-8 membered heteroalkyl ring group, and a substituted or unsubstituted 5-8 membered heteroaryl group.
  • -L1- is -(substituted or unsubstituted C6-C10 aryl)-
  • L2 is a substituted or unsubstituted 5-8 membered heteroaryl.
  • -L1- is -(substituted or unsubstituted phenyl)-
  • L2 is a substituted or unsubstituted 5-6-membered nitrogen-containing heteroaryl group (preferably pyrazolyl).
  • L1 is optionally substituted by one or more Ra , wherein Ra is halogen, C1-C6 alkyl, or C3-C6 cycloalkyl.
  • L1 is optionally substituted by one or more Ra , wherein Ra is halogen, C1-C6 alkyl, etc.
  • L2 is optionally substituted by one or more R b , wherein R b is C1-C3 alkyl.
  • R 2 is a structure of the following formula:
  • Z 1 , Z 2 , Z 3 , and Z 4 are each independently CR c or N;
  • L2 is none, C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C1-C6 alkylthio, substituted or unsubstituted 3-8 membered cycloalkyl, substituted or unsubstituted 3-8 membered heteroalkyl ring group, substituted or unsubstituted 5-8 membered heteroaryl;
  • Each R c is independently hydrogen, halogen, C1-C6 alkyl, 3-8 membered cycloalkyl, or C1-C6 alkoxy.
  • R 2 is a structure of the following formula b:
  • Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , Z 6 , Z 7 , Z 8 , and Z 9 are each independently CR c or N;
  • Each R c is independently hydrogen, halogen, C1-C6 alkyl, C3-C8 cycloalkyl, or C1-C6 alkoxy.
  • Z 1 , Z 2 , and Z 4 are each independently CR c , and Z 3 is CR c or N.
  • Z 1 , Z 2 , and Z 4 are each independently CH, and Z 3 is CR c or N.
  • Z 5 , Z 6 , and Z 7 are each independently CR c or N, and at least one of Z 5 , Z 6 , and Z 7 is N.
  • R 2 is the following structure:
  • L2 is C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C1-C6 alkylthio;
  • Each R c is independently hydrogen, halogen, C1-C6 alkyl, C3-C8 cycloalkyl, or C1-C6 alkoxy.
  • each R c is independently fluorine, bromine, chlorine, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, methoxy, or ethoxy.
  • R2 is substituted or unsubstituted
  • R2 is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • R 3a is a hydrogen atom or a C1-C3 alkyl group, and R 3b does not exist.
  • R 3a is a hydrogen atom
  • R 3b does not exist.
  • R 3a is a hydrogen atom, a hydroxyl group or a C1-C3 alkyl group, and R 3b is a hydrogen atom.
  • R 3a is a hydroxyl group
  • R 3b is a hydrogen atom
  • A is a ring selected from the group consisting of a substituted or unsubstituted four-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring, a substituted or unsubstituted six-membered heteroalkane ring, a substituted or unsubstituted seven-membered heteroalkane ring, and a substituted or unsubstituted eight-membered heteroalkane ring;
  • B is a ring selected from the group consisting of a substituted or unsubstituted four-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring, a substituted or unsubstituted six-membered heteroalkane ring, a substituted or unsubstituted seven-membered heteroalkane ring, and a substituted or unsubstituted eight-membered heteroalkane ring.
  • a and B are each independently a ring selected from the following group: a four-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring, and a substituted or unsubstituted six-membered heteroalkane ring.
  • the halogen is F, Cl, or Br, preferably F.
  • the heteroalkyl rings in the A ring and the B ring each independently contain 1-3 (preferably 1, 2, 3) nitrogen atoms.
  • the A ring and the B ring are each independently a ring selected from the following group: azetidine, substituted or unsubstituted pyrrolidine, substituted or unsubstituted piperidine ring, substituted or unsubstituted morpholine ring.
  • the substitution is preferably at the 3-position.
  • the substitution is preferably at the ortho position of the spiro atom.
  • the A ring and the B ring are each independently the following structure:
  • Y 1 is connected to the A ring through the nitrogen atom in the heteroalkyl ring.
  • the B ring is connected to Y 2 via a nitrogen atom in the heteroalkyl ring.
  • the compound is of the following formula II:
  • the number of ring atoms in the A ring is equal to or different from the number of ring atoms in the B ring.
  • the number of ring atoms in the A ring is less than, equal to or greater than the number of ring atoms in the B ring.
  • Y1 is connected to the A ring through the nitrogen atom in the heteroalkyl ring.
  • Y1 is -O-, -(C1-C6 alkylene)-O-, -NH- or -(C1-C6 alkylene)-NH-.
  • Y2 is connected to the B ring through the nitrogen atom in the heteroalkyl ring.
  • Y2 is -O-, -(C1-C6 alkylene)-O-, -NH- or -(C1-C6 alkylene)-NH-.
  • A is a 4-, 5-, 6-, 7- or 8-membered ring
  • B is a 4-, 5-, 6-, 7- or 8-membered ring.
  • the compound is of the following formula III-1:
  • the compound is of the following formula III-2:
  • the compound is of the following formula III-3:
  • the compound is of the following formula III-4:
  • the compound is of the following formula III-5:
  • the compound is of the following formula III-6:
  • R 1 , R 2 , Ring A and Ring B are as described above, and Y 1 and Y 2 are C1-C3 alkylene groups.
  • Y 1 , Y 2 , R 1 , R 2 , X, A, and B are each independently a corresponding group in Compound 1-122 prepared in Examples.
  • A is independently selected from the following rings: substituted or unsubstituted four-membered heteroalkane ring, substituted or unsubstituted five-membered heteroalkane ring, substituted or unsubstituted six-membered heteroalkane ring, substituted or unsubstituted seven-membered heteroalkane ring, substituted or unsubstituted five-membered heteroalkane ring and 6-10-membered aromatic ring, substituted or unsubstituted six-membered heteroalkane ring and 6-10-membered aromatic ring;
  • B is independently selected from the following rings: a substituted or unsubstituted four-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring, a substituted or unsubstituted six-membered heteroalkane ring, a substituted or unsubstituted seven-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring and a 6-10-membered aromatic ring, and a substituted or unsubstituted six-membered heteroalkane ring and a 6-10-membered aromatic ring.
  • A is independently selected from the following rings: substituted or unsubstituted four-membered heteroalkane ring, substituted or unsubstituted five-membered heteroalkane ring, a substituted or unsubstituted six-membered heteroalkane ring, a substituted or unsubstituted seven-membered heteroalkane ring;
  • B is a ring independently selected from the group consisting of a substituted or unsubstituted C6-C10 aromatic ring, a substituted or unsubstituted four-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring, and a substituted or unsubstituted six-membered heteroalkane ring.
  • the "substituted" means that 1 to 4 (preferably 1, 2, 3 or 4) hydrogen atoms on the group are independently replaced by substituents selected from the following group: halogen, -OH, -CN, -NO 2 , -NH 2 , -NCO, -OCN, -SCN, -NCS, -N 3 , -(C1-C3 alkylene)OH, -O(C1-C3 alkyl), -S(C1-C3 alkyl), -C(O)X'R 4 or -X'C(O)R 5 , -SO 3 R 4 , -OSO 2 OR 4 , -NR 6 SO 2 OR 5 , -NR 6 R 7 , -SO 2 NR 6 R 7 , -NH-SO 2 -R 6 , -N + R 6 R 7 R 8 , -C(O)N(R 9 ) 2 , -SO 2 R 10 , -NR 6 SO 2 OR
  • X' is a chemical bond or is oxygen or sulfur
  • R4 is independently selected from hydrogen, C1-C6 alkyl, 3-12-membered alkyl ring group, 5-10-membered aryl group, 3-12-membered heterocyclic group
  • R5 is independently selected from hydrogen, C1-C6 alkyl, 3-12-membered alkyl ring group, 5-10-membered aryl group, 3-12-membered heterocyclic group;
  • R 6 , R 7 and R 8 are each independently selected from hydrogen, C1-C6 alkyl, -C(O)X'R 4 or -X'C(O)R 5 , or, R 6 and R 7 together with the nitrogen atom to which they are attached form a 4-8 membered heterocyclic ring in the ring structure; wherein, R 6 or R 7 are not all -C(O)X'R 4 or -X'C(O)R 5 ;
  • R 9 is independently selected from hydrogen or C1-C6 alkyl, or two R 9 together with the N atom to which they are attached form a 4-8 membered heterocyclic ring;
  • R 10 is independently selected from hydrogen, C1-C6 alkyl, 3-12 membered alkyl ring group, 3-12 membered heterocyclic group, 5-10 membered aryl group;
  • R 11 is independently selected from hydrogen, C1-C6 alkyl, or two R 11 together with the N atom to which they are attached form a 4-8 membered heterocyclic ring;
  • p is an integer of 0-3 for each independently.
  • the bicyclic derivative is selected from the following group:
  • the bicyclic derivative as described in the first aspect of the present invention there is provided a method for preparing the bicyclic derivative as described in the first aspect of the present invention, and its racemate, stereoisomer, tautomer, isotope-labeled substance, nitrogen oxide, solvate, polymorph, metabolite, ester, prodrug or pharmaceutically acceptable salt thereof,
  • the method comprises one of the following methods (1) to (5):
  • a pharmaceutical composition comprising:
  • a bicyclic derivative of formula (I) as described in the first aspect of the present invention and its racemate, stereoisomer, tautomer, isotope-labeled substance, nitrogen oxide, solvate, polymorph, metabolite, ester, prodrug or pharmaceutically acceptable salt thereof, for use in the preparation of a pharmaceutical composition for treating or preventing a disease, disorder or condition associated with the activity or expression of ⁇ v ⁇ 1, ⁇ v ⁇ 6 and ⁇ v ⁇ 8 integrins.
  • the disease, disorder or condition associated with the activity or expression of ⁇ v ⁇ 1, ⁇ v ⁇ 6 and ⁇ v ⁇ 8 integrins is selected from the group consisting of autoimmune diseases, fibrotic diseases, inflammatory bowel disease (IBD), relapsing multiple sclerosis (RMS), progressive multifocal leukoencephalopathy (PML), ulcerative colitis (UC), Crohn's disease (CD), chronic viral hepatitis B and C, non-alcoholic fatty liver disease (NAFLD), age-related macular degeneration, diabetic retinopathy, retinal vascular disease, osteoporosis and cell proliferative diseases.
  • IBD inflammatory bowel disease
  • RMS relapsing multiple sclerosis
  • PML progressive multifocal leukoencephalopathy
  • UC ulcerative colitis
  • CD Crohn's disease
  • NAFLD non-alcoholic fatty liver disease
  • age-related macular degeneration diabetic retinopathy
  • retinal vascular disease retinal vascular disease
  • the fibrotic disease is selected from the following group: pulmonary fibrosis, idiopathic pulmonary fibrosis, nonspecific interstitial pneumonia (NSIP), conventional interstitial lung disease (UIP), radiation-induced pulmonary fibrosis, familial pulmonary fibrosis, airway pulmonary fibrosis, chronic group A lung disease (COPD), interstitial lung disease, liver fibrosis, chronic kidney disease, renal fibrosis, skin fibrosis, systemic sclerosis, or a combination thereof.
  • pulmonary fibrosis idiopathic pulmonary fibrosis
  • NIP nonspecific interstitial pneumonia
  • UIP conventional interstitial lung disease
  • COPD chronic group A lung disease
  • interstitial lung disease liver fibrosis, chronic kidney disease, renal fibrosis, skin fibrosis, systemic sclerosis, or a combination thereof.
  • the fibrotic disease is pulmonary fibrosis (such as IPF), liver fibrosis, skin fibrosis, scleroderma, cardiac fibrosis, renal fibrosis, intestinal fibrosis, primary sclerosing cholangitis (PSC) or biliary fibrosis (such as PBC).
  • the fibrotic disease is pulmonary fibrosis (IPF).
  • IPF pulmonary fibrosis
  • the fibrotic disease is primary sclerosing cholangitis or biliary fibrosis (such as PBC).
  • the fibrotic disease is scleroderma.
  • the fibrotic disease is psoriasis.
  • the cell proliferative disease is cancer, selected from the following group: breast cancer, cervical cancer, endometrial cancer, ovarian cancer, colon cancer, rectal cancer, pancreatic cancer, liver cancer, lung cancer, non-small cell lung cancer, brain metastasis of lung cancer, oral squamous cell carcinoma, head cancer, neck cancer, head and neck squamous cell carcinoma, oral or nasal mucosal cancer, laryngeal cancer, kidney cancer, renal cell carcinoma, ovarian cancer, spleen cancer, small intestine cancer, large intestine cancer, gastric cancer, esophageal cancer, esophageal cancer, lung squamous cell carcinoma, bile duct cancer, gallbladder cancer, endometrial cancer, melanoma, urothelial carcinoma, urogenital tract cancer, genital cancer, prostate cancer, testicular cancer, bladder cancer, blood cancer, skin cancer, bone marrow cancer, brain cancer, central nervous system cancer, muscle tissue cancer, thyroid cancer,
  • a method for treating a disease, disorder or condition associated with the activity or expression of av ⁇ 1, ⁇ v ⁇ 6 and ⁇ v ⁇ 8 integrins comprising the steps of administering an effective amount of a bicyclic derivative of formula (I) as described in the first aspect of the present invention, and its racemate, stereoisomer, tautomer, isotope label, nitrogen oxide, solvate, polymorph, metabolite, ester, prodrug or a pharmaceutically acceptable salt thereof to a patient in need thereof.
  • a bicyclic derivative is provided, and its racemate, stereoisomer, tautomer, isotope label, nitrogen oxide, solvate, polymorph, metabolite, ester, prodrug or pharmaceutically acceptable salt, wherein the bicyclic derivative has the structure shown in the following formula II-a, II-b, II-c:
  • Each Y 1 is independently C1-C6 alkylene, -O-, -(C1-C6 alkylene)-O-, -NH-, -(C1-C6 alkylene)-NH-;
  • Each Y2 is independently C1-C6 alkylene, -O-, -(C1-C6 alkylene)-O-, -C(O)-(C1-C6 alkylene)- NH-, -NH-, -NH-(C1-C6 alkylene)-, -(C1-C6 alkylene)-NH-;
  • Each R 1 is independently a substituted or unsubstituted 6-10 membered aromatic ring, a substituted or unsubstituted 5-8 membered heteroaromatic ring, a substituted or unsubstituted wherein the C ring and the D ring are each independently a substituted or unsubstituted C6-C10 aromatic ring, a substituted or unsubstituted 5-8 membered heteroaromatic ring, a substituted or unsubstituted 5-8 membered cycloalkane ring, or a substituted or unsubstituted 5-8 membered heteroalkane ring;
  • Each R 2 is independently a hydrogen atom, a substituted or unsubstituted C6-C10 aromatic ring, a substituted or unsubstituted 5-8 membered heteroaromatic ring, a substituted or unsubstituted C8-C16 condensed ring, or -L1-L2;
  • -L1- is selected from none, -(substituted or unsubstituted C1-C6 alkylene)-, -(substituted or unsubstituted C1-C6 alkyleneoxy)-, -(substituted or unsubstituted C1-C6 alkylenethio)-, -(substituted or unsubstituted C3-C8 cycloalkyl)-, -(substituted or unsubstituted C3-C8 heterocycloalkyl)-, -(substituted or unsubstituted C6-C10 aryl)-, -(substituted or unsubstituted C5-C8 heteroaryl)-,
  • L2 is selected from none, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C1-C6 alkylthio, substituted or unsubstituted C3-C8 cycloalkyl, -(C1-C3 alkyl)-substituted or unsubstituted C3-C8 cycloalkyl, -(C1-C3 alkoxy)-substituted or unsubstituted C3-C8 cycloalkyl, -O-substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C3-C8 heteroalkylcyclyl, -(C1-C3 alkyl)-substituted or unsubstituted C3-C8 heteroalkylcyclyl, -(C1-C3
  • Each R 3a ' is independently a C1-C6 alkyl group
  • each Each is independently a substituted or unsubstituted spiro ring or a substituted or unsubstituted cyclocyclic ring;
  • A is a ring selected from the group consisting of a substituted or unsubstituted four-membered cycloalkane ring, a substituted or unsubstituted five-membered cycloalkane ring, a substituted or unsubstituted six-membered cycloalkane ring, a substituted or unsubstituted seven-membered cycloalkane ring, a substituted or unsubstituted eight-membered cycloalkane ring, a substituted or unsubstituted four-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring, a substituted or unsubstituted six-membered heteroalkane ring, a substituted or unsubstituted seven-membered heteroalkane ring, a substituted or unsubstituted eight-membered heteroalkane ring,
  • B is a ring selected from the group consisting of a substituted or unsubstituted four-membered cycloalkane ring, a substituted or unsubstituted five-membered cycloalkane ring, a substituted or unsubstituted six-membered cycloalkane ring, a substituted or unsubstituted seven-membered cycloalkane ring, a substituted or unsubstituted eight-membered cycloalkane ring, a substituted or unsubstituted four-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring, a substituted or unsubstituted six-membered heteroalkane ring, a substituted or unsubstituted seven-membered heteroalkane ring, a substituted or unsubstituted eight-membered heteroalkane ring,
  • A is a ring selected from the group consisting of a substituted or unsubstituted four-membered cycloalkane ring, a substituted or unsubstituted five-membered cycloalkane ring, a substituted or unsubstituted six-membered cycloalkane ring, a substituted or unsubstituted seven-membered cycloalkane ring, a substituted or unsubstituted eight-membered cycloalkane ring, a substituted or unsubstituted four-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring, a substituted or unsubstituted six-membered heteroalkane ring, a substituted or unsubstituted seven-membered heteroalkane ring, a substituted or unsubstituted eight-membered heteroalkane ring,
  • B is a ring selected from the group consisting of a substituted or unsubstituted four-membered cycloalkane ring, a substituted or unsubstituted five-membered cycloalkane ring, a substituted or unsubstituted six-membered cycloalkane ring, a substituted or unsubstituted seven-membered cycloalkane ring, a substituted or unsubstituted eight-membered cycloalkane ring, a substituted or unsubstituted C6-C10 aromatic ring, a substituted or unsubstituted four-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring, a substituted or unsubstituted six-membered heteroalkane ring, a substituted or unsubstituted seven-membered heteroalkane ring, a substituted
  • n is independently 0, 1, 2 or 3;
  • heteroaromatic ring, heteroalkyl ring or heteroaryl group each independently has 1 to 3 (preferably 1, 2 or 3) heteroatoms selected from N, O and S;
  • Formula II-a, Formula II-b and Formula II-c are all chemically stable structures.
  • Y 1 , Y 2 , R 1 , R 2 , A, B and n are independently as described in the first aspect of the present invention.
  • the "substituted" means that 1 to 4 (preferably 1, 2, 3 or 4) hydrogen atoms on the group are independently replaced by a Boc substituent.
  • the bicyclic derivative is selected from the following group:
  • FIG. 1 shows the results of Western blotting (WB) experiments of the compounds, illustrating the degradation effects of Examples 1, 2, 7, 15, and 25 on fibrosis proteins ⁇ -SMA and Fibronectin at different concentrations. Pirfenidone and GSK-3008348 were selected as control compounds in this experiment.
  • Figure 2 shows the body weight curves of rats in the sham operation group, model group, 100 mg/kg nintedanib group, 50 mg/kg low-dose group of Example 71 and 250 mg/kg high-dose group of Example 71 (mean ⁇ SEM).
  • Figure 3 shows the body weight growth rate curves (mean ⁇ SEM) of rats in the sham operation group, model group, 100 mg/kg nintedanib group, 50 mg/kg low-dose group of Example 71 and 250 mg/kg high-dose group of Example 71.
  • Figure 4 shows the histological changes of bronchioles and pulmonary arterioles in the left lung pulmonary fibrosis lesions in the sham operation group (A), model group (B), 100 mg/kg nintedanib group (C), 50 mg/kg Example 71 low-dose group (D), and 250 mg/kg Example 71 high-dose group (E) ( ⁇ 200, H&E staining);
  • Figure A shows normal pulmonary bronchioles and accompanying pulmonary arterioles;
  • Figure B shows damage to bronchioles and pulmonary arterioles;
  • Figures C-E show that the degree of damage to bronchioles and pulmonary arterioles is reduced.
  • Figure a is a pulmonary arteriole;
  • b is a bronchiole.
  • the arrows in Figures B-E indicate inflammatory cell infiltration.
  • Figure 5 shows the injury and inflammation scores of the terminal bronchioles and the accompanying small pulmonary arteries in the left lung pulmonary fibrosis lesions in the sham operation group, the model group, the 100 mg/kg nintedanib group, the 50 mg/kg low-dose group of Example 71, and the 250 mg/kg high-dose group of Example 71.
  • *** is p ⁇ 0.001 (compared with the model group).
  • Figure 6 shows the histological changes of bronchioles and pulmonary arterioles at the edge of the left lung pulmonary fibrosis lesions in the sham operation group (A), model group (B), 100 mg/kg nintedanib group (C), 50 mg/kg Example 71 low-dose group (D), and 250 mg/kg Example 71 high-dose group (E) ( ⁇ 200, H&E staining).
  • Figure A normal pulmonary bronchioles and accompanying pulmonary arterioles can be seen
  • Figure B shows damage to bronchioles and pulmonary arterioles
  • Figures C-E show that the degree of damage to bronchioles and pulmonary arterioles is reduced.
  • a is a pulmonary arteriole
  • b is a bronchiole.
  • the arrows in Figures B-E indicate inflammatory cell infiltration.
  • Figure 7 shows the injury and inflammation scores of the terminal bronchioles and the accompanying small pulmonary arteries at the edge of the left lung pulmonary fibrosis lesions in the sham operation group, the model group, the 100 mg/kg nintedanib group, the 50 mg/kg low-dose group of Example 71, and the 250 mg/kg high-dose group of Example 71.
  • *** is p ⁇ 0.001
  • ** is p ⁇ 0.01 (compared with the model group).
  • Figure 8 shows the histological changes of alveolar tissue in the left lung pulmonary fibrosis lesions in the sham operation group (A), model group (B), 100 mg/kg nintedanib group (C), 50 mg/kg low-dose group of Example 71 (D), and 250 mg/kg high-dose group of Example 71 (E) ( ⁇ 200, H&E staining).
  • Figure A normal alveolar walls can be seen;
  • Figure B shows that the sheet-like alveolar structure in the fibrotic lesions is damaged and disappeared, filled with a large number of exuded inflammatory cells and proliferating connective tissue.
  • the alveolar structure in Figures C-E is abnormal, it is significantly better than that in the model group.
  • Group E that is, the high-dose group of Example 71, some alveoli are close to the normal alveolar wall structure.
  • the arrows in Figures B-E indicate that the alveolar structure in the fibrotic lesions is damaged and the remaining alveolar wall is thickened.
  • Figure 9 shows alveolar fibrosis lesions in the left lung pulmonary fibrosis lesions ( ⁇ 200, Masson staining) in the sham operation group (A), model group (B), 100 mg/kg nintedanib group (C), 50 mg/kg Example 71 low-dose group (D), and 250 mg/kg Example 71 high-dose group (E).
  • Normal physiological fibrous tissue without fibrous tissue deposition can be seen in Figure A; severe fibrotic lesions and obvious fibrous tissue deposition can be seen in alveolar tissue in Figure B.
  • Figures C-E also have fibrotic lesions, but are significantly better than the model group.
  • the arrows in Figures B-E indicate fibrous tissue deposition.
  • Figure 10 shows the left lung pulmonary fibrosis scores of the sham group, model group, 100 mg/kg nintedanib group, 50 mg/kg low-dose group of Example 71, and 250 mg/kg high-dose group of Example 71.
  • *** means p ⁇ 0.001
  • * means p ⁇ 0.05 (compared with the model group).
  • Figure 11 shows the proportion of different pathological scores of left lung pulmonary fibrosis in the sham operation group, model group, 100 mg/kg nintedanib group, 50 mg/kg low-dose group of Example 71, and 250 mg/kg high-dose group of Example 71.
  • *** indicates p ⁇ 0.001 (compared with the model group).
  • Figure 12 shows the degree of collagen deposition in the left lung fibrosis lesions in the sham operation group (A), model group (B), 100 mg/kg nintedanib group (C), 50 mg/kg low-dose group of Example 71 (D), and 250 mg/kg high-dose group of Example 71 (E) ( ⁇ 200, Masson staining).
  • Figure A normal physiological fibrous tissue exists, and the collagen area is ⁇ 5%;
  • Figure B shows extremely serious collagen deposition.
  • Figures C-E also show an increase in collagen deposition, but the degree is lower than that of the model group.
  • Group E that is, the high-dose group of Example 71, has the lightest collagen deposition, which is significantly lower than the model group and also lower than the nintedanib group.
  • Figure 13 shows the percentage of collagen deposition area in the sham operation group, model group, 100 mg/kg nintedanib group, 50 mg/kg low-dose group of Example 71, and 250 mg/kg high-dose group of Example 71.
  • *** means p ⁇ 0.001
  • * means p ⁇ 0.05 (compared with the model group).
  • the bicyclic derivatives have excellent activity and selectivity for integrins ⁇ v ⁇ 1, ⁇ v ⁇ 6 and ⁇ v ⁇ 8, and oral pharmacokinetic properties that are significantly better than the positive control PLN-74809, and show activity effects that are better than the positive references pirfenidone and nintedanib in in vitro and in vivo pharmacodynamic models of fibrotic diseases, so they can be used to prepare and prevent pharmaceutical compositions for fibrotic diseases. Based on the above findings, the inventors completed the present invention.
  • the terms “comprise”, “include”, and “contain” are used interchangeably and include not only closed definitions, but also semi-closed and open definitions. In other words, the terms include “consisting of”, “consisting essentially of”.
  • alkyl refers to a fully saturated cyclic or acyclic, branched or unbranched carbon chain moiety having the specified number of carbon atoms, or if not specified, up to 30 carbon atoms, such as 1 to 3, 1 to 6 carbon atoms.
  • alkyl having 1 to 8 carbon atoms refers to moieties such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl and octyl, and those moieties that are positional isomers of these moieties.
  • Alkyl having 10 to 30 carbon atoms includes decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, docosyl, docosyl, triacontyl and tetracosyl, and the alkyl group may be substituted or unsubstituted.
  • C1-C6 alkyl or “C1-C3 alkyl” refers to a straight or branched chain alkyl group having 1-6 or 1-3 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or the like.
  • alkylene refers to an alkyl group having a specified number of carbon atoms, such as 1 to 12 carbon atoms, which contains two points of connection to the rest of the compound on its longest carbon chain.
  • alkylene groups include C1-C6 alkylene groups, C1-C3 alkylene groups, specifically including methylene-(CH 2 )-, ethylene-(CH 2 CH 2 )-, n-propylene-(CH 2 CH 2 CH 2 )-, isopropylene-(CH 2 CH(CH 3 ))-, etc.
  • Alkylene groups can be cyclic or acyclic, branched or unbranched carbon chain moieties, and can be optionally substituted with one or more substituents.
  • C1-C6 alkoxy refers to a straight or branched alkoxy group having 1 to 6 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, hexyloxy, or the like.
  • C2-C4 ester group refers to a group having a C1-C3 alkyl-OC(O)- structure or a group having an -OC(O) -C1-C3 alkyl structure, wherein the alkyl group may be linear or branched, for example CH3COO-, C2H5COO- , C3H8COO- , ( CH3 ) 2CHCOO- , -COOCH3 , -COOC2H5 , -COOC3H8 , or the like.
  • C2-C4 amide group refers to a group having a C1-C3 alkyl-CO-NH-structure or a group having a A group of the structure NH-CO-C1-C3 alkyl, wherein the alkyl group may be linear or branched, for example CH3 -CO-NH-, C2H5 - CO- NH-, C3H8-CO-NH-, -COOCH3, -CO-NH-C2H5 , -CO - NH - C3H8 , or the like.
  • C2-C4 acyl refers to a group having a C1-C3 alkyl-CO- structure, wherein the alkyl group may be linear or branched, such as CH3 -CO-, C2H5 - CO-, C3H8 - CO- , or the like.
  • C1-C3 haloalkyl refers to a linear or branched alkyl group having 1 to 3 carbon atoms in which one or more hydrogen atoms are substituted by a halogen group, such as monochloromethyl, dichloroethyl, trichloropropyl, or the like.
  • C1-C4 carboxyl refers to a group of a C1-C3 alkyl-COOH structure, wherein the alkyl group may be linear or branched, such as CH3COOH , C2H5COOH , C3H8COOH , ( CH3 ) 2CHCOOH , or the like.
  • C6-C10 aromatic ring refers to a monocyclic or bicyclic aromatic hydrocarbon group having 6 to 10 carbon atoms in the ring component, such as a benzene ring, a naphthalene ring, or a similar group.
  • aryl includes 3 to 12 substituted or unsubstituted monocyclic aromatic groups, wherein each atom of the ring is carbon (i.e., carbocyclic aromatic groups) or wherein one or more atoms are heteroatoms (i.e., heteroaryl groups).
  • aryl groups include 5- to 12-membered rings, more preferably 6- to 10-membered rings.
  • aryl also includes polycyclic ring systems having two or more rings, wherein two or more carbons are shared by two adjacent rings, wherein at least one ring is aromatic, for example, the other rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl and/or heterocyclic radicals.
  • Carbocyclic aromatic groups include benzene, naphthalene, phenanthrene, phenol, aniline, etc.
  • heteroaryl refers to an optionally substituted aromatic group, for example, a 5- to 7-membered monocyclic ring system having a ring containing at least one heteroatom and at least one carbon atom, such as pyrrolyl, thienyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, furan, imidazole, thiazole, oxazole, triazole, or the like.
  • cycloalkane ring refers to a non-aromatic group (including saturated, partially saturated or unsaturated groups) having 3 to 12 carbon atoms (e.g., 3 to 8, 4, 5, or 6 carbon atoms), having a monocyclic or condensed ring (including a bridged ring system and a spirocyclic system). Therefore, the alkane ring or alkane ring group can be a saturated alkane ring or an unsaturated alkane ring. In the condensed ring system of a saturated alkane ring, one or more rings are saturated alkane rings. In the condensed ring system of an unsaturated alkane ring, one or more rings can be a saturated alkane ring or an unsaturated alkane ring.
  • the term "5-8 membered heteroaromatic ring” refers to an aromatic heterocyclic ring system having one to multiple (preferably 1, 2 or 3) heteroatoms selected from N, O and S, and having 5-8 ring atoms. It should be understood that when multiple heteroatoms are contained, the heteroatoms may be the same, partially the same, or completely different.
  • the heteroaromatic ring is preferably a heteroaromatic ring containing 1 to 2 nitrogen atoms.
  • examples of 5-membered heteroaromatic rings include (but are not limited to): pyrrole ring, furan ring, thiophene ring, imidazole ring, pyrazole ring, oxazole ring, thiazole ring
  • examples of 6-membered heteroaromatic rings include (but are not limited to) pyridine ring, pyrazine ring, pyridazine ring, pyrimidine ring, or similar groups.
  • the term "5-8 membered heteroaryl” refers to an aromatic group having one to more (preferably 1, 2 or 3) heteroatoms selected from N, O and S, and having 5 or 8 ring atoms. It should be understood that when containing multiple heteroatoms, the heteroatoms may be the same, partially the same, or completely different.
  • examples of 5-membered heteroaryl include (but are not limited to): pyrrolyl, furanyl, thienyl, imidazolyl, oxazolyl, thiazolyl, or similar groups.
  • heteroaryl refers to 3-12 membered aromatic groups, which contain one or more heteroatoms selected from nitrogen, oxygen and sulfur, including monocyclic or polycyclic systems, and the polycyclic system can be a fused ring, a bridged ring system and a spirocyclic system.
  • fused heteroaromatic ring refers to a fused ring formed by the fusion of two or more aromatic groups, which contains one or more heteroatoms selected from nitrogen, oxygen and sulfur. Among them, one or more rings in the fused heteroaromatic ring have heteroatoms.
  • Heteroaryl includes substituted or unsubstituted aromatic 3-12 membered ring structures, more preferably 5-12 membered rings, more preferably 5-10 membered rings, and its rings
  • the structure includes 1-4 heteroatoms.
  • Heteroaryl includes, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine.
  • Aryl and heteroaryl can be monocyclic, bicyclic or polycyclic. In this article, C5-C8 heteroaryl means that the number of ring atoms is 5-8.
  • heteroalkyl ring refers to a 3-12 membered non-aromatic group (including saturated, partially saturated or unsaturated groups) containing one or more heteroatoms selected from nitrogen, oxygen and sulfur, including a monocyclic or polycyclic system, and the polycyclic system can be a fused ring, a bridged ring system and a spirocyclic system. Therefore, the heteroalkyl ring or heteroalkyl ring group can be a saturated heteroalkyl ring or an unsaturated heteroalkyl ring. In a fused ring system, one or more rings can be an alkyl ring group, an aryl group or a heteroaryl group.
  • the number of ring atoms in the heteroalkyl ring can be 3 to 8, 4, 5 or 6.
  • the nitrogen atom and/or sulfur atom of the heterocyclic group is optionally oxidized to provide N-oxide, sulfinyl and sulfonyl moieties.
  • C3-C8 means that the number of ring atoms is 3-8.
  • the term "5-8 membered heteroalkyl ring” refers to any stable ring containing one or more (preferably 1, 2 or 3) heteroatoms selected from N, O and S, and a non-aromatic heterocyclic ring system with 5-7 ring atoms.
  • the heterocyclic ring may be a saturated, partially unsaturated, or unsaturated ring, but cannot be an aromatic ring.
  • the heteroalkyl ring is preferably a heteroalkyl ring containing 1 to 2 nitrogen atoms, which may optionally further include 1 or 2 oxygen atoms. It should be understood that when multiple heteroatoms are contained, the heteroatoms may be the same, partially the same, or completely different.
  • examples of 5-membered heteroalkyl rings include (but are not limited to) pyrrolidine rings, pyrroline rings, pyrazolidine rings, pyrazoline rings, 1,3-oxopentacyclic rings, and examples of 6-membered heteroalkyl rings include (but are not limited to) piperidine rings, morpholine rings, piperazine rings, 1,4-dioxane rings, or similar groups.
  • the term "four-membered heteroalkane ring” is any stable non-aromatic heterocyclic ring system containing one or more (preferably 1, 2 or 3) heteroatoms selected from N, O and S, and the number of ring atoms is 4.
  • the four-membered heteroalkane ring is preferably a four-membered heteroalkane ring containing 1 to 2 nitrogen atoms. It should be understood that when containing multiple heteroatoms, the heteroatoms can be the same, partially the same, or completely different. Examples of four-membered heteroalkane rings include (but are not limited to) oxetane, azetidine, or similar groups.
  • heterocyclic radical or “heterocyclic group” refers to a 3 to 12-membered ring structure, more preferably a 5 to 12-membered ring, more preferably a 5 to 10-membered ring, and its ring structure includes 1 to 4 heteroatoms.
  • the heterocycle can be a monocyclic, bicyclic, spirocyclic or polycyclic ring.
  • the heterocyclic radical includes, for example, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinoline, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenazine, phenothiazine, furan, phenoxazine, pyrrolidine, oxolane, thiopentane, oxazole, piperidine, piperazine, morpholine, lactone, lactams such as azetidinone and pyrrol
  • heterocycle may be substituted at one or more positions by substituents as described above, for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, amino, nitro, thiol, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, sulfamoyl, sulfinyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocyclic group, aromatic or heteroaromatic moiety, -CF3 , -CN, etc.
  • substituents for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, amino, nitro, thiol, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carb
  • halogen refers to, for example but not limited to, radioactive and non-radioactive forms of fluorine, chlorine, bromine, iodine, etc. In a preferred embodiment, the halogen is selected from fluorine, chlorine and bromine.
  • halo refers to halogen substituted
  • autoimmune disease refers to myasthenia gravis, polymyositis, autoimmune myocarditis, polymyalgia rheumatica, psoriatic arthritis, rheumatoid arthritis, Sjögren's syndrome, ankylosing spondylitis, relapsing polychondritis, inflammatory bowel disease (such as Crohn's disease, ulcerative colitis), celiac disease, autoimmune hepatitis, Primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), immune-mediated kidney disease, interstitial cystitis, Addison's disease, autoimmune thyroid disease (e.g., Hashimoto's thyroiditis, Graves' disease), diabetes mellitus, dermatomyositis, psoriasis, alopecia areata, autoimmune or immune-mediated skin diseases, bullous pemphigoid, erythe
  • compositions and methods of administration are provided.
  • the compounds of the present invention have inhibitory activity and selectivity against integrins ⁇ v ⁇ 1, ⁇ v ⁇ 6 and ⁇ v ⁇ 8, the compounds of the present invention and their various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and pharmaceutical compositions containing the compounds of the present invention as the main active ingredient can be used to treat, prevent and alleviate pulmonary fibrosis.
  • the pharmaceutical composition of the present invention comprises a safe and effective amount of the compound of the present invention or a pharmacologically acceptable salt thereof and a pharmacologically acceptable excipient or carrier.
  • safe and effective amount means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects.
  • the pharmaceutical composition contains 0.1-1000 mg of the compound of the present invention per dose, and more preferably, contains 0.5-500 mg of the compound of the present invention per dose.
  • the "one dose” is a capsule or tablet.
  • “Pharmaceutically acceptable carrier” refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility” here means that the components in the composition can be mixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds.
  • Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as Tween), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.
  • cellulose and its derivatives such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.
  • gelatin such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.
  • administrations include, but are not limited to, oral, rectal, parenteral (intravenous, intramuscular or subcutaneous), inhalation and topical administration.
  • parenteral intravenous, intramuscular or subcutaneous
  • inhalation topical administration.
  • a particularly preferred administration is oral.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
  • the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and acacia; (c) humectants, for example, glycerol; (d) disintegrators, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example,
  • Solid dosage forms such as tablets, dragees, capsules, pills and granules may be prepared with coatings and shells, such as enteric coatings and other materials known in the art. They may contain opacifying agents and the active compound or compounds in such compositions may be The release of the active compound can be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active compound can also be formed into microcapsules with one or more of the above-mentioned excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures.
  • the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.
  • an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottons
  • composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • Suspensions in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances, and the like.
  • suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances, and the like.
  • compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.
  • Dosage forms for topical administration of the compounds of the invention include ointments, powders, patches, sprays and inhalants.
  • the active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required.
  • the compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.
  • a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage during administration is a pharmaceutically effective dosage, and for a person weighing 60 kg, the daily dosage is usually 0.2 to 1000 mg, preferably 0.5 to 500 mg.
  • the specific dosage should also take into account factors such as the route of administration and the health status of the patient, which are all within the skill of a skilled physician.
  • Step 1 Dissolve 2-aminonicotinaldehyde (1 g, 8.2 mmol) and 1,1-dimethoxypropane-2-one (1.3 g, 10.6 mmol) in a 10/1 ethanol/water (22 ml) solution, slowly add sodium hydroxide solution (3 M, 3.6 ml) to the solution at 0°C under a nitrogen atmosphere, and stir the mixture at room temperature for 3 hours. Concentrate the reaction mixture, dissolve the residue in ethyl acetate (50 ml), and The organic phase was washed with saturated brine (25 ml*2), dried, filtered and concentrated to obtain 2 g of yellow solid 2-(dimethoxymethyl)-1,8-naphthyridine (A-1).
  • Step 2 A-1 (1.6 g, 7.8 mmol) was dissolved in ethanol (20 ml), platinum dioxide (50 mg, 0.2 mmol) was slowly added to the solution under nitrogen atmosphere, and the mixture was stirred at room temperature for 16 hours under hydrogen protection (15 psi). The reaction mixture was filtered, and the filtrate was concentrated to obtain 1 g of light yellow solid 7-(dimethoxymethyl)-1,2,3,4-tetrahydro-1,8-naphthyridine (A-2).
  • Step 3 A-2 (1.2 g, 5.8 mmol) was dissolved in water (10 ml), and 12 mmol/ml concentrated hydrochloric acid (1.1 ml, 13 mmol) was added to the solution at -5°C, and the mixture was stirred at 85°C for 2 hours. The mixture was cooled to room temperature, and the pH was adjusted to 11 by 3M sodium hydroxide solution. The aqueous phase was extracted with ethyl acetate (30 ml*2), and the organic phase was dried over Na 2 SO 4 , filtered and concentrated in vacuo to obtain a crude product.
  • Step 4 A-3 (3.1 g, 19.1 mmol) was dissolved in THF (60 ml), (Boc) 2 O (8.3 g, 38.2 mmol) and N, N-dimethylaminopyridine (4.7 g, 38.2 mmol) were added to the solution in sequence at room temperature, and the mixture was stirred at 80°C for 16 hours under nitrogen protection. The mixture was washed with saturated aqueous ammonium chloride solution (20 ml), extracted with ethyl acetate (20 ml*3), and the organic phase was dried over Na 2 SO 4 , filtered and concentrated in vacuo to obtain a crude product.
  • Step 5 A-4 (1 g, 3.8 mmol) was dissolved in THF (20 ml), sodium borohydride (173.1 mg, 4.6 mmol) was added at 0°C, and the mixture was stirred at 0°C for 1 hour under nitrogen protection. Water (5 ml) was added to the mixture to quench it, and ethyl acetate (10 ml*3) was extracted. The organic phase was washed with saturated brine, dried over Na 2 SO 4 , filtered and concentrated in vacuo to obtain a crude product.
  • Step 6 A-5 (600 mg, 2.3 mmol) was dissolved in DCM (10 ml), triphenylphosphine (893.1 mg, 3.4 mmol) and carbon tetrabromide (978.6 mg, 3 mmol) were added to the solution at room temperature, and the mixed liquid was stirred at room temperature for 3 hours under nitrogen protection.
  • Step 1 3-phenylacrylic acid (10 g, 67.5 mmol) was dissolved in thionyl chloride (50 ml, 689.3 mmol), the mixture was stirred at 90°C for 2 hours, and vacuum concentrated to obtain a residue. The residue was dissolved in DCM (200 ml), and sodium bicarbonate (10 g, 119. mmol) was added to the solution at room temperature to neutralize the excessive thionyl chloride. Then 4-dimethylaminopyridine (1 g, 8.2 mmol) and m-toluidine (7.2 g, 67.5 mmol) were added to the reaction solution in sequence and stirred for 14 hours.
  • the reaction solution was washed once with hydrochloric acid solution (5%, 150 ml) and sodium bicarbonate aqueous solution (150 ml), and the organic phase was dried over Na2SO4, filtered, and concentrated to obtain a crude product.
  • Step 2 Dissolve B-1 (15.7 g, 66 mmol) in chlorobenzene (200 ml), slowly add aluminum chloride (44 g, 330 mmol) under nitrogen atmosphere, and stir the reaction solution at 90°C for 2 hours under nitrogen protection. After cooling the reaction solution, pour it into ice water (300 ml), extract it with ethyl acetate (200 ml*2), wash the organic phase with saturated brine (100 ml), dry it with Na 2 SO 4 , filter it and concentrate it in vacuo to obtain a crude product.
  • Step 3 Dissolve B-2 (9.8 g, 61.4 mmol) in phosphorus oxychloride (130 ml, 1.4 mol), and stir the mixture at 100°C for 2 hours. Concentrate the mixture, slowly pour it into ice water (300 ml) and keep it in an ice bath, and add solid sodium hydroxide to adjust the pH to 7. The mixture is extracted with ethyl acetate (200 ml), and the organic phase is washed with saturated brine, dried over Na 2 SO 4 , and vacuum filtered and concentrated to obtain a crude product.
  • Step 5 Dissolve B-4 (570 mg, 1.3 mmol) in carbon tetrachloride (28 ml), add N-bromosuccinimide (258.3 mg, 1.5 mmol) and dibenzoyl peroxide (63.9 mg, 0.3 mmol) to the solution at room temperature, and stir the mixture at 80°C for 12 hours.
  • Step 1 Refer to the preparation method of intermediate A-5, filter and concentrate in vacuo to obtain 100 mg of white solid tert-butyl (6-(hydroxymethyl)pyridin-2-yl)carbamate (C-1).
  • Step 2 C-1 (100 mg, 445.9 ⁇ mol) and diisopropylethylamine (172.9 mg, 1.3 mmol) were dissolved in acetonitrile (4 ml), p-toluenesulfonyl chloride (47.2 mg, 668.9 ⁇ mol) was added at 0°C, and the mixture was stirred at room temperature for 3 hours under nitrogen protection.
  • Step 1 Dissolve 6-bromo-2-nitropyridine-3-ol (9 g, 41.1 mmol), ethyl bromoacetate (8.2 g, 49.3 mmol) and potassium carbonate (11.4 g, 82.2 mmol) in acetone (90 ml) in sequence and stir at 60°C for 2 hours under nitrogen protection. Wash the reaction solution with water (200 ml), extract with ethyl acetate (200 ml), wash the organic phase with saturated brine, dry over Na 2 SO 4 , filter and concentrate in vacuo to obtain 12 g of a brown oily crude product, ethyl 2-((6-bromo-2-nitropyridine-3-yl)oxy)acetate (D-1).
  • Step 2 Dissolve D-1 (12 g, 39.3 mmol), iron powder (11 g, 196.7 mmol) and ammonium chloride (10.5 g, 196.7 mmol) in 10/1 methanol/water (110 ml) in sequence, and stir at 60°C for 16 hours under nitrogen protection.
  • reaction solution was diluted with water (300 ml), extracted with ethyl acetate (300 ml), and the organic phase was washed with saturated brine, dried over Na 2 SO 4 , filtered and concentrated in vacuo to obtain a residue, which was dissolved in acetic acid (100 ml), stirred at 90°C for 3 hours, and then concentrated and filtered to obtain 7 g of a brown solid product 6-bromo-2H-pyrido[3,2-b][1,4]oxazine-3(4H)-one (D-2).
  • Step 3 Dissolve D-2 (1 g, 4.4 mmol) in THF (10 ml), add borane dimethyl sulfide (10 M, 1.1 ml) at room temperature, stir at 90°C for 1.5 hours, then add methanol (1 ml), stir at 90°C for 0.5 hours. Concentrate the reaction solution, wash with saturated sodium bicarbonate (50 ml), extract with ethyl acetate (50 ml), dry the organic phase over Na 2 SO 4 , filter and concentrate in vacuo to obtain 757 mg of 6-bromo-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazine (D-3) as a white solid.
  • Step 4 Dissolve D-3 (700 mg, 3.3 mmol), potassium vinyl trifluoroborate (654 mg, 4.9 mmol), palladium catalyst (119 mg, 0.2 mmol) and cesium carbonate (2.7 g, 8.1 mmol) in 1,4-dioxane (10 ml) in sequence and stir at 100 ° C for 2 hours under nitrogen protection.
  • Step 6 Dissolve D-5 (500 mg, 1.9 mmol) in 4/1 THF/H 2 O (12.5 ml), add sodium periodate (1.1 g, 5.3 mmol) and potassium osmate dihydrate (4.2 mg, 0.01 mmol) in turn at room temperature, and stir at room temperature for 1 hour.
  • the reaction solution was filtered, washed with water (30 ml), extracted with ethyl acetate (30 ml), and concentrated.
  • LC-MS ESI m/z: 289.2 [M+23] + .
  • Step 8 D-7 (50 mg, 0.2 mmol) was dissolved in DCM (3 ml), and carbon tetrabromide (93.4 mg, 0.3 mmol) and triphenylphosphine (74 mg, 0.3 mmol) were added successively at room temperature, and stirred at room temperature for 2 hours. The reaction solution was quenched by adding water (20 ml), extracted with DCM (20 ml), and the organic phase was dried, filtered, and concentrated.
  • Step 2 Refer to the preparation method of intermediate D-6 to obtain 900 mg of brown solid crude product 3-carbonyl-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazine-6-carbaldehyde (E-2).
  • Step 2 Referring to the preparation method of intermediate D-4, the crude product was purified by normal phase silica gel column chromatography (EA) to obtain 160 mg of white solid product 5-vinyl-3H-imidazo[4,5-b]pyridine (F-2).
  • EA normal phase silica gel column chromatography
  • LC-MS: ESI m/z 246.4[M+H] + .
  • Step 4 Referring to the preparation method of intermediate D-6, 120 mg of light yellow solid product tert-butyl 5-formyl-3H-imidazo[4,5-b]pyridine-3-carboxylate (F) was obtained.
  • Step 1 (3-Bromophenyl)hydrazine hydrochloride (4.4 g, 19.7 mmol), potassium acetate (2.5 g, 25.6 mmol) and acetylacetone (2.1 g, 20.7 mmol) were dissolved in ethanol solution (20 ml), stirred at 85 ° C for 3 hours under nitrogen protection. Water was added to quench, extracted with ethyl acetate, and the organic phase was washed with saturated brine, dried over Na 2 SO 4 , filtered and concentrated in vacuo to obtain 5 g of 1-(3-bromophenyl)-3,5-dimethyl-1H-pyrazole (G-1) as a yellow oily product.
  • G-1 1-(3-bromophenyl)-3,5-dimethyl-1H-pyrazole
  • Step 2 G-1 (4 g, 15.9 mmol) and triisopropyl borate (1.5 eq.) were dissolved in anhydrous THF (40 ml), and n-butyl lithium (2.5 M, 16 ml) was slowly added dropwise at -65°C under nitrogen protection, and the mixture was stirred at -65°C to -60°C for 2 hours. Dilute hydrochloric acid (3 M, 11 ml) was added dropwise to the solution at -65°C to quench the reaction, and the pH was adjusted to 7 with sodium hydroxide solution (1 M), and EA was extracted.
  • Step 1 Dissolve 3,5-dibromoaniline (5 g, 19.9 mmol) in 40 ml of concentrated hydrochloric acid, add sodium nitrite (2.1 g, 29.9 mmol) to the solution under nitrogen atmosphere at 0°C, stir at 0°C for 1 hour, and then add stannous chloride (7.6 g,
  • Step 3 H-2 (500 mg, 1.5 mmol), bis-naphthalene boronate (462 mg, 1.8 mmol), potassium acetate (446 mg, 4.6 mmol) and 1,1-bis(diphenylphosphino)ferrocenepalladium chloride (111 mg, 152 ⁇ mol) were dissolved in 5 ml of anhydrous Diox and stirred at 90°C for 2 hours under nitrogen atmosphere.
  • Step 4 H-3 (400 mg, 1.1 mmol) was dissolved in 4/1 THF/H 2 O (5 ml), sodium periodate (681 mg, 3.2 mmol) and 1M dilute hydrochloric acid (955 ⁇ l) were added to the reaction solution, and stirred at room temperature for 12 hours. The reaction solution was concentrated, and the crude product was separated by HPLC to obtain 70 mg of a white solid product (3-bromo-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)boronic acid (H). LC-MS: ESI m/z: 295.0 [M+H] + .
  • Step 3 Referring to the preparation method of intermediate H-1, the reaction solution was filtered and concentrated in vacuo to obtain 1 g of a yellow solid product (3-bromo-5-(tert-butyl)phenyl)hydrazine hydrochloride (I-3). LC-MS: ESI m/z: 243.1 [M+H] + .
  • Step 5 Referring to the preparation method of intermediate H-3, 500 mg of dark brown oily product 1-(3-(tert-butyl)-5-(4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-3,5-dimethyl-1H-pyrazole (I-5) was obtained.
  • Step 6 Referring to the preparation method of intermediate H, the crude product was separated by HPLC to obtain 55 mg of white solid (3-(tert-butyl)-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)boronic acid (I). LC-MS: ESI m/z: 273.2 [M+H] + .
  • Step 2 Dissolve J-1 (5 g, 34.7 mmol) in ethanol (50 ml) and add 10% palladium carbon (1.5 g), stir at room temperature under hydrogen atmosphere for 16 hours. Filter and concentrate the reaction solution in vacuo to obtain 5 g of 7-methyl-1,2,3,4-tetrahydro-1,8-naphthyridine (J-2) as a white solid product.
  • LC-MS: ESI m/z 249.4[M+H] + .
  • Step 4 J-3 (3.5 g, 14.1 mmol) and dimethyl carbonate (4.4 g, 49.3 mmol) were dissolved in THF (30 ml), and 2 M lithium diisopropylamine solution (10.7 ml) was added dropwise at -78 °C under nitrogen protection, and stirred for 1 hour.
  • LC-MS: ESI m/z 188.0 [M-Boc+1] + .
  • Step 2 L-1 (1.5 g, 6.3 mmol) and sodium hydride (506.1 mg, 12.7 mmol) were dissolved in THF (20 ml), stirred at 25°C for 0.5 hours, 2-(trimethylsilyl)ethoxymethyl chloride (1.6 g, 9.5 mmol) was slowly added dropwise to the solution at 0°C, and stirring was continued at 0°C for 2.5 hours. 15 ml of saturated NH 4 Cl solution was added at 0°C to quench the reaction, 20 ml of water was added to dilute, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over Na 2 SO 4 , filtered and concentrated in vacuo.
  • Step 3 Dissolve L-2 (1.2 g, 3.3 mmol), tetrahydroxydiborane (585.7 mg, 6.5 mmol), potassium acetate (961.8 mg, 9.8 mmol), 2-dicyclohexylphosphino-2'4'6-triisopropylbiphenyl (311.5 mg, 0.7 mmol) and methanesulfonic acid (2-dicyclohexylphosphino-2'4'6-triisopropyl-1,1'-biphenyl) (2'-amino-1,1'-biphenyl-2-yl) palladium (II) (276.5 mg, 0.3 mmol) in methanol (20 ml) and stir at 80°C for 3 hours under nitrogen protection. Concentrate the reaction solution and purify the crude product by HPLC to obtain 670 mg of a yellow solid product.
  • LC-MS: ESI m/z 334.6 [M+H] + .
  • Step 1 H-2 (1.2 g, 3.5 mmol), different cycloalkylboronic acids (1 equivalent), Pd(dppf)Cl 2 (0.1 equivalent), potassium carbonate (2 equivalents), dioxane/water solution (10:1, 7 ml), under nitrogen protection, heated at 93°C for 3 h; LCMS detected that the reaction was complete, quenched with water, and then ethyl acetate was added, and the yellow solid was removed by suction, extracted with ethyl acetate, and the organic phases were combined, dried over Na2SO4, filtered, and distilled under reduced pressure at 50°C to obtain a yellow oil. The crude product was purified by column chromatography (PE/EA ⁇ 20/1) to obtain the corresponding yellow oily product.
  • Step 1 Dissolve tert-butyl 2,6-diazaspiro[3.4]octane-2-carboxylate (0.5 g, 2.4 mmol) in N,N-dimethylformamide (5 ml), add diisopropylethylamine (1. ml, 5.9 mmol) and methyl (E)-4-bromobut-2-enoate (506 mg, 2.8 mmol) at room temperature and stir for 2 hours.
  • Step 2 Dissolve tert-butyl (E)-6-(4-methoxy-4-carbonylbut-2-ene-1-yl)-2,6-diazaspiro[3.4]octane-2-carboxylate (0.6 g, 1.8 mmol) in DCM (5 ml), add trifluoroacetic acid (2 ml), and stir at room temperature for 2 hours. The reaction solution is directly spin-dried to obtain 550 mg of methyl (E)-4-(2,6-diazaspiro[3.4]octane-6-yl)but-2-enoate as a yellow oily product.
  • Step 3 Dissolve methyl (E)-4-(2,6-diazaspiro[3.4]octan-6-yl)but-2-enoate (0.6 g, 1.7 mmol), intermediate A-3 (330 mg, 2 mmol) and diisopropylethylamine (657 mg, 5.1 mmol) in 1,2-dichloroethane (5 ml) in sequence. and stirred at room temperature for 30 minutes. Then sodium cyanoborohydride (160 mg, 2.5 mmol) was added and the reaction was continued for 1.5 hours. The reaction solution was washed with water (30 ml) and extracted with ethyl acetate (30 ml*2).
  • Step 4 Methyl (E)-4-(2-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-6-yl)but-2-enoate (90 mg, 0.3 mmol), intermediate G (191 mg, 0.9 mmol), (1,5-cyclooctadiene)chlororhodium(I) dimer ([Rh(COD)Cl] 2 , 6.2 mg, 0.01 mmol), (R)-(+)-2,2′-bis(diphenylphosphino)-1,1′-binaphthyl ((R)-(+)-BINAP, 15.7 mg, 0.03 mmol) and cesium carbonate (247 mg, 0.8 mmol) were dissolved in dioxane (2 ml) and water (0.2 ml) in sequence, heated to 95° C.
  • Step 1 Under nitrogen protection, at 0°C, tert-butyl 2,6-diazaspiro[3.4]octane-2-carboxylate (500 mg, 2.36 mmol, 1.0 eq) and TEA (595.7 mg, 5.9 mmol, 2.5 eq) were dissolved in DCM (5 mL), and CbzCl (803.5 mg, 4.7 mmol, 2.0 eq) was added dropwise. The reaction mixture was reacted for 16 h. The reaction solution was concentrated and the crude product was passed through a column.
  • Step 2 Under nitrogen protection, at 0°C, 6-benzyl 2-(tert-butyl) 2,6-diazaspiro[3.4]octane-2,6-dicarboxylate (295 mg, 0.9 mmol, 1.0 eq) was dissolved in DCM (6 mL), TFA (3 mL) was added dropwise, and the reaction was allowed to react for 2 h. The reaction solution was neutralized with ammonia water, and the reaction solution was concentrated to obtain 209 mg of crude yellow oily benzyl 2,6-diazaspiro[3.4]octane-6-carboxylate. LC-MS: ESI m/z: 247.1[M+H] + .
  • Step 3 Under nitrogen protection and room temperature, benzyl 2,6-diazaspiro[3.4]octane-6-carboxylate (209.7 mg, 0.85 mmol, 1.0 eq) and potassium carbonate (352.8 mg, 2.6 mmol, 3.0 eq) were dissolved in MeCN/DMF (2 mL/2 mL), and tert-butyl (E)-4-bromobut-2-enoate (178.7 mg, 0.8 mmol, 1 eq) was added dropwise at 65°C for 2 h. The reaction solution was concentrated, the crude product was dissolved in DCM, filtered, and the filtrate was concentrated.
  • Step 4 Under nitrogen protection and room temperature, benzyl (E)-2-(4-(tert-butyloxy)-4-carbonylbut-2-en-1-yl)-2,6-diazaspiro[3.4]octane-6-carboxylate (45 mg, 0.1 mmol, 1.0 eq), intermediate G (75.4 mg, 0.3 mmol, 3.0 eq), (1,5-cyclooctadiene)chlororhodium (I) dimer ([Rh(COD)Cl] 2 , 5.7 mg, 0.01 mmol, 0.1 eq), (R)-(+)-2,2′-bis(diphenylphosphino)-1,1′-binaphthyl ((R)-(+)-BINAP, 14.4 mg, 0.023 mmol, 0.2 eq) () and potassium hydroxide (19.6 mg, 0.3 mmol, 3.0 eq) in aqueous solution were dissolved
  • Step 5 Under nitrogen protection and room temperature, benzyl (S)-2-(4-(tert-butyloxy)-2-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-carbonylbutyl)-2,6-diazaspiro[3.4]octane-6-carboxylate (150 mg, 0.27 mmol, 1.0 eq) was dissolved in MeOH (2 mL), Pd/C (5 mg) and 1 drop of acetic acid were added, and the mixture was reacted at room temperature for 16 h. Celite was used for filtration, and the reaction solution was concentrated.
  • Step 6 Under nitrogen protection and room temperature, tert-butyl (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(2,6-diazaspiro[3.4]octan-2-yl)butanoate (60 mg, 0.1 mmol, 1.0 eq) and potassium carbonate (58.4 mg, 0.4 mmol, 3.0 eq) were dissolved in DMF (6 mL), and a DMF solution of tert-butyl 7-(bromomethyl)-3,4-dihydro-1,8-naphthyridin-1(2H)-carboxylate (A) (30.5 mg, 0.13 mmol, 0.95 eq) was added dropwise at 65°C and reacted for 2 h.
  • Step 1 Dissolve tert-butyl 5-carbonyl-2,6-diazaspiro[3.4]octane-2-carboxylate (220 mg, 972.3 ⁇ mol) in THF (10 ml), add sodium hydride (110. mg, 2.8 mmol) and intermediate A (450 mg, 1.4 mmol) at 0°C, and stir at 60°C for 3 hours under nitrogen protection. Add saturated aqueous ammonium chloride solution (5 ml) to the reaction solution, and extract with ethyl acetate (15 ml*3). The organic phase is washed with saturated brine, dried over Na 2 SO 4 , filtered and concentrated in vacuo to obtain a crude product.
  • Step 2 Dissolve tert-butyl 7-((2-(tert-butoxycarbonyl)-5-carbonyl-2,6-diazaspiro[3.4]octan-6-yl)methyl)-3,4-dihydro-1,8-naphthyridin-1(2H)-carboxylate (220 mg, 465.5 ⁇ mol) in methanol (2 ml), add 4M hydrochloric acid dioxane (5 ml) at room temperature, and then stir at room temperature for 1 hour under nitrogen protection. LCMS detected that the raw material reacted completely.
  • reaction solution was concentrated to obtain 200 mg of a light yellow solid product 6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-5-one.
  • Step 3 6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-5-one (150 mg, 485.7 ⁇ mol) was dissolved in acetonitrile (5 ml), and methyl (E)-4-bromobut-2-enoate (34.78 mg, 194.3 ⁇ mol) and diisopropylethylamine (188.3 mg, 1.5 mmol) were added in sequence at room temperature, and stirred at 80°C for 16 hours under nitrogen protection. The reaction solution was concentrated to obtain a crude product.
  • Step 4 See the preparation method of intermediate 1-INTB step 4.
  • Step 1 Refer to the preparation method of intermediate 1-INTB step 3.
  • Step 2 See the preparation method of intermediate 3-INTB step 2.
  • the reaction solution was concentrated to obtain 1 g of a yellow solid product 7-((2,7-diazaspiro[3.5]nonan-7-yl)methyl)-1,2,3,4-tetrahydro-1,8-naphthyridine.
  • LC-MS ESI m/z: 273.2 [M+H] + .
  • Step 3 Refer to the preparation method of intermediate 1-INT step 1.
  • Step 4 See the preparation method of intermediate 1-INTB step 4.
  • the reaction solution was concentrated and purified by high performance liquid chromatography to obtain 45 mg of reddish brown oily product.
  • 1 H NMR (400 MHz, METHANOL-d4) ⁇ ppm 7.57-7.41 (m, 2H) 7.39-7.26 (m, 3H) 6.72 (d, J 7.2 Hz, 1H) 6.04 (s, 1H) 4.11 (s, 2H) 3.98-3.78 (m, 2H) 3.65-3.58 (m, 2H) 3.55-3.50 (m, 3H) 3.48-3.40 (m, 3H) 3.35-3.28 (m, 3H) 3.15-3.08 (m, 3H) 2.83-2.73 (m, 3H) 2.72-2.64 (m, 1H) 2.22 (s, 3H) 2.20 (s, 3H) 2.12-1.95 (m, 4
  • Step 1 See the preparation method of intermediate 1-INT step 1.
  • Step 2 Refer to the preparation method of intermediate 3-INTB step 2. Obtain 810 mg of methyl (E)-4-(2,7-diazaspiro[3.5]nonan-7-yl)but-2-enoate as a pale yellow solid product.
  • Step 3 Dissolve intermediate A-3 (604.6 mg, 3.0 mmol) and TEA (628.7 mg, 6.2 mmol) in DCM (20 ml), add sodium acetate borohydride (987.5 mg, 4.7 mmol) and acetic acid (18.7 mg, 0.3 mmol) under nitrogen protection, and stir at room temperature overnight.
  • Step 4 See the preparation method of intermediate 1-INTB step 4.
  • Step 1 Dissolve tert-butyl 2,7-diazaspiro [3.5] nonane-7-carboxylate (500 mg, 2.2 mmol) and methyl (E)-4-bromobut-2-enoate (395.5 mg, 2.2 mmol) in 10 ml of acetonitrile, and then add potassium carbonate (610.7 mg, 4.4 mmol). The reaction solution is stirred at 80 ° C for 16 hours.
  • reaction solution is filtered and dried to obtain a crude product, which is purified by normal phase silica gel column chromatography (PE/EA from 100% to 40%) to obtain a yellow oily product tert-butyl (E)-2-(4-methoxy-4-carbonylbut-2-ene-1-yl)-2,7-diazaspiro [3.5] nonane-7-carboxylate 610 mg.
  • PE/EA normal phase silica gel column chromatography
  • Step 2 Refer to the preparation method of intermediate 1-INTB step 4.
  • the crude product was purified by normal phase silica gel column chromatography (PE/EA from 100% to 20%) to obtain 940 mg of yellow oily product tert-butyl (S)-2-(2-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-methoxy-4-carbonylbutyl)-2,7-diazaspiro[3.5]nonane-7-carboxylate.
  • LC-MS ESI m/z: 497.7 [M+H] + .
  • Step 3 Refer to the preparation method of intermediate 3-INTB in step 2.
  • the reaction solution was concentrated and dried to obtain 1 g of crude methyl (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(2,7-diazaspiro[3.5]nonane-2-yl)butanoate (6-INTA).
  • LC-MS ESI m/z: 397.5 [M+H] + .
  • Step 3 Refer to the preparation method of intermediate 5-INTB in step 3.
  • the crude product was purified by normal phase silica gel column chromatography (DCM/MeOH from 100% to 90%) to obtain 30 mg of a yellow oily product.
  • Step 1 Refer to the preparation method of intermediate 3-INTB step 3.
  • the crude product was purified by normal phase silica gel column chromatography (PE/EA from 100% to 0%) to obtain 1 g of yellow oily product tert-butyl (E)-7-(4-methoxy-4-carbonylbut-2-en-1-yl)-2,7-diazaspiro[4.4]nonane-2-carboxylate.
  • Step 2 Refer to the preparation method of intermediate 3-INTB step 2.
  • the reaction solution was directly concentrated to obtain 1 g of white solid methyl (E)-4-(2,7-diazaspiro[4.4]nonane-2-yl)but-2-enoate.
  • Step 3 See the preparation method of intermediate 5-INTB step 3.
  • the crude product was separated by high performance liquid chromatography to obtain a yellow oily product methyl (E)-4-(7-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,7-diazaspiro[4.4]nonane-2-yl)but-2-ene Acid ester (7-INTA) 740mg.
  • Step 4 Refer to the preparation method of intermediate 1-INTB step 4.
  • the crude product was purified by normal phase silica gel column chromatography (DCM/MeOH from 100% to 70%) to obtain 77 mg of a yellow oily product.
  • Step 1 Refer to the preparation method of intermediate 1-INTB step 1.
  • the crude product was purified by normal phase silica gel column chromatography (PE/EA from 100% to 30%) to obtain 1 g of yellow oily product tert-butyl (E)-9-(4-methoxy-4-carbonylbut-2-en-1-yl)-3,9-diazaspiro[5.5]undecane-3-carboxylate.
  • Step 2 Refer to the preparation method of intermediate 3-INTB step 2. 500 mg of white solid methyl (E)-4-(3,9-diazaspiro[5.5]undec-3-yl)but-2-enoate was obtained.
  • Step 3 See the preparation method of intermediate 5-INTB step 3.
  • the crude product was separated by high performance liquid chromatography (formic acid) to obtain 60 mg of yellow oily product methyl (E)-4-(9-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-3,9-diazaspiro[5.5]undecane-3-yl)but-2-enoate (8-INTA).
  • Step 4 Refer to the preparation method of intermediate 1-INTB step 4.
  • the crude product was purified by normal phase silica gel column chromatography (DCM/MeOH from 100% to 70%) to obtain 50 mg of a yellow oily product.
  • Step 1 Refer to the preparation method of intermediate 3-INTB step 3.
  • Step 3 See the preparation method of intermediate 5-INTB step 3.
  • the crude product was purified by HPLC to obtain 70 mg of a brown product, methyl (E)-4-(8-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,8-diazaspiro[4.5]decane-2-yl)but-2-enoate (9-INTA).
  • Step 4 See the preparation method of intermediate 1-INTB step 4.
  • Step 1 Refer to the preparation method of intermediate 3-INTB step 3.
  • Step 2 refers to the preparation method of intermediate 3-INTB step 2.
  • 120 mg of methyl (E)-4-(2,8-diazaspiro[4.5]decane-8-yl)but-2-enoate was obtained as a white solid product.
  • Step 3 See the preparation method of intermediate 5-INTB step 3.
  • the crude product was separated by high performance liquid chromatography to obtain 70 mg of yellow oily product methyl (E)-4-(2-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,8-diazaspiro[4.5]decane-8-yl)but-2-enoate (10-INTA).
  • Step 4 See the preparation method of intermediate 1-INTB step 4.
  • Step 1 See the preparation method of intermediate 5-INTB in step 3.
  • Step 2 Dissolve tert-butyl 1-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)spiro[indoline-3,4'-piperidine]-1'-carboxylate (140 mg, 0.32 mmol) in DCM (2 ml), add hydrochloric acid/methanol (2 ml, 4 M) to the reaction system, and stir at room temperature for 1 hour. The reaction solution is directly concentrated to obtain 80 mg of a yellow solid product 1-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)spiro[indoline-3,4'-piperidine].
  • Step 3 1-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)spiro[indoline-3,4'-piperidine] (40 mg, 0.15 mmol), methyl (E)-4-bromobut-2-enoate (18.73 mg, 0.10 mmol) and potassium carbonate (41 mg, 0.30 mmol) were added to DMF and stirred at 85°C for 4 hours. Water (10 ml) was added to the reaction solution and extracted with ethyl acetate (10 ml).
  • Step 4 Refer to the preparation method of intermediate 1-INTB step 4.
  • Step 1 Dissolve intermediate B (300 mg, 0.5 mmol) in acetonitrile (5 ml), add diisopropylethylamine (0.3 ml, 1.4 mmol) and intermediate 6-INTA (373.5 mg, 0.9 mmol) in turn at room temperature, heat the reaction solution to 80°C and stir for 6 hours.
  • Step 2 Refer to the preparation method of intermediate 3-INTB step 2. Obtain 230 mg of yellow solid product.
  • Step 1 See the preparation method of intermediate 12-INTB step 1.
  • Step 2 Refer to the preparation method of intermediate 3-INTB step 2. Obtain 100 mg of light yellow oily product.
  • Step 1 Dissolve intermediate D (30 mg, 0.01 mmol), intermediate 6-INTA (36.1 mg, 0.1 mmol) and N,N-diisopropylethylamine (35.3 mg, 0.3 mmol) in DCM (5 ml) in turn and stir at room temperature for 2 hours. The reaction solution was quenched with water (20 ml) and extracted with DCM (20 ml).
  • Step 2 Dissolve tert-butyl (S)-6-((2-(2-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-methoxy-4-carbonylbutyl)-2,7-diazaspiro[3.5]nonan-7-yl)methyl)-2,3-dihydro-4H-pyrido[3,2-b][1,4]oxazine-4-carboxylate (30 mg, 0.05 mmol) in DCM (5 ml), add methanolic hydrochloric acid (4 M, 5 ml) at room temperature, and stir for 1 hour. The reaction solution was directly spin-dried to obtain 25 mg of a yellow solid product.
  • Step 1 Refer to the preparation method of intermediate 1-INTB step 1.
  • 1 H NMR 400 MHz, CDCl 3 -d) ⁇ 6.82 (m, 1H) 5.93 (m, 1H) 3.99 (s, 4H) 3.74 (s, 3H) 3.33 (s, 4H) 3.17 (m, 2H) 1.43 (s, 9H).
  • Step 2 Refer to the preparation method of intermediate 1-INTB step 4.
  • Step 3 Refer to the preparation method of intermediate 14-INTB step 2.
  • LC-MS: ESI m/z 369.6 [M+H] + .
  • Step 4 Refer to the preparation method of intermediate 5-INTB in step 3.
  • LC-MS: ESI m/z 515.7 [M+H] + .
  • Step 1 Dissolve intermediate J (45 mg, 104 ⁇ mol) and 19-INTA (50.5 mg, 114.4 ⁇ mol) in acetonitrile (5 ml), then add diisopropylethylamine (40.3 mg, 312.1 ⁇ mol) and sodium iodide (31.2 mg, 208.1 ⁇ mol). The reaction solution was stirred at 60 ° C for 16 hours.
  • Step 1 Dissolve tert-butyl 4-formylpiperidine-1-carboxylate (5g, 23.4mmol) and 4-bromophenylhydrazine hydrochloride (5.2g, 23.4mmol) in chloroform (50ml) and ethanol (0.1ml). Cool to 0°C under nitrogen protection, slowly add trifluoroacetic acid (8g, 70.3mmol), and heat to 50°C after the addition is complete and stir for 16 hours. The reaction solution is quenched with water (30ml) and hydroxylamine aqueous solution (10ml), and extracted with DCM (30ml).
  • LC-MS:ESI m/z 365.4[M+H] + .
  • Step 2 Dissolve tert-butyl 5-bromospiro[indole-3,4'-piperidine]-1'-carboxylate (2.4 g, 6.6 mmol) in ethanol (30 ml), and slowly add sodium borohydride (1.2 g, 30.2 mmol) at room temperature. Stir at room temperature for 16 hours. The reaction solution was spin-dried, washed with water (50 ml), and extracted with DCM (50 ml).
  • Step 3 Dissolve tert-butyl 5-bromospiro[dihydroindole-3,4'-piperidine]-1'-carboxylate (750 mg, 2 mmol) and aldehyde in 1,2-dichloroethane (10 ml), add 2 drops of acetic acid at room temperature and stir for 30 minutes. Add sodium acetate borohydride (1.3 g, 6.1 mmol) and react for 16 hours.
  • LC-MS: ESI m/z 467.5[M+H] + .
  • Step 5 Refer to the preparation method of intermediate 5-INTB in step 3.
  • LC-MS: ESI m/z 513.5 [M+H] + .
  • Step 1 See the preparation method of intermediate 14-INTB step 1.
  • Step 1 Refer to the preparation method of intermediate 1-INTB step 4.
  • LC-MS ESI m/z: 575.6 [M+H] + .
  • Step 2 Refer to the preparation method of intermediate 14-INTB step 2.
  • Step 3 Refer to the preparation method of intermediate 14-INTB in step 1.
  • Step 1 Refer to the preparation method of intermediate 1-INTB step 4.
  • Step 2 Refer to the preparation method of intermediate 14-INTB step 2. 30 mg of methyl (S)-3-(3-(tert-butyl)-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(2,7-diazaspiro[3.5]non-2-yl)butanoate (25-INTA) was obtained as a yellow oily product. LC-MS: ESI m/z: 453.7 [M+H] + .
  • Step 3 Refer to the preparation method of intermediate 14-INTB in step 1.
  • Step 1 Add 3-bromo-4-methylpyridine (20 g, 116.3 mmol), palladium catalyst (5.3 g, 5.8 mmol), zinc cyanide (8.2 g, 69.8 mmol), zinc powder (0.8 g, 11.6 mmol) and DPPF (6.5 g, 11.6 mmol) to DMF (200 ml), replace nitrogen three times, and stir at 100 ° C for 16 hours under nitrogen atmosphere.
  • LC-MS: ESI m/z 119.0[M+H] + .
  • Step 2 Dissolve 4-methylnicotinonitrile (7.4 g, 62.6 mmol) and diethyl carbonate (74 g, 626.4 mmol) in THF (200 ml), replace with nitrogen three times, add NaH (12.5 g, 313.2 mmol) to the reaction system at 0°C, and stir at 60°C for 16 hours. Quench the reaction solution with saturated ammonium chloride (100 ml) and extract with ethyl acetate (100 ml).
  • Step 3 Add ethyl 2-(3-cyanopyridin-4-yl)acetate (5 g, 26.3 mmol) and Raney-Ni (5 g, 26.3 mmol) to ethanol (100 ml) and water (100 ml), replace with hydrogen three times, and heat to 50 ° C under hydrogen atmosphere and stir for 16 hours.
  • the reaction solution is filtered.
  • the filtrate is concentrated to obtain 3.8 g of yellow oily product 1,4-dihydro-2,7-diazinaphan-3(2H)-one.
  • LC-MS: ESI m/z 149.1[M+H] + .
  • Step 5 Dissolve 1,2,3,4-tetrahydro-2,7-naphthyridine (3.4 g, 25.3 mmol), (Boc) 2 O (8.3 g, 38 mmol), TEA (7.7 g, 76 mmol) and DMAP (309.6 mg, 2.5 mmol) in DCM (50 ml) and stir at room temperature for 16 hours. Quench the reaction solution with water (20 ml) and extract with DCM (20 ml).
  • Step 6 Dissolve tert-butyl 3,4-dihydro-2,7-naphthyridine-2(1H)-carboxylate (1.4g, 6.0mmol) and palladium hydroxide (557.6mg, 0.8mmol) in acetic acid (100ml), replace with hydrogen three times, heat to 80°C and stir for 12 hours under hydrogen atmosphere. Filter the reaction solution, add water (20ml) after concentrating the filtrate, adjust the pH to 9 with ammonia water, and extract with DCM (20ml).
  • Step 7 Dissolve tert-butyl octahydro-2,7-naphthyridine-2(1H)-carboxylate (800 mg, 3.3 mmol), potassium carbonate (1.4 g, 4.4 mmol) and methyl 4-bromocrotonate (595 mg, 3.3 mmol) in acetonitrile (15 ml) and stir at room temperature for 8 hours.
  • Step 8 Refer to the preparation method of intermediate 1-INTB in step 4.
  • Step 9 Refer to the preparation method of intermediate 14-INTB in step 2. 80 mg of yellow solid product methyl (3S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(octahydro-2,7-naphthyridin-2(1H)-yl)butanoate (29-INTA) was obtained.
  • Step 10 Refer to the preparation method of intermediate 5-INTB in step 3.
  • LC-MS: ESI m/z 557.5 [M+H] + .
  • Step 1 Refer to the preparation method of intermediate 3-INTB step 3.
  • Step 2 Refer to the preparation method of intermediate 1-INTB step 4.
  • LC-MS ESI m/z: 483.3 [M+H] + .
  • Step 3 Refer to the preparation method of intermediate 14-INTB in step 2.
  • the reaction solution was directly concentrated to obtain 150 mg of yellow oily product methyl (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(2,6-diazaspiro[3.4]octan-6-yl)butanoate (30-INTA).
  • Step 4 Refer to the preparation method of intermediate 5-INTB in step 3.
  • Step 1 Refer to the preparation method of step 1 of intermediate 12-INTB, wherein the preparation of 31-INTA refers to the preparation of 30-INTA.
  • Step 1 Refer to the preparation method of step 1 of intermediate 12-INTB, wherein the preparation of 32-INTA refers to the preparation of 30-INTA.
  • Step 1 Refer to the preparation method of intermediate 5-INTB in step 3.
  • LC-MS: ESI m/z 513.1 [M+H] + .
  • Step 1 Refer to the preparation method of intermediate 5-INTB in step 3.
  • LC-MS: ESI m/z 513.6 [M+H] + .
  • Step 1 Refer to the preparation method of intermediate 5-INTB in step 3.
  • LC-MS: ESI m/z 527.7 [M+H] + .
  • Step 1 Refer to the preparation method of intermediate 1-INTB step 1.
  • Step 2 Refer to the preparation method of intermediate 1-INTB step 4.
  • Step 3 Refer to the preparation method of intermediate 14-INTB in step 2.
  • the reaction solution was spin-dried to obtain 40 mg of a yellow oily product (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(2,7-diazaspiro[3.5]nonanoic acid-7-yl)butanoic acid methyl ester (37-INTA).
  • Step 4 Refer to the preparation method of intermediate 5-INTB in step 3.
  • Step 1 Refer to the preparation method of step 3 of intermediate 5-INTB, wherein the preparation of 38-INTA refers to the preparation of 19-INTA.
  • LC-MS: ESI m/z 593.6 [M+H] + .
  • Step 1 Refer to the preparation method of step 3 of intermediate 5-INTB, wherein the preparation of 39-INTA refers to the preparation of 19-INTA.
  • LC-MS: ESI m/z 570.8 [M+H] + .
  • Step 1 See the preparation method of intermediate 5-INTB step 3.
  • Step 1 Refer to the preparation method of 5-INTB step 3.
  • Step 1 Refer to the preparation method of intermediate 1-INTB step 4.
  • Step 3 See the preparation method of intermediate 5-INTB in step 3.
  • the crude product was purified by HPLC to obtain 15 mg of a colorless oily product.
  • LC-MS: ESI m/z 607.5 [M+H] + .
  • Step 1 See the preparation method of intermediate 1-INTB step 4.
  • Step 2 Refer to the preparation method of intermediate 2-INTB step 5.
  • the reaction solution was filtered and concentrated to obtain 520 mg of crude product methyl (S)-3-(3-(tert-butyl)-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(2,6-diazaspiro[3.4]octan-2-yl)butanoate (43-INTA).
  • LC-MS: ESI m/z 439.6[M+H] + .
  • Step 3 See the preparation method of intermediate 5-INTB in step 3.
  • the crude product was purified by HPLC to obtain 210 mg of a yellow oily product.
  • LC-MS: ESI m/z 585.5 [M+H] + .
  • Step 1 Refer to the preparation method of intermediate 1-INTB step 4.
  • LC-MS: ESI m/z 484.4[M+H] + .
  • Step 2 Dissolve tert-butyl (S)-2-(2-(5-bromopyridin-3-yl)-4-methoxy-4-carbonylbutyl)-2,7-diazaspiro[3.5]nonane-7-carboxylate (65 mg, 134.7 ⁇ mol), 3,5-dimethyl-1H-pyrazole (19.4 mg, 202.1 ⁇ mol), methanesulfonic acid-2-(di-tert-butylphosphino)-3,6-dimethoxy-2,4,6-triisopropyl-1,1-biphenyl(2-amino-1,1-biphenyl-2-yl)palladium(II) (11.5 mg) and cesium carbonate (109.8 mg, 336.9 ⁇ mol) in 1,4-dioxane (4 ml) in sequence, heat to 110°C under nitrogen protection and stir for 16 hours.
  • Step 4 Refer to the preparation method of intermediate 5-INTB in step 3.
  • LC-MS: ESI m/z 544.3 [M+H] + .
  • Step 1 Refer to the preparation method of step 3 of intermediate 5-INTB, wherein the preparation of 24-INTA is the same as 6-INTA.
  • Step 1 Refer to the preparation method of step 3 of intermediate 5-INTB, wherein the preparation of 25-INTA is the same as 6-INTA.
  • Step 1 Refer to the preparation method of intermediate 5-INTB step 3.
  • Step 1 Refer to the preparation method of intermediate 5-INTB step 3.
  • LC-MS: ESI m/z 701.4 [M+H] + .
  • Step 2 Refer to the preparation method of intermediate 3-INTB step 2. Obtain 70 mg of light yellow oily product.
  • LC-MS: ESI m/z 601.4 [M+H] + .
  • Step 1 Refer to the preparation method of step 3 of intermediate 5-INTB, wherein the preparation of 49-INTA is the same as 30-INTA.
  • Step 1 Refer to the preparation method of step 3 of intermediate 5-INTB, wherein the preparation of 50-INTA is the same as 30-INTA.
  • Step 1 Refer to the preparation method of step 3 of intermediate 5-INTB, wherein the preparation of 51-INTA is the same as 32-INTA.
  • Step 1 Refer to the preparation method of intermediate 5-INTB step 3, wherein the preparation of 52-INTA is the same as 32-INTA.
  • LC-MS: ESI m/z 599.6 [M+H] + .
  • Step 1 Refer to the preparation method of intermediate 1-INTB step 4.
  • Step 1 Refer to the preparation method of intermediate 1-INTB step 4.
  • Step 1 Dissolve tert-butyl 2,7-diazaspiro[4.4]nonane-2-carboxylate (100 mg, 441.9 ⁇ mol) in DCM (5 ml), then add TEA (184.5 ⁇ l, 1.3 mol) and methyl chloroacetate (62 mg, 574.4 ⁇ mol) in sequence, and react at room temperature for 2 hours.
  • reaction solution was spin-dried, washed with water (10 ml), extracted with ethyl acetate (10 ml), and the organic phase was dried and spin-dried to obtain 130 mg of yellow oily product tert-butyl 7-(2-methoxy-2-carbonylethyl)-2,7-diazaspiro[4.4]nonane-2-carboxylate.
  • Step 3 Refer to the preparation method of intermediate 5-INTB in step 3.
  • the crude product was purified by HPLC to obtain 100 mg of a white solid product.
  • LC-MS: ESI m/z 345.5 [M+H] + .
  • Step 1 Refer to the preparation method of intermediate 3-INTB step 3.
  • Step 2 Refer to the preparation method of intermediate 1-INTB step 4.
  • Step 3 Refer to the preparation method of intermediate 3-INTB in step 2.
  • 500 mg of methyl (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(1-oxa-4,9-diazaspiro[5.5]undec-4-yl)butanoate (63-INTA) was obtained as a light yellow oily product.
  • LC-MS: ESI m/z 427.6 [M+H] + .
  • Step 4 Refer to the preparation method of intermediate 5-INTB in step 3.
  • LC-MS: ESI m/z 573.7 [M+H] + .
  • Step 1 Refer to the preparation method of intermediate 5-INTB step 3.
  • Step 2 Refer to the preparation method of intermediate 3-INTB step 2. Concentration afforded 30 mg of a light yellow oily product. LC-MS: ESI m/z: 528.6 [M+H] + .
  • Step 1 Dissolve tert-butyl 7-hydroxy-2-azaspiro[3.5]nonane-2-carboxylate (200 mg, 0.8 mmol), 1,8-naphthyridin-2-ol (1 equivalent) and triphenylphosphine (1.2 equivalents) in dry THF (20 ml) in sequence. Add di-tert-butyl azodicarboxylate (1.2 equivalents) dropwise to the solution at 0°C under nitrogen protection, and stir the reaction solution for 1 hour.
  • Step 2 Refer to the preparation method of intermediate J step 2. 50 mg of white solid product tert-butyl 7-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)oxy)-2-azaspiro[3.5]nonane-2-carboxylate was obtained. LC-MS: ESI m/z: 374.2 [M+H] + .
  • Step 3 Refer to the preparation method of intermediate 14-INTB in step 2. Obtain 40 mg of white solid product 7-((2-azaspiro[3.5]nonan-7-yl)oxy)-1,2,3,4-tetrahydro-1,8-naphthyridine. LC-MS: ESI m/z: 274.2 [M+H] + .
  • Step 4 Refer to the preparation method of intermediate 3-INTB in step 3.
  • Step 5 Refer to the preparation method of intermediate 1-INTB in step 4.
  • the crude product was purified by HPLC to obtain 26 mg of a yellow solid product.
  • Step 1 Refer to the preparation method of intermediate 3-INTB step 3.
  • Step 2 Refer to the preparation method of intermediate 14-INTB step 2. 640 mg of methyl (E)-4-((7-azaspiro[3.5]nonan-2-yl)amino)but-2-enoate was obtained as a yellow solid product.
  • Step 3 See the preparation method of intermediate 5-INTB step 3.
  • the crude product was subjected to high performance liquid chromatography to obtain a brown solid product methyl (E)-4-((7-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-7-azaspiro[3.5]nonane-2-yl)amino)butane-2-yl Enoic acid ester (66-INTA) 250 mg.
  • LC-MS: ESI m/z 385.5 [M+H] + .
  • Step 4 See the preparation method of intermediate 1-INTB step 4.
  • the crude product was purified by HPLC to obtain 50 mg of a brown solid product.
  • LC-MS: ESI m/z 557.4 [M+H] + ;
  • Step 1 Refer to the preparation method of intermediate 3-INTB step 3.
  • Step 2 Refer to the preparation method of intermediate 1-INTB step 4.
  • Step 3 Refer to the preparation method of intermediate 14-INTB in step 2. 110 mg of methyl (S)-4-(8,8-difluoro-2,6-diazaspiro[3.4]octan-6-yl)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)butanoate (67-INTA) was obtained as a light yellow oily product.
  • LC-MS: ESI m/z 419.4 [M+H] + .
  • Step 4 Refer to the preparation method of intermediate 5-INTB in step 3.
  • LC-MS: ESI m/z 565.5 [M+H] + .
  • Step 1 Refer to the preparation method of intermediate 5-INTB in step 3.
  • LC-MS: ESI m/z 409.5 [M+H] + .
  • Step 2 Refer to the preparation method of intermediate 3-INTB step 2. 500 mg of a light yellow oily product 7-((5,5-difluoro-2,7-diazaspiro[3.5]nonan-7-yl)methyl)-1,2,3,4-tetrahydro-1,8-naphthyridine was obtained.
  • Step 3 See the preparation method of intermediate 3-INTB step 3.
  • Step 4 See the preparation method of intermediate 1-INTB step 4.
  • LC-MS: ESI m/z 579.6 [M+H] + .
  • Step 1 Refer to the preparation method of step 4 of intermediate 1-INTB, wherein the preparation of 72-INTA is the same as 71-INTA.
  • LC-MS: ESI m/z 561.3 [M+H] + .
  • Step 1 Refer to the preparation method of intermediate 1-INTB step 4.
  • the crude product was purified by HPLC to obtain 110 mg of a yellow oily product.
  • LC-MS: ESI m/z 527.4 [M+H] + .
  • Step 1 Refer to the preparation method of intermediate 1-INTB step 4.
  • the crude product was purified by HPLC to obtain 210 mg of colorless oily product tert-butyl (S)-2-(2-(3-bromo-5-(tert-butyl)phenyl)-4-methoxy-4-carbonylbutyl)-2,7-diazaspiro[3.5]nonane-7-carboxylate.
  • Step 2 Refer to the preparation method of intermediate 14-INTB step 2.
  • the reaction solution was directly concentrated to obtain 75 mg of yellow oily product methyl (S)-3-(3-bromo-5-(tert-butyl)phenyl)-4-(2,7-diazaspiro[3.5]nonan-2-yl)butanoate (76-INTA).
  • Step 3 See the preparation method of intermediate 5-INTB in step 3.
  • LC-MS: ESI m/z 583.6 [M+H] + .
  • Step 1 Refer to the preparation method of intermediate 1-INTB step 4.
  • LC-MS: ESI m/z 500.6 [M+H] + .
  • Step 1 Refer to the preparation method of intermediate 1-INTB step 4.
  • the crude product was purified by HPLC to obtain 180 mg of yellow solid product tert-butyl (S)-2-(4-methoxy-2-(3-(2-methoxyethoxy)phenyl)-4-carbonylbutyl)-2,7-diazaspiro[3.5]nonane-7-carboxylate.
  • Step 3 See the preparation method of intermediate 5-INTB step 3.
  • the crude product was purified by HPLC to obtain 90 mg of a yellow oily product.
  • LC-MS: ESI m/z 523.5 [M+H] + ; 1 H NMR (400 MHz, METHANOL-d 4 ) 8.42 (s, 2H), 7.34-7.24 (m, 2H), 6.94-6.85 (m, 3H), 6.59-6.52 (m, 1H), 5.28-5.05 (m, 4H), 4.17-4.11 (m, 2H), 3.78-3.75 (m, 2H), 3.72 (s, 2H), 3.61 (s, 3H), 3.57-3.53 (m, 4H), 3.44 (s, 3H), 3.44-3.42 (m, 1H), 2.82-2.61 (m, 8H), 1.97-1.87 (m, 6H).
  • Step 1 Refer to the preparation method of intermediate 1-ITNB step 4.
  • Step 2 Dissolve tert-butyl (S)-2-(2-(5-bromo-2-fluorophenyl)-4-methoxy-4-carbonylbutyl)-2,6-diazaspiro[3.4]octane-6-carboxylate (380 mg, 0.8 mmol), (5-bromo-2-fluorophenyl)boric acid (90.3 mg, 0.9 mmol), palladium catalyst (66.9 mg, 0.1 mmol) and cesium carbonate (765.2 mg, 2.4 mmol) in dioxane (10 ml), replace with nitrogen three times, and add under nitrogen protection. Heat to 120°C and stir for 16 hours.
  • Step 3 Refer to the preparation method of intermediate 3-INTB in step 2.
  • the crude product was purified by HPLC to obtain 60 mg of methyl (S)-3-(5-(3,5-dimethyl-1H-pyrazol-1-yl)-2-fluorophenyl)-4-(2,6-diazaspiro[3.4]octan-2-yl)butanoate (80-INTA) as a yellow oily product.
  • LC-MS: ESI m/z 401.5[M+H] + .
  • Step 4 Refer to the preparation method of intermediate 5-INTB in step 3.
  • the crude product was purified by HPLC to obtain 40 mg of a yellow oily product.
  • LC-MS: ESI m/z 547.4 [M+H] + .
  • Step 1 Refer to the preparation method of intermediate 1-INTB step 4.
  • Step 3 See the preparation method of intermediate 5-INTB step 3.
  • the crude product was purified by high performance liquid chromatography to obtain 80 mg of colorless oily product tert-butyl (S)-2-(3-(4-methoxy-4-carbonyl-1-(6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-2-yl)butan-2-yl)phenyl)-1H-pyrrole-1-carboxylate.
  • Step 1 Refer to the preparation method of intermediate 1-INTB step 4.
  • Step 2 Dissolve benzyl (S)-2-(2-(3-bromo-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-methoxy-4-carbonylbutyl)-2,6-diazaspiro[3.4]octane-6-carboxylate (400 mg, 0.7 mmol), cyclopropylboronic acid (87 mg, 1 mmol), cesium carbonate (547 mg, 1.7 mmol) and palladium catalyst (49 mg, 0.1 mmol) in dioxane (10 ml) in sequence. Heat to 100°C and stir for 3 hours under nitrogen protection.
  • Step 4 See the preparation method of intermediate 1-INTB in step 3.
  • LC-MS: ESI m/z 569.4 [M+H] + .
  • Step 1 See the preparation method of intermediate 1-INTB step 4.
  • LC-MS: ESI m/z 357.3[M+H] + .
  • Step 2 Refer to the preparation method of intermediate 1-INTB step 4.
  • the crude product was purified by HPLC to obtain 21 mg of a colorless oily product.
  • LC-MS: ESI m/z 509.6 [M+H] + .
  • Step 1 See the preparation method of intermediate 20-INTB step 1.
  • Step 2 See the preparation method of intermediate 20-INTB step 2.
  • the crude product was purified by HPLC to obtain 50 mg of a yellow solid product.
  • LC-MS: ESI m/z 543.5 [M+H] + .
  • the intermediate 43-INTA was used as the starting material, and the preparation method and steps were the same as those for 90-INTB to obtain 400 mg of a yellow oily product.
  • LC-MS: ESI m/z 599.6 [M+H] + .
  • the intermediate 82-INTA was used as the starting material, and the preparation method and steps were the same as those for 90-INTB to obtain 350 mg of a yellow oily product.
  • LC-MS: ESI m/z 583.4 [M+H] + .
  • Step 1 Refer to the preparation method of intermediate 3-INTB step 3.
  • Step 2 Refer to the preparation method of intermediate 1-INTB step 4.
  • the crude product was purified by normal phase silica gel column chromatography (EA) to obtain 140 mg of yellow oily product tert-butyl 7-((S)-2-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-methoxy-4-carbonylbutyl)-1,7-diazaspiro[4.4]nonane-1-carboxylate.
  • Step 4 Refer to the preparation method of intermediate 5-INTB in step 3.
  • LC-MS: ESI m/z 543.6 [M+H] + .
  • Step 1 Dissolve pyridine-2,6-diamine (5g, 45.8mmol) and 1,1,3,3-tetramethoxypropane (7.5g, 45.8mmol) in phosphoric acid (50ml), heat to 70°C under nitrogen protection, and stir for 2 hours. Adjust the pH of the reaction solution to 10 with 5M sodium hydroxide aqueous solution at 0°C, filter and rinse the filter cake with DCM (50ml*3), continue to extract the obtained solution with DCM (200ml*3), wash the organic phase with saturated brine (100ml*2), dry with Na 2 SO 4 , filter, and spin dry to obtain a crude product.
  • phosphoric acid 50ml
  • Step 2 Referring to the preparation method of intermediate A-2, 290 mg of crude product 5,6,7,8-tetrahydro-1,8-naphthyridin-2-amine was obtained.
  • LC-MS: ESI m/z 151.4 [M+H] + .
  • Step 4 Refer to the preparation method of intermediate 6-INTB in step 1.
  • LC-MS: ESI m/z 312.3 [M+H] + .
  • Step 5 Refer to the preparation method of intermediate 1-INTB in step 4.
  • LC-MS: ESI m/z 481.6 [M+H] + .
  • LC-MS: ES m/z 452.4[M+Na] + .
  • Step 7 Referring to the preparation method of intermediate D-6, the crude product was purified by HPLC to obtain 70 mg of yellow oily product methyl (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(7-formyl-3,4-dihydroisoquinolin-2(1H)-yl)butanoate (95-INTA).
  • Step 8 Refer to the preparation method of intermediate 1-INTB in step 3.
  • the crude product was purified by HPLC to obtain 10 mg of a yellow solid product.
  • LC-MS: ESI m/z 565.4 [M+H] + .
  • Step 1 Refer to the preparation method of intermediate 1-INTB step 4.
  • the crude product was purified by HPLC to obtain 10 mg of a yellow solid product.
  • LC-MS: ESI m/z 635.4 [M+H] + .
  • Step 1 Refer to the preparation method of intermediate 1-INTB step 4.
  • the crude product was purified by HPLC to obtain 10 mg of a yellow solid product.
  • LC-MS: ESI m/z 605.2 [M+H] + .
  • Step 1 Refer to the preparation method of intermediate 1-INTB step 4.
  • the crude product was purified by HPLC to obtain 10 mg of a yellow solid product.
  • LC-MS: ESI m/z 563.2 [M+H] + .
  • Step 1 Refer to the preparation method of intermediate 1-INTB step 4.
  • the crude product was purified by HPLC to obtain 10 mg of a yellow solid product.
  • LC-MS: ESI m/z 581.2 [M+H] + .
  • Step 1 Refer to the preparation method of intermediate 1-INTB step 4.
  • the crude product was purified by HPLC to obtain 10 mg of a yellow solid product.
  • LC-MS: ESI m/z 559.3 [M+H] + .
  • Step 1 See the preparation method of intermediate 1-INTB step 4.
  • the crude product was purified by HPLC to obtain 50 mg of methyl (S)-3-(5-bromopyridin-3-yl)-4-(5,5-difluoro-7-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,7-diazaspiro[3.5]nonane-2-yl)butanoate as a yellow solid product.
  • Step 2 See the preparation method of intermediate 44-INTB step 2.
  • the crude product was purified by HPLC to obtain 15 mg of a yellow solid product.
  • LC-MS: ESI m/z 580.3 [M+H] + .
  • Step 2 Refer to the preparation method of intermediate 3-INTB step 2. Obtain yellow oily product 7-(2-(5,5-difluoro-2,7-diazaspiro[3.5]nonan-7-yl)ethyl)-1,2,3,4-tetrahydro-1,8-naphthyridine.
  • LC-MS: ESI m/z 323.3 [M+H] + .
  • Step 3 Refer to the preparation method of intermediate 3-INTB step 3.
  • LC-MS: ESI m/z 421.2 [M+H] + .
  • Step 4 Refer to the preparation method of intermediate 1-INTB step 4. A pale yellow solid product is obtained.
  • LC-MS: ESI m/z 593.3 [M+H] + .
  • Step 1 Refer to the preparation method of intermediate 1-INTB step 4.
  • the crude product was purified by HPLC to obtain 50 mg of a yellow solid product.
  • LC-MS: ESI m/z 617.4 [M+H] + .
  • Step 1 Refer to the preparation method of intermediate 1-INTB step 4.
  • the crude product was purified by HPLC to obtain 50 mg of yellow solid product methyl (S)-3-(5-bromo-2-fluorophenyl)-4-(8,8-difluoro-6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-2-yl)butanoate.
  • Step 2 See the preparation method of intermediate 80-INTB step 2.
  • LC-MS: ESI m/z 583.3 [M+H] + .
  • Step 1 Refer to the preparation method of intermediate 1-INTB step 4.
  • the crude product was purified by HPLC to obtain 50 mg of a yellow solid product.
  • LC-MS: ESI m/z 621.4 [M+H] + .
  • Step 1 Refer to the preparation method of intermediate 1-INTB step 4.
  • the crude product was purified by HPLC to obtain 50 mg of a yellow solid product.
  • LC-MS: ESI m/z 605.4 [M+H] + .
  • Step 1 Refer to the preparation of intermediate 1-INTB in step 4.
  • the crude product was purified by HPLC to obtain 50 mg of a yellow solid product.
  • LC-MS: ESI m/z 561.3 [M+H] + .
  • Step 1 Using tert-butyl (E)-8,8-difluoro-2-(4-methoxy-4-carbonylbut-2-en-1-yl)-2,6-diazaspiro[3.4]octane-6-carboxylate as the starting material, refer to the preparation method of intermediate 1-INTB step 4.
  • the principle is to detect the change in molecular weight of the fluorescently labeled small molecule before and after the interaction with other molecules, and calculate the fluorescence polarization values in the horizontal and vertical directions for correlation analysis. If the binding equilibrium between the fluorescently labeled small molecule and the macromolecule is established, it moves slowly when excited, and the measured fluorescence polarization light value will increase. If the binding between the fluorescently labeled small molecule and the macromolecule is replaced by other ligands, its rotation or flipping speed in the free state will become faster, the emitted light will be depolarized relative to the excitation light plane, and the measured polarization light value will decrease, thereby calculating the fluorescence anisotropy of the sample.
  • the experimental reaction system was 40ul, and duplicate wells were set up.
  • the buffer for ⁇ V ⁇ 6 was: 50mM HEPES PH7.4, 150mM Nacl, 0.5mM CHAPS, 0.4mM MgCl2, and the buffer for ⁇ V ⁇ 1 and ⁇ V ⁇ 8 was: 50mM HEPES PH7.4, 150mM Nacl, 0.5mM CHAPS, 0.1mM MnCl2.
  • the working concentration of the fluorescent substrate is 1nM
  • the working concentrations of ⁇ V ⁇ 6 (recombinant human integrin produced by R&D systems, catalog number 3817-AV) and ⁇ V ⁇ 8 (recombinant human integrin produced by R&D systems, catalog number 4135-AV) are both 8nM
  • the working concentration of ⁇ V ⁇ 1 (catalog number 6579-AVB) is 12.5nM
  • the initial screening concentrations of the compound are 1uM and 100nM
  • the final concentration of DMSO is 0.2%. All components are mixed in a 384-well plate (corning catalog number: CLS3575) and reacted at room temperature (about 24°C) for 2 hours before determining the Anisotropy value.
  • the testing instrument is the BioTek brand SYNERGY neo2 microplate reader, with an Excitation of 485nM and an emission of 530nM.
  • the wells with only buffer added are used as blank controls for system readings.
  • the inhibition rate is calculated by taking the average value of the duplicate wells, and the IC 50 value of the compound with an inhibition rate greater than 50% under the condition of 100nM of the compound is determined.
  • PLN-74809 and GSK-3008348 were selected as control compounds in this experiment.
  • PLN-74809 (CAS: 2376257-44-0) and GSK-3008348 (CAS: 1629249-33-7) are two very potential integrin ligand molecules.
  • the experimental treatment method is the same as above.
  • C is the Anisotropy value of complete binding of the fluorescent substrate to the protein
  • F is the Anisotropy value at the corresponding concentration of the compound
  • B is the background value of the Anisotropy of the fluorescent substrate.
  • the S curve is drawn with the compound concentration and the corresponding inhibition rate value (Table 1), and the corresponding IC 50 value is calculated (Table 2).
  • the a-level embodiment has excellent inhibitory activity against the target subtype; the b-level embodiment has a strong inhibitory activity; the c-level embodiment has a general inhibitory activity.
  • the A-level embodiment has strong selectivity for subtype 2 compared with subtype 1; the B-level embodiment has moderate selectivity; and the C-level has no selectivity.
  • IC50 IC50 ⁇ 100 nM
  • c IC50 > 1000 nM
  • B: Fold 1.0-4.0, indicating that the compound has moderate selectivity for a certain subtype
  • N/A means no test was performed;
  • the examples show different inhibitory effects on one, two or three subtypes of ⁇ v ⁇ 1, ⁇ v ⁇ 6 and ⁇ v ⁇ 8, respectively, and especially show excellent inhibitory effects and selectivity on the single subtype of ⁇ v ⁇ 6 and the two subtypes of ⁇ v ⁇ 1 and ⁇ v ⁇ 6, such as Examples 43, 44, 71, 72, 96-103 (aaaCA type) and the like have excellent inhibitory effects on both ⁇ v ⁇ 1 and ⁇ v ⁇ 6 and good selectivity for ⁇ v ⁇ 8, and the selectivity is much better than the positive reference PLN-74809.
  • Examples 1, 5, 49-50, 52-54, 75, 77-78 and the like have excellent inhibitory activity and specific selectivity for ⁇ v ⁇ 6.
  • Fibroblasts are the main effector cells in fibrotic diseases. When activated, they can undergo functional and phenotypic changes and transdifferentiate into myofibroblasts (MFb), which have contractile potential and strong collagen synthesis ability. This is the main cellular process that induces fibrotic lesions.
  • the phenotypic characteristics of activated cells are the expression of ⁇ -smooth muscle actin ( ⁇ -Smooth muscle actin, ⁇ -SMA) and fibronectin (Fibronectin, FN), so they can be activated by these two proteins.
  • the changes in protein expression levels were used to verify the inhibitory effect of integrin ligands on fibroblast activation.
  • Cell line human fetal lung fibroblasts (HFL-1); TGF- ⁇ : R&D; culture medium: F12K culture medium from Hyclone; serum: Gibco; Fibronectin antibody: abcam, catalog number: ab45688; ⁇ -SMA antibody: abcam, catalog number: ab124964.
  • HFL-1 cells were inoculated in F12K medium containing 10% FBS and cultured routinely. When treated with different compounds, 1 ⁇ 106 cells/well were inoculated in 6-well plates, cultured under normal culture conditions for 24 hours after inoculation, and then replaced with serum-free medium. The excipient DMSO and different concentrations of compounds (3 concentrations for each compound, 3 replicates for each concentration) were pretreated for 2 hours, and then 10 ng/ml of TGF- ⁇ recombinant protein was added for 24 hours. After treatment, the cells were recovered for protein immunoblotting (Western blotting, WB) detection of Fibronectin and ⁇ -SMA.
  • WB Western blotting
  • Pirfenidone and GSK-3008348 were selected as control compounds for this experiment.
  • Pirfenidone (CAS: 53179-13-8) is a widely used anti-fibrotic drug
  • GSK-3008348 (CAS: 1629249-33-7) is a highly potential integrin ligand molecule developed by GlaxoSmithKline (GSK Plc.).
  • the drug concentration of pirfenidone was 0.5 mg/mL, and the drug concentration of GSK-3008348 was the same as that of the test compound (3 concentrations, 3 replicates for each concentration), and the treatment method was the same as above.
  • Protein extraction Wash the cells with PBS, add 100ul RIPA lysis buffer to each well, scrape the cells with a cell scraper and collect them in an EP tube, place on ice for 30 minutes, centrifuge at 13000g for 10 minutes after sufficient lysis, and take the supernatant and place it in a new EP tube. Protein quantification is performed according to the instructions of the BCA protein concentration assay kit (enhanced). According to the quantitative results, dilute the sample to make the protein concentration consistent. Add the loading buffer and mix well, heat in a boiling water bath for 3-5 minutes to fully denature the protein, and load the sample after cooling to room temperature.
  • Make gel inject separation gel, leave 1/5 of the space, and fill it up with deionized water. After standing at room temperature for 1 hour, pour out the deionized water, dry it with absorbent filter paper, and then fill it up with concentrated gel; then quickly insert the comb, and pay attention to remove bubbles with the remaining concentrated gel, and then stand at room temperature for 30 minutes.
  • Sample loading/electrophoresis/transfer The total protein loading amount of each sample is 25ug, which is added to the sample well with a sample gun.
  • Electrophoresis Run at 70V to the lower layer of the concentrated gel and the upper layer of the separation gel, and then run at 120V to the end.
  • Transfer First soak the 0.45um PVDF membrane, filter paper, and sponge in the transfer buffer, and mark the PVDF membrane; after the electrophoresis is completed, select the appropriate gel; make a transfer sandwich, put it on the rack, fill it with electrotransfer buffer, and run at a constant current of 230mA for 120min.
  • Antigen blocking and antibody incubation First wash the membrane with PBS, add Western blocking solution (5% nonfat-milk), and incubate on a shaker at room temperature for 1 hour.
  • Primary antibody incubation Add primary antibodies ⁇ -SMA, Fibronectin and internal reference Actin respectively, overnight at 4°C; wash the primary antibody, add TBST, and wash 3 times on a shaker at room temperature, 5 minutes each time.
  • Secondary antibody incubation Add the corresponding secondary antibody and incubate at room temperature with shaking for 1 hour. Wash the secondary antibody, add TBST, and wash 4 times on a shaker at room temperature, 6 minutes each time. Finally, the fluorescence imager is used for detection, and the optical density value of the protein band is quantitatively read. The optical density value of the actin band is used as the internal reference to compare the expression of each target protein.
  • this experiment uses the Antibody Stripping method, first detecting the ⁇ -SMA protein, then using the stripping buffer to remove the ⁇ -SMA primary antibody, and then detecting the Actin protein.
  • the incubation methods for the two antibodies are the same as above.
  • Embodiments 1, 2, 7, 15, and 25 have significant promoting degradation effects on fibrosis proteins ⁇ -SMA and Fibronectin (as shown in Figure 1).
  • Embodiments 2, 7, 15, and 25 significantly reduce the expression of ⁇ -SMA protein, have an excellent promoting degradation effect on fibrosis protein ⁇ -SMA, and are significantly better than the positive references pirfenidone and GSK-3008348, showing excellent anti-fibrosis effects
  • Embodiments 1, 2, 7, and 25 significantly reduce the expression of Fibronectin protein, have an excellent promoting degradation effect on fibrosis protein Fibronectin, and are also better than the positive references pirfenidone and GSK-3008348.
  • Embodiments 2, 7, and 25 all have excellent promoting degradation effects on both ⁇ -SMA and Fibronectin proteins, and the effects are significantly better than the positive references GSK-3008348 and pirfenidone. This indicates that the ligands of integrins ⁇ v ⁇ 1, 6, and 8 in the present invention have a significant inhibitory effect on the secretion of ⁇ -SMA protein and Fibronectin protein as well as the activation of fibroblasts, and have the potential to become anti-fibrosis drugs.
  • Substrate, liver microsomes, MgCl 2 , potassium phosphate buffer, and water were added to a 96-well plate and gently vortexed to mix.
  • the microsome mixture was pre-incubated in a 37°C water bath for 5 min, and the reaction was initiated by adding the NADPH generating system. After incubation for 0, 10, 20, and 30 min, 40 ⁇ L of the reaction mixture was mixed with 100 ⁇ L of acetonitrile (with internal standard 100 ng/mL) to terminate the reaction. After the incubation, the mixture was centrifuged at 4600 rpm for 10 min at 4°C. 60 ⁇ L of the supernatant was taken, diluted 1:1 with water, and 5 ⁇ L was injected for LC-MS/MS analysis.
  • a Waters XBridge C18 column (50 mm ⁇ 2.1 mm, 5 ⁇ m) was used with an injection volume of 5 ⁇ L. The flow rate was 1 mL/min and the running time was 1.5 min. The mobile phase A was water containing 0.1% formic acid, and B was acetonitrile containing 0.1% formic acid.
  • the gradient elution program is shown in Table 4 below:
  • LC-MS/MS High performance liquid chromatography tandem mass spectrometry
  • model API 4500 (AB Sciex).
  • Electrospray ionization (ESI) conditions and multiple reaction monitoring (MRM) mode were used to detect the eluted compounds.
  • the dry gas temperature was 450°C
  • the pressure was 50 psi
  • the nebulizing gas pressure was 20 psi.
  • the compounds of the present invention are metabolically stable in the liver microsomes of humans, mice, rats, dogs and monkeys, among which Examples 1, 2, 6, 72 and 73 have better half-lives in human liver microsomes than the positive reference PLN-74809, and Examples 2, 6, 73 and 117 have better half-lives in dog liver microsomes than PLN-74809.
  • Other compounds of the present invention also have similar physicochemical properties.
  • mice Male SD rats, weighing about 200 g, were purchased from Shanghai Xipuer-Bikai Experimental Animal Co., Ltd., with 3 rats in each group. Each test compound was administered orally (PO) and intravenously (IV).
  • the injection dose was 1 mg/kg, and the test compound was prepared into a 0.1 mg/mL solution using the same solvent and injected into the tail vein at a volume of 10 mL/kg. Blood was collected at 5, 15, 30, 60, 120, 240, 480, 720, and 1440 minutes after administration to determine the blood drug concentration.
  • the oral dose was 10 mg/kg.
  • Each blood collection volume was about 200 ⁇ L, stored in an EDTA-2K anticoagulation tube, and centrifuged at 5500 rpm for 10 min to separate plasma. Take 20 ⁇ L of the test plasma sample, standard plasma sample, and QC plasma sample at each time point, mix them with 100 ⁇ L acetonitrile (with internal standard 100 ng/mL), shake at room temperature for 10 min, and then centrifuge at 3700 rpm for 18 min at 4 ° C. Take 60 ⁇ L of the supernatant, dilute it 1:1 with water, shake it at room temperature for 10 min, and then inject 5 ⁇ L for LC-MS/MS analysis.
  • LC-MS/MS High performance liquid chromatography tandem mass spectrometry
  • model API 4500 (AB Sciex)
  • Electrospray ionization (ESI) conditions and multiple reaction monitoring (MRM) mode were used to detect the eluted compounds.
  • the ion source temperature was 450°C
  • the spray gas and auxiliary heating gas pressures were both 50 psi
  • the curtain gas pressure was 20 psi
  • the collision chamber outlet voltage was 13.0 V.
  • the declustering voltages of the internal standard and the test compound were 79, 130, and 135 eV, respectively, and the collision energies were 19, 24, and 41 eV, respectively.
  • the terminal elimination half-life was 1.5 and 0.4 hours, respectively, and the injection AUC INF was 4644h*nM and 6511h*nM, respectively.
  • the terminal elimination half-life of the two was 3.0 and 0.7 hours, the peak time was 0.5 and 0.4 hours, the peak concentration was 4622 and 15946nM, respectively, and the oral AUC INF was 12994h*nM and 31406h*nM, respectively.
  • the rats absorbed well, and the oral bioavailability was 28% and 48%, respectively, which was significantly better than the oral bioavailability of the positive reference PLN-74809 (the actual measured data of rats was only 2%).
  • Other compounds in the present invention also have similar physical and chemical properties, and have excellent drug-forming potential and excellent in vivo metabolic characteristics.
  • the experimental test indicators include animal weight and histopathological score.
  • the animal weight is observed and recorded once a day, and the weight growth rate is calculated.
  • the histopathological scoring criteria are as follows:
  • Example 71 helps to improve the overall state of the animals, helps rats recover their weight faster, and no obvious side effects are observed.
  • Example 71 can reduce bleomycin-induced bronchial and arteriolar damage and inflammatory infiltration in the left lung tissue.
  • Example 71 H&E stained sections were observed under a microscope to compare the lesions around the left lung bronchi, pulmonary small blood vessels, and alveolar tissue and the degree of inflammatory cell infiltration. As shown in Figure 4, the low-dose group (D) and high-dose group (E) of Example 71 were significantly different from the model group.
  • the low-dose group (D) and high-dose group (E) of Example 71 can reduce the degree of inflammation of the lung tissue at the edge of the lesion, which is significantly better than the model group (B), and the high-dose group (E) of Example 71 is better than the low-dose group and the positive reference nintedanib group (C) in reducing inflammation.
  • the alveolar wall thickening in the high-dose group (E) of Example 71 was the mildest, with slight alveolar wall thickening at the arrow point, and the degree of pathological changes was significantly lower than that in the model group (B), and also lower than that in the low-dose group (D) and the nintedanib group (C) of Example 71.
  • the degree of reduction in the low-dose group of Example 71 was comparable to that in the nintedanib group. This indicates that the high-dose group of Example 71 is more advantageous than the positive reference nintedanib group, and the drug effect is more significant.
  • Example 71 can alleviate bleomycin-induced pulmonary fibrosis changes
  • the deposition degree of fibrotic collagen in lung tissue was observed by Masson staining under a microscope, and fibrosis scores were performed. As shown in Figure 9, the degree of fibrosis in the low-dose group (D) of Example 71 and the high-dose group (E) of Example 71 was significantly reduced compared with the model group (B), and the degree of reduction in the two dosage groups of Example 71 was better than that in the nintedanib group (C).
  • Figure 11 is the result of statistical analysis of the proportion of mild and severe fibrosis in the sham operation group, model group, nintedanib group, low-dose group of Example 71, and high-dose group of Example 71.
  • fibrosis scores ⁇ 3 points can be regarded as mild; scores ⁇ 4 points can be regarded as severe.
  • the proportion of severe cases in the low-dose group of Example 71 was significantly reduced (p ⁇ 0.001), and the proportion of severe cases in the high-dose group of Example 71 was significantly reduced (p ⁇ 0.001).
  • Example 71 Compared with the positive reference nintedanib group, the proportion of severe cases in the high and low dose groups of Example 71 was significantly lower than that in the nintedanib group, and the proportion of severe cases in the high-dose group of Example 71 was lower than that in the low-dose group, and significantly lower than that in the nintedanib group. This shows that more than 80% of the rats in the high-dose group of Example 71 were mild cases, and the efficacy was extremely significant. Figures 9, 10 and 11 illustrate that Example 71 has a significant anti-pulmonary fibrosis effect.
  • FIG12 shows the degree of collagen deposition in the alveolar tissue of rats in each group in Masson staining.
  • the collagen deposition in the low-dose group (D) of Example 71 and the high-dose group (E) of Example 71 was the lightest, and the degree was significantly lower than that in the model group (B) and the nivolumab group (E). Nintedanib group (C).
  • the collagen deposition area of the high and low dose groups of Example 71 was significantly reduced (both p ⁇ 0.001), and the degree of reduction was significantly better than that of the nintedanib group (p ⁇ 0.05).
  • the degree of reduction in collagen deposition area in the high dose group of Example 71 was more significant, indicating that Example 71 can also effectively reduce the degree of lung collagen deposition.
  • mice (C57BL/6J) were randomly divided into groups according to body weight. From day 2, the normal feed in all cages except the normal control group was replaced with feed containing 0.1% DDC (3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine) to induce mouse sclerosing cholangitis (PSC).
  • the normal control group was provided with normal diet and drinking water for 3 consecutive weeks (Day 2-Day 22). From day 2, the corresponding drugs were given to different groups every day for 3 consecutive weeks (Day 2-Day 22). The day after the last administration (Day 23), the mice were bled, and the blood samples were centrifuged at 4°C and 1500 ⁇ g to separate the serum and frozen at -80°C.
  • mice After the blood was drawn, the mice were euthanized, the whole liver was separated and weighed, and then 1/2 of the liver lobe was cut and placed in 10% formalin (formaldehyde) solution for fixation for 48h, and then pathological examination was performed.
  • formalin formalin
  • the experimental test indicators included animal body weight, liver weight, serum liver function indexes, and histopathological scores. The animal body weight was observed and recorded once a day, and the percentage of body weight change was compared. An automatic biochemical detector was used to measure the levels of alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), total bile acid (TBA), and total bilirubin (TBIL) in mouse blood samples.
  • ALT alanine aminotransferase
  • ALP alkaline phosphatase
  • AST aspartate aminotransferase
  • TAA total bile acid
  • TBIL total bilirubin
  • Example 71 After mice ingested DDC, liver damage gradually worsened, and obvious symptoms of bile duct sclerosis appeared. The body weight of all mice fed with DDC-containing feed decreased to varying degrees, and became more serious over time. The liver weight/body weight ratio increased significantly in the model group, and Example 71 had a significant therapeutic effect compared with the model group, indicating that Example 71 has excellent anti-liver fibrosis, anti-bile duct sclerosis and anti-inflammatory effects.

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Abstract

A bicyclic αvβ1, αvβ6 and αvβ8 integrin inhibitor compound represented by formula (I), and a racemate, stereoisomer, tautomer, solvate, polymorph, metabolite, ester, prodrug or pharmaceutically acceptable salt thereof, and a pharmaceutical composition comprising same, a preparation method therefor, and a pharmaceutical use thereof. The structure of formula (I) is shown below.

Description

一类双环衍生物的整合素抑制剂A class of bicyclic derivative integrin inhibitors 技术领域Technical Field
本发明涉及药物化学领域，具体涉及一类双环衍生物作为αvβ1、αvβ6和αvβ8整联蛋白抑制剂、包含此类化合物的药物组合物以及它们在治疗和/或预防需要αvβ1、αvβ6和αvβ8整联蛋白抑制剂的病症中的用途。The present invention relates to the field of medicinal chemistry, and in particular to a class of bicyclic derivatives as inhibitors of αvβ1, αvβ6 and αvβ8 integrins, pharmaceutical compositions comprising such compounds, and their use in the treatment and/or prevention of conditions requiring inhibitors of αvβ1, αvβ6 and αvβ8 integrins.
背景技术Background technique
整合素又称整联蛋白、接合蛋白，是一大类细胞表面受体家族，是由α亚基和β亚基结合形成的异二聚体跨膜糖蛋白。在哺乳动物中，已经发现18种α和8种β亚基，这些亚基非共价键结合形成至少24种不同的整合素，由各类细胞差异表达并识别多种配体，介导细胞内与细胞外基质以及相邻细胞间的信息传递。整合素是一类细胞黏附分子，由胞外区、跨膜区和胞内区三部分组成，胞外N端结构域与特异性配体结合，胞内区通过α辅肌动蛋白、踝蛋白以及黏着斑蛋白与细胞骨架蛋白相连接，形成配体-整合素-细胞骨架跨膜系统。整合素作为细胞表面受体可以通过细胞膜实现双向传递信号。一种为外向内信号传递。整合素与ECM中的配体结合，导致整合素构象变化，并在细胞膜上聚集，形成局灶性黏附，激活多个细胞内信号通路，将信号从胞外转导入胞内，从而调节细胞黏附、扩散、迁移、增殖、分化以及重塑。另一种为内向外信号传递。细胞内信号诱导踝蛋白构象发生改变，导致踝蛋白与整合素β亚基胞内结构域结合，这种结合解开了胞质内α亚基链尾和β亚基链尾之间的链接，整合素头部构象发生变化，使其与胞外配体结合的亲和力增加，增强了细胞的黏附能力。根据整合素配体的特异性可将整合素分为3种:结合层粘连蛋白类整合素、结合胶原蛋白类整合素以及结合精氨酸-甘氨酸-天冬氨酸序列(Arg-Gly-Asp，RGD)类整合素。Integrins, also known as integrins and junction proteins, are a large family of cell surface receptors. They are heterodimeric transmembrane glycoproteins formed by the combination of α subunits and β subunits. In mammals, 18 α and 8 β subunits have been found. These subunits are non-covalently bound to form at least 24 different integrins, which are differentially expressed by various cells and recognize a variety of ligands, mediating information transmission between cells and the extracellular matrix and between adjacent cells. Integrins are a type of cell adhesion molecules, consisting of three parts: the extracellular region, the transmembrane region, and the intracellular region. The extracellular N-terminal domain binds to specific ligands, and the intracellular region is connected to the cytoskeleton protein through α-actinin, talin, and focal adhesion proteins to form a ligand-integrin-cytoskeleton transmembrane system. As a cell surface receptor, integrin can transmit signals bidirectionally through the cell membrane. One is outside-in signal transmission. Integrins bind to ligands in the ECM, causing conformational changes in integrins, which aggregate on the cell membrane to form focal adhesions, activate multiple intracellular signaling pathways, and transduce signals from the extracellular to the intracellular, thereby regulating cell adhesion, diffusion, migration, proliferation, differentiation, and remodeling. The other is inside-out signaling. Intracellular signals induce conformational changes in talin, causing talin to bind to the intracellular domain of the integrin β subunit. This binding unties the link between the α subunit tail and the β subunit tail in the cytoplasm, and the conformation of the integrin head changes, increasing its affinity for binding to extracellular ligands and enhancing the cell's adhesion ability. According to the specificity of integrin ligands, integrins can be divided into three types: laminin-binding integrins, collagen-binding integrins, and arginine-glycine-aspartic acid sequence (Arg-Gly-Asp, RGD) integrins.
不同亚型的RGD类整合素参与机体不同部位的纤维化进展，在众多αv家组蛋白中，整合素αvβ1在活化的成纤维细胞和系膜细胞上表达；整合素αvβ6能够与TGF-β1的RGD序列结合，实现细胞-细胞间接触从而激活TGF-β1；αvβ8则能与TGF-β3LAP中RGD序列结合，将LAP复合物呈递给细胞表面的基质金属蛋白酶，从而激活TGF-β1。在肾和肺组织纤维化中avβ6及avβ1发挥主要作用，而在肝纤维化中avβ1占主导作用。整合素avβ1是一种低亲和力的纤连蛋白受体，在基底上皮细胞中高表达，具有促进角化细胞在底层纤维连接蛋白EDA上迁移的作用。阻断整合素avβ1与TGF-β1相互作用有助于抑制TGF-β1活性，阻断纤维化进程。有报道称，在正常的肺组织中整合素αvβ6表达量很低，但是当肺损伤发生炎症和纤维化时，avβ6快速高表达(Hatley et al,Angewandte Chemie International Edition,2018,57(13):3298.)。在患有原发性胆汁性肝硬化(PBC)、酒精性脂肪肝、乙肝、丙肝等疾病的患者中，整合素avβ6的mRNA表达量升高。在肾疾病相关的慢性炎症和纤维化疾病中整合素avβ6的表达较于正常肾组织显著提高。此外，在糖尿病、肺出血肾炎综合症、Alport综合症、狼疮性肾炎等病人活检样品中整合素avβ6都显著高表达(Koivisto et al,The international journal of biochemistry&cell biology,2018,99:186)。整合素αvβ8对于TGFβ的调控极为重要，唯一的结合配体为L-TGFβ，在多种癌症细胞尤其是Treg高的肿瘤细胞中高表达，L-TGFβ则主要在表达在T细胞等免疫细胞。研究人员发现，αvβ8促进肿瘤微环境Treg的分化，进而促进肿瘤的生长。作用机制为T细胞GARP/L-TGFbeta结合αvβ8之后， 发生构象变化，释放出活化TGFβ，与T细胞上TGFβR结合，促进T细胞向Treg分化。另外，在IL-1β和白蛋白诱导的小鼠气道纤维化模型中，肺成纤维细胞αvβ8的条件性缺失可抑制小鼠气道纤维化。Different subtypes of RGD integrins participate in the progression of fibrosis in different parts of the body. Among the many αv family proteins, integrin αvβ1 is expressed on activated fibroblasts and mesangial cells; integrin αvβ6 can bind to the RGD sequence of TGF-β1, achieve cell-cell contact and activate TGF-β1; αvβ8 can bind to the RGD sequence in TGF-β3LAP, present the LAP complex to matrix metalloproteinases on the cell surface, and activate TGF-β1. avβ6 and avβ1 play a major role in renal and pulmonary tissue fibrosis, while avβ1 plays a dominant role in liver fibrosis. Integrin avβ1 is a low-affinity fibronectin receptor that is highly expressed in basal epithelial cells and has the function of promoting the migration of keratinocytes on the underlying fibronectin EDA. Blocking the interaction between integrin avβ1 and TGF-β1 helps inhibit TGF-β1 activity and block the process of fibrosis. It has been reported that the expression of integrin αvβ6 is very low in normal lung tissue, but when inflammation and fibrosis occur in lung injury, avβ6 is rapidly expressed (Hatley et al, Angewandte Chemie International Edition, 2018, 57(13): 3298.). In patients with primary biliary cirrhosis (PBC), alcoholic fatty liver disease, hepatitis B, hepatitis C and other diseases, the mRNA expression of integrin avβ6 is increased. In chronic inflammatory and fibrotic diseases related to kidney disease, the expression of integrin avβ6 is significantly increased compared with normal kidney tissue. In addition, integrin avβ6 is significantly highly expressed in biopsy samples of patients with diabetes, Goodpasture's syndrome, Alport syndrome, lupus nephritis, etc. (Koivisto et al, The international journal of biochemistry & cell biology, 2018, 99: 186). Integrin αvβ8 is extremely important for the regulation of TGFβ. Its only binding ligand is L-TGFβ, which is highly expressed in a variety of cancer cells, especially tumor cells with high Tregs. L-TGFβ is mainly expressed in immune cells such as T cells. Researchers have found that αvβ8 promotes the differentiation of Tregs in the tumor microenvironment, thereby promoting tumor growth. The mechanism of action is that after T cell GARP/L-TGFbeta binds to αvβ8, Conformational changes occur, releasing activated TGFβ, which binds to TGFβR on T cells and promotes the differentiation of T cells into Tregs. In addition, in the IL-1β and albumin-induced mouse airway fibrosis model, conditional deletion of lung fibroblasts αvβ8 can inhibit mouse airway fibrosis.
组织纤维化可发生于多种器官，是一种较为常见的纤维化疾病包括特发性肺纤维化(IPF)、非酒精性脂肪肝(NASH)、肝硬化、肾纤维化、硬皮症、心肌纤维化等。组织损伤和炎症是纤维化的重要诱因。由于炎症导致器官实质细胞发生坏死，局部免疫细胞被激活，多种血细胞进入损伤部位。被激活的免疫细胞产生大量具有高度生物活性的细胞因子和趋化因子，导致间充质细胞的局部激活，这些细胞产生细胞外基质(ECM)，破坏细胞外微环境，并进一步增加促炎细胞因子、趋化因子和血管生成因子的产生。最终，细胞外基质异常增多和过度沉积而发生病变致使组织纤维化(Ricard-Blumet al,Matrix Biology,2018,68:122.)。纤维化的主要特征是形成和沉积过多的纤维结缔组织。慢性的伴纤维化损伤会使组织结构被破坏、器官功能发生障碍最终导致器官衰竭。Tissue fibrosis can occur in many organs and is a common fibrotic disease, including idiopathic pulmonary fibrosis (IPF), non-alcoholic fatty liver disease (NASH), cirrhosis, renal fibrosis, scleroderma, myocardial fibrosis, etc. Tissue damage and inflammation are important causes of fibrosis. Due to inflammation, organ parenchymal cells undergo necrosis, local immune cells are activated, and a variety of blood cells enter the site of injury. The activated immune cells produce a large number of highly biologically active cytokines and chemokines, leading to local activation of mesenchymal cells. These cells produce extracellular matrix (ECM), destroy the extracellular microenvironment, and further increase the production of proinflammatory cytokines, chemokines, and angiogenic factors. Ultimately, the abnormal increase and excessive deposition of extracellular matrix lead to pathological changes and tissue fibrosis (Ricard-Blumet al, Matrix Biology, 2018, 68: 122.). The main feature of fibrosis is the formation and deposition of excessive fibrous connective tissue. Chronic fibrotic damage can destroy tissue structure, impair organ function, and eventually lead to organ failure.
整合素家族作为慢性炎症、纤维化以及肿瘤免疫等关键调控因子备受关注，亟需开发一类结构新颖的、安全有效的整合素抑制剂。The integrin family has attracted much attention as a key regulatory factor in chronic inflammation, fibrosis, and tumor immunity. There is an urgent need to develop a class of integrin inhibitors with novel structures that are safe and effective.
发明内容Summary of the invention
本发明的目的在于提供一类结构新颖的双环衍生物的整合素抑制剂，此类化合物结构中含有双环结构，对整合素αvβ1、αvβ6和αvβ8具有较好的抑制活性和一定的药物选择性，优异的口服药代性质，且能够有效抑制纤维连接蛋白和胶原蛋白在纤维化疾病体内外药效模型中的表达量升高。The purpose of the present invention is to provide a class of integrin inhibitors with novel structures of bicyclic derivatives. Such compounds contain a bicyclic structure, have good inhibitory activity and certain drug selectivity against integrins αvβ1, αvβ6 and αvβ8, excellent oral pharmacokinetic properties, and can effectively inhibit the increased expression of fibronectin and collagen in in vitro and in vivo pharmacodynamic models of fibrotic diseases.
在本发明的第一方面，提供了一种如式I所示的一类双环衍生物，及其消旋体、立体异构体、互变异构体、同位素标记物、氮氧化物、溶剂化物、多晶型物、代谢产物、酯、前药或其药学上可接受的盐：
In the first aspect of the present invention, there is provided a class of bicyclic derivatives as shown in Formula I, and racemates, stereoisomers, tautomers, isotope-labeled products, nitrogen oxides, solvates, polymorphs, metabolites, esters, prodrugs or pharmaceutically acceptable salts thereof:
其中，in,
Y1为C1～C6亚烷基，-O-，-(C1～C6亚烷基)-O-，-NH-，-(C1～C6亚烷基)-NH-； Y1 is C1~C6 alkylene, -O-, -(C1~C6 alkylene)-O-, -NH-, -(C1~C6 alkylene)-NH-;
Y2为C1～C6亚烷基，-O-，-(C1～C6亚烷基)-O-，-C(O)-(C1～C6亚烷基)-NH-，-NH-，-NH-(C1～C6亚烷基)-，-(C1～C6亚烷基)-NH-； Y2 is C1~C6 alkylene, -O-, -(C1~C6 alkylene)-O-, -C(O)-(C1~C6 alkylene)-NH-, -NH-, -NH-(C1~C6 alkylene)-, -(C1~C6 alkylene)-NH-;
R1为取代或未取代的6-10元芳环，取代或未取代的5-8元杂芳环，取代或未取代的其中C环、D环各自独立地为取代或未取代的C6-C10芳环、取代或未取代的5-8元杂芳环，取代或未取代的5-8元环烷环，或取代或未取代的5-8元杂烷环； R1 is a substituted or unsubstituted 6-10 membered aromatic ring, a substituted or unsubstituted 5-8 membered heteroaromatic ring, a substituted or unsubstituted wherein the C ring and the D ring are each independently a substituted or unsubstituted C6-C10 aromatic ring, a substituted or unsubstituted 5-8 membered heteroaromatic ring, a substituted or unsubstituted 5-8 membered cycloalkane ring, or a substituted or unsubstituted 5-8 membered heteroalkane ring;
R2为氢原子，取代或未取代的C6-C10芳环，取代或未取代的5-8元杂芳环，取代或未取代的C8-C16稠环，或-L1-L2； R2 is a hydrogen atom, a substituted or unsubstituted C6-C10 aromatic ring, a substituted or unsubstituted 5-8 membered heteroaromatic ring, a substituted or unsubstituted C8-C16 condensed ring, or -L1-L2;
其中，-L1-选自无、-(取代或未取代的C1-C6亚烷基)-、-(取代或未取代的C1-C6亚烷氧 基)-、-(取代或未取代的C1-C6亚烷硫基)-、-(取代或未取代的C3-C8环烷基)-、-(取代或未取代的C3-C8杂环烷基)-、-(取代或未取代的C6-C10芳基)-、-(取代或未取代的C5-C8杂芳基)-，Wherein, -L1- is selected from none, -(substituted or unsubstituted C1-C6 alkylene)-, -(substituted or unsubstituted C1-C6 alkyleneoxy) -(substituted or unsubstituted C1-C6 alkylenethio)-, -(substituted or unsubstituted C3-C8 cycloalkyl)-, -(substituted or unsubstituted C3-C8 heterocycloalkyl)-, -(substituted or unsubstituted C6-C10 aryl)-, -(substituted or unsubstituted C5-C8 heteroaryl)-,
L2选自无、取代或未取代的C1-C6烷基、取代或未取代的C1-C6烷氧基、取代或未取代的C1-C6烷硫基、取代或未取代的C3-C8环烷基、-(C1-C3烷基)-取代或未取代的C3-C8环烷基、-(C1-C3烷氧基)-取代或未取代的C3-C8环烷基、-O-取代或未取代的C3-C8环烷基、取代或未取代的C3-C8杂烷环基、-(C1-C3烷基)-取代或未取代的C3-C8杂烷环基、-(C1-C3烷氧基)-取代或未取代的C3-C8杂烷环基、-O-取代或未取代的C3-C8杂烷环基、取代或未取代的5-8元杂芳基；L2 is selected from none, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C1-C6 alkylthio, substituted or unsubstituted C3-C8 cycloalkyl, -(C1-C3 alkyl)-substituted or unsubstituted C3-C8 cycloalkyl, -(C1-C3 alkoxy)-substituted or unsubstituted C3-C8 cycloalkyl, -O-substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C3-C8 heteroalkylcyclyl, -(C1-C3 alkyl)-substituted or unsubstituted C3-C8 heteroalkylcyclyl, -(C1-C3 alkoxy)-substituted or unsubstituted C3-C8 heteroalkylcyclyl, -O-substituted or unsubstituted C3-C8 heteroalkylcyclyl, substituted or unsubstituted 5-8 membered heteroaryl;
X为氧原子或氮原子；X is an oxygen atom or a nitrogen atom;
其中，当X为氧原子时，R3a为氢原子，C1～C6烷基，取代或未取代的C6-C10芳环，R3b不存在；当X为氮原子时，R3a为氢原子，羟基，C1～C6烷基，取代或未取代的C6-C10芳环，R3b为氢原子；Wherein, when X is an oxygen atom, R 3a is a hydrogen atom, a C1-C6 alkyl group, a substituted or unsubstituted C6-C10 aromatic ring, and R 3b is absent; when X is a nitrogen atom, R 3a is a hydrogen atom, a hydroxyl group, a C1-C6 alkyl group, a substituted or unsubstituted C6-C10 aromatic ring, and R 3b is a hydrogen atom;
为取代或未取代的螺环或取代或未取代的并环； is a substituted or unsubstituted spiro ring or a substituted or unsubstituted cyclocyclic ring;
其中，当为取代或未取代的螺环，如式Ia所示：
Among them, when is a substituted or unsubstituted spiro ring, as shown in Formula Ia:
A为选自下组的环：取代或未取代的四元环烷环，取代或未取代的五元环烷环，取代或未取代的六元环烷环，取代或未取代的七元环烷环，取代或未取代的八元环烷环、取代或未取代的四元杂烷环，取代或未取代的五元杂烷环，取代或未取代的六元杂烷环，取代或未取代的七元杂烷环，取代或未取代的八元杂烷环，取代或未取代的五元杂烷环并C6-C10芳香环，取代或未取代的六元杂烷环并C6-C10芳香环；A is a ring selected from the group consisting of a substituted or unsubstituted four-membered cycloalkane ring, a substituted or unsubstituted five-membered cycloalkane ring, a substituted or unsubstituted six-membered cycloalkane ring, a substituted or unsubstituted seven-membered cycloalkane ring, a substituted or unsubstituted eight-membered cycloalkane ring, a substituted or unsubstituted four-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring, a substituted or unsubstituted six-membered heteroalkane ring, a substituted or unsubstituted seven-membered heteroalkane ring, a substituted or unsubstituted eight-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring and a C6-C10 aromatic ring, and a substituted or unsubstituted six-membered heteroalkane ring and a C6-C10 aromatic ring;
B为选自下组的环：取代或未取代的四元环烷环，取代或未取代的五元环烷环，取代或未取代的六元环烷环，取代或未取代的七元环烷环，取代或未取代的八元环烷环、取代或未取代的四元杂烷环，取代或未取代的五元杂烷环，取代或未取代的六元杂烷环，取代或未取代的七元杂烷环，取代或未取代的八元杂烷环，取代或未取代的五元杂烷环并C6-C10芳香环，取代或未取代的六元杂烷环并C6-C10芳香环；B is a ring selected from the group consisting of a substituted or unsubstituted four-membered cycloalkane ring, a substituted or unsubstituted five-membered cycloalkane ring, a substituted or unsubstituted six-membered cycloalkane ring, a substituted or unsubstituted seven-membered cycloalkane ring, a substituted or unsubstituted eight-membered cycloalkane ring, a substituted or unsubstituted four-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring, a substituted or unsubstituted six-membered heteroalkane ring, a substituted or unsubstituted seven-membered heteroalkane ring, a substituted or unsubstituted eight-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring and a C6-C10 aromatic ring, and a substituted or unsubstituted six-membered heteroalkane ring and a C6-C10 aromatic ring;
当为取代或未取代的并环，如式Ib所示：
when is a substituted or unsubstituted cyclic ring, as shown in Formula Ib:
为单键或者双键； is a single bond or a double bond;
A为选自下组的环：取代或未取代的四元环烷环，取代或未取代的五元环烷环，取代或未取代的六元环烷环，取代或未取代的七元环烷环，取代或未取代的八元环烷环、取代或未取代的四元杂烷环，取代或未取代的五元杂烷环，取代或未取代的六元杂烷环，取代或未取 代的七元杂烷环，取代或未取代的八元杂烷环，取代或未取代的五元杂烷环并C6-C10芳香环，取代或未取代的六元杂烷环并C6-C10芳香环；A is a ring selected from the group consisting of a substituted or unsubstituted four-membered cycloalkane ring, a substituted or unsubstituted five-membered cycloalkane ring, a substituted or unsubstituted six-membered cycloalkane ring, a substituted or unsubstituted seven-membered cycloalkane ring, a substituted or unsubstituted eight-membered cycloalkane ring, a substituted or unsubstituted four-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring, a substituted or unsubstituted six-membered heteroalkane ring, a substituted or unsubstituted a substituted seven-membered heteroalkane ring, a substituted or unsubstituted eight-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring and a C6-C10 aromatic ring, a substituted or unsubstituted six-membered heteroalkane ring and a C6-C10 aromatic ring;
B为选自下组的环：取代或未取代的四元环烷环，取代或未取代的五元环烷环，取代或未取代的六元环烷环，取代或未取代的七元环烷环，取代或未取代的八元环烷环、取代或未取代的C6-C10芳香环，取代或未取代的四元杂烷环，取代或未取代的五元杂烷环，取代或未取代的六元杂烷环，取代或未取代的七元杂烷环，取代或未取代的八元杂烷环，取代或未取代的五元杂烷环并C6-C10芳香环，取代或未取代的六元杂烷环并C6-C10芳香环；B is a ring selected from the group consisting of a substituted or unsubstituted four-membered cycloalkane ring, a substituted or unsubstituted five-membered cycloalkane ring, a substituted or unsubstituted six-membered cycloalkane ring, a substituted or unsubstituted seven-membered cycloalkane ring, a substituted or unsubstituted eight-membered cycloalkane ring, a substituted or unsubstituted C6-C10 aromatic ring, a substituted or unsubstituted four-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring, a substituted or unsubstituted six-membered heteroalkane ring, a substituted or unsubstituted seven-membered heteroalkane ring, a substituted or unsubstituted eight-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring and a C6-C10 aromatic ring, and a substituted or unsubstituted six-membered heteroalkane ring and a C6-C10 aromatic ring;
n为0、1、2或3；n is 0, 1, 2 or 3;
其中，所述“取代”是指基团上的1-4个(优选为1、2、3或4个)氢原子各自独立地被选自下组的取代基所取代：C1-C6烷基、C3-C8环烷基、C3-C8杂环烷基、C1-C3卤代烷基、卤素、硝基、氰基、氨基、羟基、硫醇基、＝O、C1-C4羧基、C2-C4酯基、C2-C4酰胺基、C1-C6烷氧基、羧酸、C1-C4醇基、C1-C4烷胺基、-O-(CH2)m-C3-C8环烷、-O-(CH2)m-C3-C8杂烷环、-NH-(CH2)m-C3-C8环烷环、-NH-(CH2)m-C3-C8杂烷环；其中各个m各自独立地为0～3的整数；Wherein, the "substituted" means that 1 to 4 (preferably 1, 2, 3 or 4) hydrogen atoms on the group are independently replaced by substituents selected from the following group: C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, C1-C3 haloalkyl, halogen, nitro, cyano, amino, hydroxyl, thiol, =O, C1-C4 carboxyl, C2-C4 ester, C2-C4 amide, C1-C6 alkoxy, carboxylic acid, C1-C4 alcohol, C1-C4 alkylamino, -O-(CH 2 ) m -C3-C8 cycloalkane, -O-(CH 2 ) m -C3-C8 heteroalkyl ring, -NH-(CH 2 ) m -C3-C8 cycloalkane ring, -NH-(CH 2 ) m -C3-C8 heteroalkyl ring; wherein each m is independently an integer of 0 to 3;
其中，所述的杂芳环、杂烷环或杂芳基各自独立地具有1-3个(优选为1、2或3个)选自N、O和S杂原子。Wherein, the heteroaromatic ring, heteroalkyl ring or heteroaryl group each independently has 1-3 (preferably 1, 2 or 3) heteroatoms selected from N, O and S.
在另一优选例中，Y1为无。In another preferred embodiment, Y1 is none.
在另一优选例中，Y1为C1～C3亚烷基，-(C1～C3亚烷基)-NH-。In another preferred embodiment, Y1 is C1~C3 alkylene, -(C1~C3 alkylene)-NH-.
在另一优选例中，Y1为C1-C3亚烷基。In another preferred embodiment, Y 1 is C1-C3 alkylene.
在另一优选例中，Y1为亚甲基。In another preferred embodiment, Y1 is a methylene group.
在另一优选例中，Y2为C1～C3亚烷基，-O-，-(C1～C3亚烷基)-O-，-C(O)-(C1～C3亚烷基)-NH-，-NH-，-NH-(C1～C3亚烷基)-，-(C1～C3亚烷基)-NH-。在另一优选例中，Y2为C1-C3亚烷基、-C(O)-(C1-C3亚烷基)-NH-、-(C1-C3亚烷基)-NH-。In another preferred embodiment, Y2 is C1-C3 alkylene, -O-, -(C1-C3 alkylene)-O-, -C(O)-(C1-C3 alkylene)-NH-, -NH-, -NH-(C1-C3 alkylene)-, -(C1-C3 alkylene)-NH-. In another preferred embodiment, Y2 is C1-C3 alkylene, -C(O)-(C1-C3 alkylene)-NH-, -(C1-C3 alkylene)-NH-.
在另一优选例中，Y2为-O-，-(C1～C3亚烷基)-O-。In another preferred embodiment, Y2 is -O-, -(C1-C3 alkylene)-O-.
在另一优选例中，Y2为C1-C3亚烷基。In another preferred embodiment, Y2 is C1-C3 alkylene.
在另一优选例中，Y2为亚甲基、亚乙基或-O-。In another preferred embodiment, Y2 is methylene, ethylene or -O-.
在另一优选例中，R1为取代或未取代的其中C环为C6-C10芳环或5-8元杂芳环，D环为取代或未取代的5-8元杂芳环或取代或未取代的5-8元杂烷环。In another preferred embodiment, R 1 is substituted or unsubstituted The C ring is a C6-C10 aromatic ring or a 5-8 membered heteroaromatic ring, and the D ring is a substituted or unsubstituted 5-8 membered heteroaromatic ring or a substituted or unsubstituted 5-8 membered heteroalkyl ring.
在另一优选例中，R1为取代或未取代的其中C环为C6芳环或5-6元含氮杂芳环，D环为取代或未取代的5-6元含氮杂芳环或取代或未取代的5-6元含氮杂烷环。In another preferred embodiment, R 1 is substituted or unsubstituted The C ring is a C6 aromatic ring or a 5-6-membered nitrogen-containing heteroaromatic ring, and the D ring is a substituted or unsubstituted 5-6-membered nitrogen-containing heteroaromatic ring or a substituted or unsubstituted 5-6-membered nitrogen-containing heteroalkane ring.
在另一优选例中，所述C环或D环的含氮杂芳环或含氮杂烷环任选地还包括1或2个O原子。In another preferred embodiment, the nitrogen-containing heteroaromatic ring or nitrogen-containing heteroalkyl ring of the C ring or the D ring optionally further includes 1 or 2 O atoms.
在另一优选例中，R1为取代或未取代的其中D环为取代或未取代的5-6元含氮杂芳环或取代或未取代的5-6元含氮杂烷环。In another preferred embodiment, R 1 is substituted or unsubstituted The D ring is a substituted or unsubstituted 5-6 membered nitrogen-containing heteroaromatic ring or a substituted or unsubstituted 5-6 membered nitrogen-containing heteroalkane ring.
在另一优选例中，R1为取代或未取代的5-8元含氮杂芳环。In another preferred embodiment, R 1 is a substituted or unsubstituted 5-8 membered nitrogen-containing heteroaromatic ring.
在另一优选例中，R1为取代或未取代的选自下组的基团：
In another preferred embodiment, R 1 is a substituted or unsubstituted group selected from the following group:
在另一优选例中，R1为取代或未取代的吡啶环，取代或未取代的1,2,3,4-四氢萘啶环，取代或未取代的喹啉环，或取代或未取代的氮杂吲哚环。In another preferred embodiment, R 1 is a substituted or unsubstituted pyridine ring, a substituted or unsubstituted 1,2,3,4-tetrahydronaphthyridine ring, a substituted or unsubstituted quinoline ring, or a substituted or unsubstituted azaindole ring.
在另一优选例中，R1为 In another preferred embodiment, R 1 is
在另一优选例中，R2为被Rc取代的C6-C10芳环(优选为C6芳环)，其中Rc为氢、卤素、C1-C6烷基、C3-C8环烷基、C1-C6烷氧基。In another preferred embodiment, R 2 is a C6-C10 aromatic ring (preferably a C6 aromatic ring) substituted by R c , wherein R c is hydrogen, halogen, C1-C6 alkyl, C3-C8 cycloalkyl, or C1-C6 alkoxy.
在另一优选例中，R2中，所述C8-C16稠环是指两个或三个环稠合的环碳原子数为8个至16个的环，稠合环中的各个环各自独立地为6元芳环、5-6元杂芳环、5-6元环烷环、5-6元杂烷环。In another preferred embodiment, in R 2 , the C8-C16 fused ring refers to a ring with 8 to 16 carbon atoms in which two or three rings are fused, and each ring in the fused ring is independently a 6-membered aromatic ring, a 5-6-membered heteroaromatic ring, a 5-6-membered cycloalkane ring, or a 5-6-membered heteroalkane ring.
在另一优选例中，R2为C9-C10稠环。In another preferred embodiment, R 2 is a C9-C10 fused ring.
在另一优选例中，R2中，所述C9-C10稠环优选为6元芳环与5-6元杂芳环稠合，更优选为6元芳环与6元杂芳环稠合。In another preferred embodiment, in R 2 , the C9-C10 fused ring is preferably a 6-membered aromatic ring fused with a 5-6-membered heteroaromatic ring, more preferably a 6-membered aromatic ring fused with a 6-membered heteroaromatic ring.
在另一优选例中，R2为氢原子或-L1-L2；其中，-L1-为-(取代或未取代的C6-C10芳基)-，L2为取代或未取代的5-8元杂芳基。In another preferred embodiment, R2 is a hydrogen atom or -L1-L2; wherein -L1- is -(substituted or unsubstituted C6-C10 aryl)-, and L2 is a substituted or unsubstituted 5-8 membered heteroaryl.
在另一优选例中，R2为-L1-L2。In another preferred embodiment, R2 is -L1-L2.
在另一优选例中，-L1-选自-(取代或未取代的C3-C8环烷基)-、-(取代或未取代的C3-C8杂环烷基)-、-(取代或未取代的C6-C10芳基)-、-(取代或未取代的C5-C8杂芳基)-。In another preferred embodiment, -L1- is selected from -(substituted or unsubstituted C3-C8 cycloalkyl)-, -(substituted or unsubstituted C3-C8 heterocycloalkyl)-, -(substituted or unsubstituted C6-C10 aryl)-, and -(substituted or unsubstituted C5-C8 heteroaryl)-.
在另一优选例中，-L1-选自-(取代或未取代的C6-C10芳基)-、-(取代或未取代的5-8元杂芳基)-。In another preferred embodiment, -L1- is selected from -(substituted or unsubstituted C6-C10 aryl)-, -(substituted or unsubstituted 5-8 membered heteroaryl)-.
在另一优选例中，-L1-选自-(取代或未取代的C6芳基)-、-(取代或未取代的6元杂芳基)-。In another preferred embodiment, -L1- is selected from -(substituted or unsubstituted C6 aryl)-, -(substituted or unsubstituted 6-membered heteroaryl)-.
在另一优选例中，L2为无、取代或未取代的C1-C6烷基、取代或未取代的C1-C6烷氧基、取代或未取代的C1-C6烷硫基、取代或未取代的C3-C8环烷基、-(C1-C3亚烷基)-取代或未取代的C3-C8环烷基、-(C1-C3亚烷氧基)-取代或未取代的C3-C8环烷基、-O-取代或未取代的C3-C8环烷基、取代或未取代3-8元杂烷环基、-(C1-C3亚烷基)-取代或未取代的3-8元杂烷环基、-(C1-C3亚烷氧基)-取代或未取代的3-8元杂烷环基、-O-取代或未取代的3-8元杂烷环基、取代或未取代的5-8元杂芳基。In another preferred embodiment, L2 is none, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C1-C6 alkylthio, substituted or unsubstituted C3-C8 cycloalkyl, -(C1-C3 alkylene)-substituted or unsubstituted C3-C8 cycloalkyl, -(C1-C3 alkyleneoxy)-substituted or unsubstituted C3-C8 cycloalkyl, -O-substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted 3-8 membered heteroalkyl ring group, -(C1-C3 alkylene)-substituted or unsubstituted 3-8 membered heteroalkyl ring group, -(C1-C3 alkyleneoxy)-substituted or unsubstituted 3-8 membered heteroalkyl ring group, -O-substituted or unsubstituted 3-8 membered heteroalkyl ring group, substituted or unsubstituted 5-8 membered heteroaryl.
在另一优选例中，L2选自取代或未取代的3-8元环烷基、-(C1-C3亚烷基)-取代或未取代的C3-C8环烷基、-(C1-C3亚烷氧基)-取代或未取代的3-8元环烷基、-O-取代或未取代的3-8元环烷基、取代或未取代的3-8元杂烷环基、-(C1-C3亚烷基)-取代或未取代的3-8元杂烷环基、-(C1-C3亚烷氧基)-取代或未取代的3-8元杂烷环基、-O-取代或未取代的3-8元杂烷环基、取代或未取代的5-8元杂芳基。In another preferred embodiment, L2 is selected from substituted or unsubstituted 3-8 membered cycloalkyl, -(C1-C3 alkylene)-substituted or unsubstituted C3-C8 cycloalkyl, -(C1-C3 alkyleneoxy)-substituted or unsubstituted 3-8 membered cycloalkyl, -O-substituted or unsubstituted 3-8 membered cycloalkyl, substituted or unsubstituted 3-8 membered heteroalkyl ring group, -(C1-C3 alkylene)-substituted or unsubstituted 3-8 membered heteroalkyl ring group, -(C1-C3 alkyleneoxy)-substituted or unsubstituted 3-8 membered heteroalkyl ring group, -O-substituted or unsubstituted 3-8 membered heteroalkyl ring group, substituted or unsubstituted 5-8 membered heteroaryl group.
在另一优选例中，L2选自取代或未取代的3-8元杂烷环基、取代或未取代的5-8元杂芳基。 In another preferred embodiment, L2 is selected from a substituted or unsubstituted 3-8 membered heteroalkyl ring group, and a substituted or unsubstituted 5-8 membered heteroaryl group.
在另一优选例中，-L1-为-(取代或未取代的C6-C10芳基)-，L2为取代或未取代的5-8元杂芳基。In another preferred embodiment, -L1- is -(substituted or unsubstituted C6-C10 aryl)-, and L2 is a substituted or unsubstituted 5-8 membered heteroaryl.
在另一优选例中，-L1-为-(取代或未取代的苯基)-，L2为取代或未取代的5-6元含氮杂芳基(优选吡唑基)。In another preferred embodiment, -L1- is -(substituted or unsubstituted phenyl)-, and L2 is a substituted or unsubstituted 5-6-membered nitrogen-containing heteroaryl group (preferably pyrazolyl).
在另一优选例中，L1任选地被一个或多个Ra取代，其中Ra为卤素、C1-C6烷基、C3-C6环烷基。In another preferred embodiment, L1 is optionally substituted by one or more Ra , wherein Ra is halogen, C1-C6 alkyl, or C3-C6 cycloalkyl.
在另一优选例中，L1任选地被一个或多个Ra取代，其中Ra为卤素、C1-C6烷基、。In another preferred embodiment, L1 is optionally substituted by one or more Ra , wherein Ra is halogen, C1-C6 alkyl, etc.
在另一优选例中，L2任选地被一个或多个Rb取代，其中Rb为C1-C3烷基。In another preferred embodiment, L2 is optionally substituted by one or more R b , wherein R b is C1-C3 alkyl.
在另一优选例中，R2为如下式a结构：
In another preferred embodiment, R 2 is a structure of the following formula:
其中，Z1、Z2、Z3、Z4各自独立地为CRc或N；wherein Z 1 , Z 2 , Z 3 , and Z 4 are each independently CR c or N;
L2为无、C1-C6烷基、取代或未取代的C1-C6烷氧基、取代或未取代的C1-C6烷硫基、取代或未取代的3-8元环烷基、取代或未取代的3-8元杂烷环基、取代或未取代的5-8元杂芳基； L2 is none, C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C1-C6 alkylthio, substituted or unsubstituted 3-8 membered cycloalkyl, substituted or unsubstituted 3-8 membered heteroalkyl ring group, substituted or unsubstituted 5-8 membered heteroaryl;
各个Rc各自独立地为氢、卤素、C1-C6烷基、3-8元环烷基、C1-C6烷氧基。Each R c is independently hydrogen, halogen, C1-C6 alkyl, 3-8 membered cycloalkyl, or C1-C6 alkoxy.
在另一优选例中，R2为如下式b结构：
In another preferred embodiment, R 2 is a structure of the following formula b:
其中，Z1、Z2、Z3、Z4、Z5、Z6、Z7、Z8、Z9各自独立地为CRc或N；wherein Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , Z 6 , Z 7 , Z 8 , and Z 9 are each independently CR c or N;
各个Rc各自独立地为氢、卤素、C1-C6烷基、C3-C8环烷基、C1-C6烷氧基。Each R c is independently hydrogen, halogen, C1-C6 alkyl, C3-C8 cycloalkyl, or C1-C6 alkoxy.
在另一优选例中，Z1、Z2、Z4各自独立地为CRc，Z3为CRc或N。In another preferred embodiment, Z 1 , Z 2 , and Z 4 are each independently CR c , and Z 3 is CR c or N.
在另一优选例中，Z1、Z2、Z4各自独立地为CH，Z3为CRc或N。In another preferred embodiment, Z 1 , Z 2 , and Z 4 are each independently CH, and Z 3 is CR c or N.
在另一优选例中，Z5、Z6、Z7各自独立地为CRc或N，且Z5、Z6、Z7中的至少一个为N。在另一优选例中，R2为如下结构：
In another preferred embodiment, Z 5 , Z 6 , and Z 7 are each independently CR c or N, and at least one of Z 5 , Z 6 , and Z 7 is N. In another preferred embodiment, R 2 is the following structure:
其中，L2为C1-C6烷基、取代或未取代的C1-C6烷氧基、取代或未取代的C1-C6烷硫基；Wherein, L2 is C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C1-C6 alkylthio;
各个Rc各自独立地为氢、卤素、C1-C6烷基、C3-C8环烷基、C1-C6烷氧基。 Each R c is independently hydrogen, halogen, C1-C6 alkyl, C3-C8 cycloalkyl, or C1-C6 alkoxy.
在另一优选例中，各个Rc各自独立地为氟、溴、氯、甲基、乙基、正丙基、异丙基、正丁基、叔丁基、异丁基、环丙基、环丁基、环戊基、环己基、甲氧基、乙氧基。In another preferred embodiment, each R c is independently fluorine, bromine, chlorine, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, methoxy, or ethoxy.
在另一优选例中，R2为取代或未取代的 In another preferred embodiment, R2 is substituted or unsubstituted
在另一优选例中，R2为 In another preferred embodiment, R2 is
在另一优选例中，X为氧原子时，R3a为氢原子或C1～C3烷基，R3b不存在。In another preferred embodiment, when X is an oxygen atom, R 3a is a hydrogen atom or a C1-C3 alkyl group, and R 3b does not exist.
在另一优选例中，X为氧原子时，R3a为氢原子，R3b不存在。In another preferred embodiment, when X is an oxygen atom, R 3a is a hydrogen atom, and R 3b does not exist.
在另一优选例中，X为氮原子时，R3a为氢原子、羟基或C1～C3烷基，R3b为氢原子。In another preferred embodiment, when X is a nitrogen atom, R 3a is a hydrogen atom, a hydroxyl group or a C1-C3 alkyl group, and R 3b is a hydrogen atom.
另一优选例中，X为氮原子时，R3a为羟基，R3b为氢原子。In another preferred embodiment, when X is a nitrogen atom, R 3a is a hydroxyl group, and R 3b is a hydrogen atom.
在另一优选例中，为取代或未取代的螺环，如式Ia所示：
In another preferred embodiment, is a substituted or unsubstituted spiro ring, as shown in Formula Ia:
在另一优选例中，A为选自下组的环：取代或未取代的四元杂烷环，取代或未取代的五元杂烷环，取代或未取代的六元杂烷环，取代或未取代的七元杂烷环，取代或未取代的八元杂烷环；In another preferred embodiment, A is a ring selected from the group consisting of a substituted or unsubstituted four-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring, a substituted or unsubstituted six-membered heteroalkane ring, a substituted or unsubstituted seven-membered heteroalkane ring, and a substituted or unsubstituted eight-membered heteroalkane ring;
B为选自下组的环：取代或未取代的四元杂烷环，取代或未取代的五元杂烷环，取代或未取代的六元杂烷环，取代或未取代的七元杂烷环，取代或未取代的八元杂烷环。B is a ring selected from the group consisting of a substituted or unsubstituted four-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring, a substituted or unsubstituted six-membered heteroalkane ring, a substituted or unsubstituted seven-membered heteroalkane ring, and a substituted or unsubstituted eight-membered heteroalkane ring.
在另一优选例中，A、B各自独立地为选自下组的环：四元杂烷环，取代或未取代的五元杂烷环，取代或未取代的六元杂烷环。In another preferred embodiment, A and B are each independently a ring selected from the following group: a four-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring, and a substituted or unsubstituted six-membered heteroalkane ring.
在另一优选例中，所述A环和B环中的取代是指一个或多个(例如1、2、3个)氢原子被卤素或＝O取代。In another preferred embodiment, the substitution in the A ring and the B ring refers to one or more (eg, 1, 2, 3) hydrogen atoms being replaced by halogen or =O.
在另一优选例中，所述卤素为F、Cl、Br，优选为F。In another preferred embodiment, the halogen is F, Cl, or Br, preferably F.
在另一优选例中，所述A环和B环中的杂烷环各自独立地包含1-3(优选1、2、3)个氮原子。In another preferred embodiment, the heteroalkyl rings in the A ring and the B ring each independently contain 1-3 (preferably 1, 2, 3) nitrogen atoms.
在另一优选例中，A环和B环各自独立地为选自下组的环：氮杂环丁烷、取代或未取代的吡咯烷、取代或未取代的哌啶环、取代或未取代的吗啉环。In another preferred embodiment, the A ring and the B ring are each independently a ring selected from the following group: azetidine, substituted or unsubstituted pyrrolidine, substituted or unsubstituted piperidine ring, substituted or unsubstituted morpholine ring.
在另一优选例中，所述A环和B环中，所述取代优选为3位取代。In another preferred embodiment, in the A ring and the B ring, the substitution is preferably at the 3-position.
在另一优选例中，所述A环和B环中，所述取代优选在螺原子的邻位取代。In another preferred embodiment, in the A ring and the B ring, the substitution is preferably at the ortho position of the spiro atom.
在另一优选例中，所述A环和B环各自独立地为以下结构：
In another preferred embodiment, the A ring and the B ring are each independently the following structure:
其中，“*”表示该碳原子为螺原子。Here, “*” indicates that the carbon atom is a spiro atom.
在另一优选例中，Y1通过杂烷环中的氮原子连接到A环。In another preferred embodiment, Y 1 is connected to the A ring through the nitrogen atom in the heteroalkyl ring.
在另一优选例中，B环通过杂烷环中的氮原子连接到Y2。In another preferred embodiment, the B ring is connected to Y 2 via a nitrogen atom in the heteroalkyl ring.
在另一优选例中，所述化合物为如下式II结构：
In another preferred embodiment, the compound is of the following formula II:
在另一优选例中，所述A环的环原子数与所述B环的环原子数相等或不相等。In another preferred embodiment, the number of ring atoms in the A ring is equal to or different from the number of ring atoms in the B ring.
在另一优选例中，所述A环的环原子数小于、等于或大于B环的环原子数。In another preferred embodiment, the number of ring atoms in the A ring is less than, equal to or greater than the number of ring atoms in the B ring.
在另一优选例中，A为杂烷环时，Y1通过杂烷环中的氮原子连接到A环。In another preferred embodiment, when A is a heteroalkyl ring, Y1 is connected to the A ring through the nitrogen atom in the heteroalkyl ring.
在另一优选例中，当A为环烷环时，Y1为-O-，-(C1～C6亚烷基)-O-，-NH-或-(C1～C6亚烷基)-NH-。In another preferred embodiment, when A is a cycloalkane ring, Y1 is -O-, -(C1-C6 alkylene)-O-, -NH- or -(C1-C6 alkylene)-NH-.
在另一优选例中，B为杂烷环时，Y2通过杂烷环中的氮原子连接到B环。In another preferred embodiment, when B is a heteroalkyl ring, Y2 is connected to the B ring through the nitrogen atom in the heteroalkyl ring.
在另一优选例中，当B为烷环时，Y2为-O-，-(C1～C6亚烷基)-O-，-NH-或-(C1～C6亚烷基)-NH-。In another preferred embodiment, when B is an alkyl ring, Y2 is -O-, -(C1-C6 alkylene)-O-, -NH- or -(C1-C6 alkylene)-NH-.
在另一优选例中，当A为4、5、6、7、8元环时，B为4、5、6、7、8元环。In another preferred embodiment, when A is a 4-, 5-, 6-, 7- or 8-membered ring, B is a 4-, 5-, 6-, 7- or 8-membered ring.
在另一优选例中，所述化合物为如下式III-1结构：
In another preferred embodiment, the compound is of the following formula III-1:
在另一优选例中，所述化合物为如下式III-2结构：
In another preferred embodiment, the compound is of the following formula III-2:
在另一优选例中，所述化合物为如下式III-3结构：
In another preferred embodiment, the compound is of the following formula III-3:
在另一优选例中，所述化合物为如下式III-4结构：
In another preferred embodiment, the compound is of the following formula III-4:
在另一优选例中，所述化合物为如下式III-5结构：
In another preferred embodiment, the compound is of the following formula III-5:
在另一优选例中，所述化合物为如下式III-6结构：
In another preferred embodiment, the compound is of the following formula III-6:
在另一优选例中，在上述式III-1～III-6中，R1、R2、A环、B环如上文所述，Y1、Y2为C1-C3亚烷基。In another preferred embodiment, in the above formulae III-1 to III-6, R 1 , R 2 , Ring A and Ring B are as described above, and Y 1 and Y 2 are C1-C3 alkylene groups.
在另一优选例中，Y1、Y2、R1、R2、X、A、B各自独立地为实施例中所制备的化合物1-122中的对应基团。In another preferred embodiment, Y 1 , Y 2 , R 1 , R 2 , X, A, and B are each independently a corresponding group in Compound 1-122 prepared in Examples.
在另一优选例中，当为取代或未取代的螺环，如式Ia所示：
In another preferred embodiment, when is a substituted or unsubstituted spiro ring, as shown in Formula Ia:
A独立地选自下组的环：取代或未取代的四元杂烷环，取代或未取代的五元杂烷环，取代或未取代的六元杂烷环，取代或未取代的七元杂烷环，取代或未取代的五元杂烷环并6-10元芳香环，取代或未取代的六元杂烷环并6-10元芳香环；A is independently selected from the following rings: substituted or unsubstituted four-membered heteroalkane ring, substituted or unsubstituted five-membered heteroalkane ring, substituted or unsubstituted six-membered heteroalkane ring, substituted or unsubstituted seven-membered heteroalkane ring, substituted or unsubstituted five-membered heteroalkane ring and 6-10-membered aromatic ring, substituted or unsubstituted six-membered heteroalkane ring and 6-10-membered aromatic ring;
B独立地选自下组的环：取代或未取代的四元杂烷环，取代或未取代的五元杂烷环，取代或未取代的六元杂烷环，取代或未取代的七元杂烷环，取代或未取代的五元杂烷环并6-10元芳香环，取代或未取代的六元杂烷环并6-10元芳香环。B is independently selected from the following rings: a substituted or unsubstituted four-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring, a substituted or unsubstituted six-membered heteroalkane ring, a substituted or unsubstituted seven-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring and a 6-10-membered aromatic ring, and a substituted or unsubstituted six-membered heteroalkane ring and a 6-10-membered aromatic ring.
在另一优选例中，当为取代或未取代的并环，如式Ib所示：
In another preferred embodiment, when is a substituted or unsubstituted cyclic ring, as shown in Formula Ib:
为单键或者双键； is a single bond or a double bond;
A独立地选自下组的环：取代或未取代的四元杂烷环，取代或未取代的五元杂烷环，取 代或未取代的六元杂烷环，取代或未取代的七元杂烷环；A is independently selected from the following rings: substituted or unsubstituted four-membered heteroalkane ring, substituted or unsubstituted five-membered heteroalkane ring, a substituted or unsubstituted six-membered heteroalkane ring, a substituted or unsubstituted seven-membered heteroalkane ring;
B独立地选自下组的环：取代或未取代的C6-C10芳香环，取代或未取代的四元杂烷环，取代或未取代的五元杂烷环，取代或未取代的六元杂烷环。B is a ring independently selected from the group consisting of a substituted or unsubstituted C6-C10 aromatic ring, a substituted or unsubstituted four-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring, and a substituted or unsubstituted six-membered heteroalkane ring.
在另一优选例中，所述“取代”是指基团上的1-4个(优选为1、2、3或4个)氢原子各自独立地被选自下组的取代基所取代：卤素、-OH、-CN、-NO2、-NH2、-NCO、-OCN、-SCN、-NCS、-N3、-(C1-C3亚烷基)OH、-O(C1-C3烷基)、-S(C1-C3烷基)、-C(O)X’R4或-X’C(O)R5、-SO3R4、-OSO2OR4、-NR6SO2OR5、-NR6R7、-SO2NR6R7、-NH-SO2-R6、-N+R6R7R8、-C(O)N(R9)2、-SO2R10、-NR11C(O)N(R11)2、-B(OH)2、-B(O(C1-C6烷基或亚烷基))2、-P(O)(OH)3、-OP(O)(OC1-C3烷基)2、C1-C6烷基、C1-C6卤代烷基、C1-C6杂烷基、氧代(＝O)、3-12元烷环基、3-12元杂烷环基、5-10元芳基、5-10元芳杂环基、3-12元杂环基、-O-(CH2)p-3-8元环烷、-O-(CH2)p-3-8元杂烷环、-NH-(CH2)p-3-8元环烷、-NH-(CH2)p-3-8元杂烷环；In another preferred embodiment, the "substituted" means that 1 to 4 (preferably 1, 2, 3 or 4) hydrogen atoms on the group are independently replaced by substituents selected from the following group: halogen, -OH, -CN, -NO 2 , -NH 2 , -NCO, -OCN, -SCN, -NCS, -N 3 , -(C1-C3 alkylene)OH, -O(C1-C3 alkyl), -S(C1-C3 alkyl), -C(O)X'R 4 or -X'C(O)R 5 , -SO 3 R 4 , -OSO 2 OR 4 , -NR 6 SO 2 OR 5 , -NR 6 R 7 , -SO 2 NR 6 R 7 , -NH-SO 2 -R 6 , -N + R 6 R 7 R 8 , -C(O)N(R 9 ) 2 , -SO 2 R 10 , -NR 11 C(O)N(R 11 ) 2 , -B(OH) 2 , -B(O(C1-C6 alkyl or alkylene)) 2 , -P(O)(OH) 3 , -OP(O)(OC1-C3 alkyl) 2 , C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 heteroalkyl, oxo(═O), 3-12 membered alkyl ring group, 3-12 membered heteroalkyl ring group, 5-10 membered aryl group, 5-10 membered aromatic heterocyclic group, 3-12 membered heterocyclic group, -O-(CH 2 ) p -3-8 membered cycloalkane, -O-(CH 2 ) p -3-8 membered heteroalkyl ring, -NH-(CH 2 ) p -3-8 membered cycloalkane, -NH-(CH 2 ) p -3-8 membered heteroalkyl ring;
X'为化学键或为氧或硫，R4独立选自氢、C1-C6烷基、3-12元烷环基、5-10元芳基、3-12元杂环基，R5独立地选自氢、C1-C6烷基、3-12元烷环基、5-10元芳基、3-12元杂环基；X' is a chemical bond or is oxygen or sulfur, R4 is independently selected from hydrogen, C1-C6 alkyl, 3-12-membered alkyl ring group, 5-10-membered aryl group, 3-12-membered heterocyclic group, R5 is independently selected from hydrogen, C1-C6 alkyl, 3-12-membered alkyl ring group, 5-10-membered aryl group, 3-12-membered heterocyclic group;
R6、R7和R8各自独立地选自氢、C1-C6烷基、-C(O)X’R4或-X’C(O)R5，或者，R6和R7与它们所连接的N原子一起构成在环结构中具有4-8元杂环；其中，R6或R7不全为-C(O)X’R4或-X’C(O)R5；R 6 , R 7 and R 8 are each independently selected from hydrogen, C1-C6 alkyl, -C(O)X'R 4 or -X'C(O)R 5 , or, R 6 and R 7 together with the nitrogen atom to which they are attached form a 4-8 membered heterocyclic ring in the ring structure; wherein, R 6 or R 7 are not all -C(O)X'R 4 or -X'C(O)R 5 ;
R9独立地选自氢或C1-C6烷基，或两个R9与它们所连接的N原子一起构成4-8元杂环；R 9 is independently selected from hydrogen or C1-C6 alkyl, or two R 9 together with the N atom to which they are attached form a 4-8 membered heterocyclic ring;
R10独立地选自氢、C1-C6烷基、3-12元烷环基、3-12元杂环基、5-10元芳基；R 10 is independently selected from hydrogen, C1-C6 alkyl, 3-12 membered alkyl ring group, 3-12 membered heterocyclic group, 5-10 membered aryl group;
R11独立地选自氢、C1-C6烷基、或两个R11与它们所连接的N原子一起构成具有4-8元杂环；R 11 is independently selected from hydrogen, C1-C6 alkyl, or two R 11 together with the N atom to which they are attached form a 4-8 membered heterocyclic ring;
p各自独立地为0～3的整数。p is an integer of 0-3 for each independently.
在另一优选例中，所述的双环衍生物选自下组：



In another preferred embodiment, the bicyclic derivative is selected from the following group:



在本发明的第二方面，提供了一种如本发明第一方面所述的双环衍生物，及其消旋体、立体异构体、互变异构体、同位素标记物、氮氧化物、溶剂化物、多晶型物、代谢产物、酯、前药或其药学上可接受的盐的制备方法，In the second aspect of the present invention, there is provided a method for preparing the bicyclic derivative as described in the first aspect of the present invention, and its racemate, stereoisomer, tautomer, isotope-labeled substance, nitrogen oxide, solvate, polymorph, metabolite, ester, prodrug or pharmaceutically acceptable salt thereof,
(i)当为取代或未取代的螺环时，所述方法包括以下方法(1)至(5)中的其中一个：(i) When When is a substituted or unsubstituted spiro ring, the method comprises one of the following methods (1) to (5):
(1)以化合物i为起始原料，与试剂4-溴丁-2-烯酸酯通过亲核取代反应得到中间体ii，中间体ii去保护后得到中间体iii，中间体iii通过与卤代物或醛的衍生物反应得到中间体iv,在铑催化剂和手性配体催化下发生共轭加成反应得到中间体v，最后通过碱的水解反应得到目标产品，如路线一：

(1) Compound i is used as a starting material, and reacts with a reagent 4-bromobut-2-enoate to obtain intermediate ii through a nucleophilic substitution reaction. Intermediate ii is deprotected to obtain intermediate iii. Intermediate iii is reacted with a halogenated compound or an aldehyde derivative to obtain intermediate iv. A conjugate addition reaction is carried out under the catalysis of a rhodium catalyst and a chiral ligand to obtain intermediate v. Finally, a base hydrolysis reaction is carried out to obtain the target product, as shown in route 1:

(2)中间体ii在铑的催化剂和手性配体催化下共轭加成反应得到中间体vi,去保护基后生成中间体vii,中间体vii通过与卤代物或醛的衍生物反应得到中间体v，最后通过碱的水解反应得到目标产品，如路线二：
(2) Intermediate ii undergoes conjugate addition reaction under the catalysis of rhodium catalyst and chiral ligand to obtain intermediate vi, and after removing the protecting group, intermediate vii is generated. Intermediate vii is reacted with a halogenated compound or an aldehyde derivative to obtain intermediate v, and finally the target product is obtained by alkaline hydrolysis reaction, as shown in Route 2:
(3)以化合物i为起始原料，首先通过与卤代物或醛的衍生物反应得到中间体viii，中间体viii去保护基后得到中间体ix，ix与试剂4-溴丁-2-烯酸酯通过亲核取代反应得到中间体iv，在铑催化剂和手性配体催化下共轭加成得到中间体v,最后通过碱的水解反应得到目标产品，如路线三：

(3) Compound i is used as a starting material, and is first reacted with a halogenated compound or an aldehyde derivative to obtain an intermediate viii. After the intermediate viii is deprotected, an intermediate ix is obtained. ix is reacted with a reagent 4-bromobut-2-enoate to obtain an intermediate iv through a nucleophilic substitution reaction. Under the catalysis of a rhodium catalyst and a chiral ligand, a conjugate addition reaction is performed to obtain an intermediate v. Finally, a base hydrolysis reaction is performed to obtain the target product, as shown in Route 3:

(4)以化合物vii为起始原料，首先通过亲核取代反应得到中间体x，最后通过碱的水解反应得到目标产品，如路线三：
(4) Using compound vii as the starting material, firstly, the intermediate x is obtained by a nucleophilic substitution reaction, and finally the target product is obtained by an alkaline hydrolysis reaction, as shown in route 3:
(5)化合物v与羟胺反应得到目标产物，如路线四：
(5) Compound v reacts with hydroxylamine to obtain the target product, as shown in Scheme 4:
n1,n2,n3,n4＝0～3；Ra,Rb＝X,C＝O；Rc＝X；w＝1-2；R’可以为甲基或叔丁基；R1可以为 R2可以为 n1, n2, n3, n4 = 0 to 3; Ra, Rb = X, C = O; Rc = X; w = 1-2; R' can be methyl or tert-butyl; R 1 can be R2 can be
(ii)当为取代或未取代的并环时，所述方法包括以下步骤：(ii) When When it is a substituted or unsubstituted cyclized ring, the method comprises the following steps:
化合物x与4-溴丁-2-烯酸酯通过亲核取代反应得到中间体xi，去除保护基后得到中间体 xii，中间体xii的NH引入R1CH2-得到化合物xiii，之后在铑催化剂与手性配体的催化下共轭加成得到化合物xiv，最后通过碱性条件水解得到目标产品，如下路线：
Compound x reacts with 4-bromobut-2-enoate to obtain intermediate xi through nucleophilic substitution reaction, and the intermediate xi is obtained after removing the protecting group. xii, the NH of intermediate xii is introduced into R 1 CH 2 - to obtain compound xiii, which is then subjected to conjugate addition under the catalysis of a rhodium catalyst and a chiral ligand to obtain compound xiv, and finally hydrolyzed under alkaline conditions to obtain the target product, as follows:
Z为C或N；M为H、保护基、醛基、卤素；n1,n2＝0～3；Ra,Rb＝X,C＝O；w＝1-2；R’可以为甲基或叔丁基；R1可以为R2可以为 Z is C or N; M is H, a protecting group, an aldehyde group, or a halogen; n1, n2 = 0 to 3; Ra, Rb = X, C = O; w = 1-2; R' can be a methyl group or a tert-butyl group; R 1 can be R2 can be
在本发明的第三方面，提供了一种药物组合物，所述的药物组合物包括：In a third aspect of the present invention, a pharmaceutical composition is provided, comprising:
(a)治疗有效量的如本发明第一方面所述的双环衍生物，及其消旋体、立体异构体、互变异构体、同位素标记物、氮氧化物、溶剂化物、多晶型物、代谢产物、酯、前药或其药学上可接受的盐；和(a) a therapeutically effective amount of the bicyclic derivative as described in the first aspect of the present invention, and its racemate, stereoisomer, tautomer, isotope-labeled product, nitrogen oxide, solvate, polymorph, metabolite, ester, prodrug or pharmaceutically acceptable salt thereof; and
(b)药学上可接受的载体。(b) a pharmaceutically acceptable carrier.
在本发明的第四方面，提供了如本发明第一方面所述的式(I)所示的双环衍生物，及其消旋体、立体异构体、互变异构体、同位素标记物、氮氧化物、溶剂化物、多晶型物、代谢产物、酯、前药或其药学上可接受的盐，用于制备治疗或预防与αvβ1、αvβ6和αvβ8整联蛋白活性或表达量相关的疾病、病症或病状的药物组合物。In the fourth aspect of the present invention, provided is a bicyclic derivative of formula (I) as described in the first aspect of the present invention, and its racemate, stereoisomer, tautomer, isotope-labeled substance, nitrogen oxide, solvate, polymorph, metabolite, ester, prodrug or pharmaceutically acceptable salt thereof, for use in the preparation of a pharmaceutical composition for treating or preventing a disease, disorder or condition associated with the activity or expression of αvβ1, αvβ6 and αvβ8 integrins.
在另一优选例中，所述与αvβ1、αvβ6和αvβ8整联蛋白活性或表达量相关的疾病、病症或病状选自下组：自身免疫疾病、纤维化疾病、炎症性肠病(IBD)、复发型多发性硬化症(RMS)、进展性多灶性白质脑病(PML)、溃疡性结肠炎(UC)、克罗恩病(CD)、慢性病毒性乙肝和丙肝，非酒精性脂肪肝(NAFLD)、老年性黄斑变性、糖尿病型视网膜症、视网膜血管性疾病、骨质疏松和细胞增殖性疾病。In another preferred embodiment, the disease, disorder or condition associated with the activity or expression of αvβ1, αvβ6 and αvβ8 integrins is selected from the group consisting of autoimmune diseases, fibrotic diseases, inflammatory bowel disease (IBD), relapsing multiple sclerosis (RMS), progressive multifocal leukoencephalopathy (PML), ulcerative colitis (UC), Crohn's disease (CD), chronic viral hepatitis B and C, non-alcoholic fatty liver disease (NAFLD), age-related macular degeneration, diabetic retinopathy, retinal vascular disease, osteoporosis and cell proliferative diseases.
在另一优选例中，所述的纤维化疾病选自下组：肺纤维化、特发性肺纤维化、非特异性间质性肺炎(NSIP)、常规间质性肺疾病(UIP)、辐射诱发性肺纤维化、家族性肺纤维化、气道肺纤维化、慢性组赛性肺疾病(COPD)、间质性肺病、肝纤维化、慢性肾病、肾纤维化、皮肤纤维化、系统性硬化症，或其组合。 In another preferred embodiment, the fibrotic disease is selected from the following group: pulmonary fibrosis, idiopathic pulmonary fibrosis, nonspecific interstitial pneumonia (NSIP), conventional interstitial lung disease (UIP), radiation-induced pulmonary fibrosis, familial pulmonary fibrosis, airway pulmonary fibrosis, chronic group A lung disease (COPD), interstitial lung disease, liver fibrosis, chronic kidney disease, renal fibrosis, skin fibrosis, systemic sclerosis, or a combination thereof.
在另一优选例中，所述纤维化疾病是肺纤维化(如IPF)、肝纤维化、皮肤纤维化、硬皮病、心脏纤维化、肾纤维化、肠道纤维化、原发性硬化性胆管炎(PSC)或胆汁性纤维化(如PBC)。In another preferred embodiment, the fibrotic disease is pulmonary fibrosis (such as IPF), liver fibrosis, skin fibrosis, scleroderma, cardiac fibrosis, renal fibrosis, intestinal fibrosis, primary sclerosing cholangitis (PSC) or biliary fibrosis (such as PBC).
在另一优选例中，所述纤维化疾病是肺纤维化(IPF)。In another preferred embodiment, the fibrotic disease is pulmonary fibrosis (IPF).
在另一优选例中，所述纤维化疾病是原发性硬化性胆管炎或胆汁性纤维化(如PBC)。In another preferred embodiment, the fibrotic disease is primary sclerosing cholangitis or biliary fibrosis (such as PBC).
在另一优选例中，所述纤维化疾病是硬皮病。In another preferred embodiment, the fibrotic disease is scleroderma.
在另一优选例中，所述纤维化疾病是牛皮癣。In another preferred embodiment, the fibrotic disease is psoriasis.
在另一优选例中，所述细胞增值性疾病为癌症，选自下组：乳腺癌、宫颈癌、子宫内膜癌、卵巢癌、结肠癌、直肠癌、胰腺癌、肝癌、肺癌、非小细胞肺癌、肺癌脑转移、口腔鳞状细胞癌、头癌、颈癌、头颈部鳞状细胞癌、口或鼻黏膜癌、喉癌、肾癌、肾细胞癌、卵巢癌、脾癌、小肠癌、大肠癌、胃癌、食道癌、食管癌、肺鳞状细胞癌、胆管癌、胆囊癌、子宫内膜癌、黑色素瘤、尿路上皮癌、泌尿生殖道癌、生殖器癌、前列腺癌、睾丸癌、膀胱癌、血液癌、皮肤癌、骨髓癌、脑癌、中枢神经系统癌、肌肉组织癌、甲状腺癌，或其组合。In another preferred embodiment, the cell proliferative disease is cancer, selected from the following group: breast cancer, cervical cancer, endometrial cancer, ovarian cancer, colon cancer, rectal cancer, pancreatic cancer, liver cancer, lung cancer, non-small cell lung cancer, brain metastasis of lung cancer, oral squamous cell carcinoma, head cancer, neck cancer, head and neck squamous cell carcinoma, oral or nasal mucosal cancer, laryngeal cancer, kidney cancer, renal cell carcinoma, ovarian cancer, spleen cancer, small intestine cancer, large intestine cancer, gastric cancer, esophageal cancer, esophageal cancer, lung squamous cell carcinoma, bile duct cancer, gallbladder cancer, endometrial cancer, melanoma, urothelial carcinoma, urogenital tract cancer, genital cancer, prostate cancer, testicular cancer, bladder cancer, blood cancer, skin cancer, bone marrow cancer, brain cancer, central nervous system cancer, muscle tissue cancer, thyroid cancer, or a combination thereof.
在本发明的第五方面，提供了一种治疗与avβ1、αvβ6和αvβ8整联蛋白活性或表达量相关的疾病、病症或病状的方法，所述方法包括步骤：将有效量的如本发明第一方面所述的式(I)所示的双环衍生物，及其消旋体、立体异构体、互变异构体、同位素标记物、氮氧化物、溶剂化物、多晶型物、代谢产物、酯、前药或其药学上可接受的盐施用于有此需要的患者。In the fifth aspect of the present invention, there is provided a method for treating a disease, disorder or condition associated with the activity or expression of avβ1, αvβ6 and αvβ8 integrins, the method comprising the steps of administering an effective amount of a bicyclic derivative of formula (I) as described in the first aspect of the present invention, and its racemate, stereoisomer, tautomer, isotope label, nitrogen oxide, solvate, polymorph, metabolite, ester, prodrug or a pharmaceutically acceptable salt thereof to a patient in need thereof.
在本发明的第六方面，提供了一种双环衍生物，及其消旋体、立体异构体、互变异构体、同位素标记物、氮氧化物、溶剂化物、多晶型物、代谢产物、酯、前药或其药学上可接受的盐，所述双环衍生物具有如下式II-a、II-b、II-c所示的结构：
In a sixth aspect of the present invention, a bicyclic derivative is provided, and its racemate, stereoisomer, tautomer, isotope label, nitrogen oxide, solvate, polymorph, metabolite, ester, prodrug or pharmaceutically acceptable salt, wherein the bicyclic derivative has the structure shown in the following formula II-a, II-b, II-c:
其中，in,
各个Y1各自独立地为C1～C6亚烷基，-O-，-(C1～C6亚烷基)-O-，-NH-，-(C1～C6亚烷基)-NH-；Each Y 1 is independently C1-C6 alkylene, -O-, -(C1-C6 alkylene)-O-, -NH-, -(C1-C6 alkylene)-NH-;
各个Y2各自独立地为C1～C6亚烷基，-O-，-(C1～C6亚烷基)-O-，-C(O)-(C1～C6亚烷基)- NH-，-NH-，-NH-(C1～C6亚烷基)-，-(C1～C6亚烷基)-NH-；Each Y2 is independently C1-C6 alkylene, -O-, -(C1-C6 alkylene)-O-, -C(O)-(C1-C6 alkylene)- NH-, -NH-, -NH-(C1-C6 alkylene)-, -(C1-C6 alkylene)-NH-;
各个R1各自独立地为取代或未取代的6-10元芳环，取代或未取代的5-8元杂芳环，取代或未取代的其中C环、D环各自独立地为取代或未取代的C6-C10芳环、取代或未取代的5-8元杂芳环，取代或未取代的5-8元环烷环，或取代或未取代的5-8元杂烷环；Each R 1 is independently a substituted or unsubstituted 6-10 membered aromatic ring, a substituted or unsubstituted 5-8 membered heteroaromatic ring, a substituted or unsubstituted wherein the C ring and the D ring are each independently a substituted or unsubstituted C6-C10 aromatic ring, a substituted or unsubstituted 5-8 membered heteroaromatic ring, a substituted or unsubstituted 5-8 membered cycloalkane ring, or a substituted or unsubstituted 5-8 membered heteroalkane ring;
各个R2各自独立地为氢原子，取代或未取代的C6-C10芳环，取代或未取代的5-8元杂芳环，取代或未取代的C8-C16稠环，或-L1-L2；Each R 2 is independently a hydrogen atom, a substituted or unsubstituted C6-C10 aromatic ring, a substituted or unsubstituted 5-8 membered heteroaromatic ring, a substituted or unsubstituted C8-C16 condensed ring, or -L1-L2;
其中，-L1-选自无、-(取代或未取代的C1-C6亚烷基)-、-(取代或未取代的C1-C6亚烷氧基)-、-(取代或未取代的C1-C6亚烷硫基)-、-(取代或未取代的C3-C8环烷基)-、-(取代或未取代的C3-C8杂环烷基)-、-(取代或未取代的C6-C10芳基)-、-(取代或未取代的C5-C8杂芳基)-，Wherein, -L1- is selected from none, -(substituted or unsubstituted C1-C6 alkylene)-, -(substituted or unsubstituted C1-C6 alkyleneoxy)-, -(substituted or unsubstituted C1-C6 alkylenethio)-, -(substituted or unsubstituted C3-C8 cycloalkyl)-, -(substituted or unsubstituted C3-C8 heterocycloalkyl)-, -(substituted or unsubstituted C6-C10 aryl)-, -(substituted or unsubstituted C5-C8 heteroaryl)-,
L2为选自无、取代或未取代的C1-C6烷基、取代或未取代的C1-C6烷氧基、取代或未取代的C1-C6烷硫基、取代或未取代的C3-C8环烷基、-(C1-C3烷基)-取代或未取代的C3-C8环烷基、-(C1-C3烷氧基)-取代或未取代的C3-C8环烷基、-O-取代或未取代的C3-C8环烷基、取代或未取代的C3-C8杂烷环基、-(C1-C3烷基)-取代或未取代的C3-C8杂烷环基、-(C1-C3烷氧基)-取代或未取代的C3-C8杂烷环基、-O-取代或未取代的C3-C8杂烷环基、取代或未取代的5-8元杂芳基；L2 is selected from none, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C1-C6 alkylthio, substituted or unsubstituted C3-C8 cycloalkyl, -(C1-C3 alkyl)-substituted or unsubstituted C3-C8 cycloalkyl, -(C1-C3 alkoxy)-substituted or unsubstituted C3-C8 cycloalkyl, -O-substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C3-C8 heteroalkylcyclyl, -(C1-C3 alkyl)-substituted or unsubstituted C3-C8 heteroalkylcyclyl, -(C1-C3 alkoxy)-substituted or unsubstituted C3-C8 heteroalkylcyclyl, -O-substituted or unsubstituted C3-C8 heteroalkylcyclyl, substituted or unsubstituted 5-8 membered heteroaryl;
各个R3a’各自独立地为C1-C6烷基；Each R 3a ' is independently a C1-C6 alkyl group;
各个各自独立地为取代或未取代的螺环或取代或未取代的并环；each Each is independently a substituted or unsubstituted spiro ring or a substituted or unsubstituted cyclocyclic ring;
其中，当为取代或未取代的螺环，如式Ia所示：
Among them, when is a substituted or unsubstituted spiro ring, as shown in Formula Ia:
A为选自下组的环：取代或未取代的四元环烷环，取代或未取代的五元环烷环，取代或未取代的六元环烷环，取代或未取代的七元环烷环，取代或未取代的八元环烷环、取代或未取代的四元杂烷环，取代或未取代的五元杂烷环，取代或未取代的六元杂烷环，取代或未取代的七元杂烷环，取代或未取代的八元杂烷环，取代或未取代的五元杂烷环并C6-C10芳香环，取代或未取代的六元杂烷环并C6-C10芳香环；A is a ring selected from the group consisting of a substituted or unsubstituted four-membered cycloalkane ring, a substituted or unsubstituted five-membered cycloalkane ring, a substituted or unsubstituted six-membered cycloalkane ring, a substituted or unsubstituted seven-membered cycloalkane ring, a substituted or unsubstituted eight-membered cycloalkane ring, a substituted or unsubstituted four-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring, a substituted or unsubstituted six-membered heteroalkane ring, a substituted or unsubstituted seven-membered heteroalkane ring, a substituted or unsubstituted eight-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring and a C6-C10 aromatic ring, and a substituted or unsubstituted six-membered heteroalkane ring and a C6-C10 aromatic ring;
B为选自下组的环：取代或未取代的四元环烷环，取代或未取代的五元环烷环，取代或未取代的六元环烷环，取代或未取代的七元环烷环，取代或未取代的八元环烷环、取代或未取代的四元杂烷环，取代或未取代的五元杂烷环，取代或未取代的六元杂烷环，取代或未取代的七元杂烷环，取代或未取代的八元杂烷环，取代或未取代的五元杂烷环并C6-C10芳香环，取代或未取代的六元杂烷环并C6-C10芳香环；B is a ring selected from the group consisting of a substituted or unsubstituted four-membered cycloalkane ring, a substituted or unsubstituted five-membered cycloalkane ring, a substituted or unsubstituted six-membered cycloalkane ring, a substituted or unsubstituted seven-membered cycloalkane ring, a substituted or unsubstituted eight-membered cycloalkane ring, a substituted or unsubstituted four-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring, a substituted or unsubstituted six-membered heteroalkane ring, a substituted or unsubstituted seven-membered heteroalkane ring, a substituted or unsubstituted eight-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring and a C6-C10 aromatic ring, and a substituted or unsubstituted six-membered heteroalkane ring and a C6-C10 aromatic ring;
当为取代或未取代的并环，如式Ib所示：
when is a substituted or unsubstituted cyclic ring, as shown in Formula Ib:
为单键或者双键； is a single bond or a double bond;
A为选自下组的环：取代或未取代的四元环烷环，取代或未取代的五元环烷环，取代或未取代的六元环烷环，取代或未取代的七元环烷环，取代或未取代的八元环烷环、取代或未取代的四元杂烷环，取代或未取代的五元杂烷环，取代或未取代的六元杂烷环，取代或未取代的七元杂烷环，取代或未取代的八元杂烷环，取代或未取代的五元杂烷环并C6-C10芳香环，取代或未取代的六元杂烷环并C6-C10芳香环；A is a ring selected from the group consisting of a substituted or unsubstituted four-membered cycloalkane ring, a substituted or unsubstituted five-membered cycloalkane ring, a substituted or unsubstituted six-membered cycloalkane ring, a substituted or unsubstituted seven-membered cycloalkane ring, a substituted or unsubstituted eight-membered cycloalkane ring, a substituted or unsubstituted four-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring, a substituted or unsubstituted six-membered heteroalkane ring, a substituted or unsubstituted seven-membered heteroalkane ring, a substituted or unsubstituted eight-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring and a C6-C10 aromatic ring, and a substituted or unsubstituted six-membered heteroalkane ring and a C6-C10 aromatic ring;
B为选自下组的环：取代或未取代的四元环烷环，取代或未取代的五元环烷环，取代或未取代的六元环烷环，取代或未取代的七元环烷环，取代或未取代的八元环烷环、取代或未取代的C6-C10芳香环，取代或未取代的四元杂烷环，取代或未取代的五元杂烷环，取代或未取代的六元杂烷环，取代或未取代的七元杂烷环，取代或未取代的八元杂烷环，取代或未取代的五元杂烷环并C6-C10芳香环，取代或未取代的六元杂烷环并C6-C10芳香环；B is a ring selected from the group consisting of a substituted or unsubstituted four-membered cycloalkane ring, a substituted or unsubstituted five-membered cycloalkane ring, a substituted or unsubstituted six-membered cycloalkane ring, a substituted or unsubstituted seven-membered cycloalkane ring, a substituted or unsubstituted eight-membered cycloalkane ring, a substituted or unsubstituted C6-C10 aromatic ring, a substituted or unsubstituted four-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring, a substituted or unsubstituted six-membered heteroalkane ring, a substituted or unsubstituted seven-membered heteroalkane ring, a substituted or unsubstituted eight-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring and a C6-C10 aromatic ring, and a substituted or unsubstituted six-membered heteroalkane ring and a C6-C10 aromatic ring;
各个n各自独立地为0、1、2或3；Each n is independently 0, 1, 2 or 3;
其中，所述“取代”是指基团上的1-4个(优选为1、2、3或4个)氢原子各自独立地被选自下组的取代基所取代：C1-C6烷基、C3-C8环烷基、C3-C8杂环烷基、C1-C3卤代烷基、卤素、硝基、氰基、氨基、羟基、硫醇基、＝O、C1-C4羧基、C2-C4酯基、C2-C4酰胺基、C1-C6烷氧基、羧酸、C1-C4醇基、C1-C4烷胺基、-O-(CH2)m-C3-C8环烷、-O-(CH2)m-C3-C8杂烷环、-NH-(CH2)m-C3-C8环烷、-NH-(CH2)m-C3-C8杂烷环或-N＝Ph2；其中各个m各自独立地为0～3的整数；Wherein, the "substituted" means that 1 to 4 (preferably 1, 2, 3 or 4) hydrogen atoms on the group are independently replaced by a substituent selected from the following group: C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, C1-C3 haloalkyl, halogen, nitro, cyano, amino, hydroxyl, thiol, =O, C1-C4 carboxyl, C2-C4 ester, C2-C4 amide, C1-C6 alkoxy, carboxylic acid, C1-C4 alcohol, C1-C4 alkylamino, -O-(CH 2 ) m -C3-C8 cycloalkane, -O-(CH 2 ) m -C3-C8 heteroalkyl ring, -NH-(CH 2 ) m -C3-C8 cycloalkane, -NH-(CH 2 ) m -C3-C8 heteroalkyl ring or -N=Ph 2 ; wherein each m is independently an integer of 0 to 3;
其中，所述的杂芳环、杂烷环或杂芳基各自独立地具有1-3个(优选为1、2或3个)选自N、O和S杂原子；wherein the heteroaromatic ring, heteroalkyl ring or heteroaryl group each independently has 1 to 3 (preferably 1, 2 or 3) heteroatoms selected from N, O and S;
其中，所述式II-a、式II-b、式II-c的结构均为化学稳定的结构。Among them, the structures of Formula II-a, Formula II-b and Formula II-c are all chemically stable structures.
在另一优选例中，Y1、Y2、R1、R2、A、B、n各自独立地如本发明第一方面所述。In another preferred embodiment, Y 1 , Y 2 , R 1 , R 2 , A, B and n are independently as described in the first aspect of the present invention.
在另一优选例中，所述“取代”是指基团上的1-4个(优选为1、2、3或4个)氢原子各自独立地被Boc取代基所取代。In another preferred embodiment, the "substituted" means that 1 to 4 (preferably 1, 2, 3 or 4) hydrogen atoms on the group are independently replaced by a Boc substituent.
在另一优选例中，所述双环衍生物选自下组：





In another preferred embodiment, the bicyclic derivative is selected from the following group:





应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。 It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1显示了化合物的蛋白免疫印迹(WB)实验结果，说明实施例1、2、7、15、25在不同浓度下，分别对纤维化蛋白α-SMA、Fibronectin的降解作用。选用吡非尼酮(Pirfenidone)和GSK-3008348作为本实验的对照化合物。Figure 1 shows the results of Western blotting (WB) experiments of the compounds, illustrating the degradation effects of Examples 1, 2, 7, 15, and 25 on fibrosis proteins α-SMA and Fibronectin at different concentrations. Pirfenidone and GSK-3008348 were selected as control compounds in this experiment.
图2显示了假手术组、模型组、100mg/kg尼达尼布组、50mg/kg实施例71低剂量组和250mg/kg实施例71高剂量组的大鼠体重曲线(平均值±SEM)。Figure 2 shows the body weight curves of rats in the sham operation group, model group, 100 mg/kg nintedanib group, 50 mg/kg low-dose group of Example 71 and 250 mg/kg high-dose group of Example 71 (mean ± SEM).
图3显示了假手术组、模型组、100mg/kg尼达尼布组、50mg/kg实施例71低剂量组和250mg/kg实施例71高剂量组的大鼠体重增长率曲线(平均值±SEM)。Figure 3 shows the body weight growth rate curves (mean ± SEM) of rats in the sham operation group, model group, 100 mg/kg nintedanib group, 50 mg/kg low-dose group of Example 71 and 250 mg/kg high-dose group of Example 71.
图4显示了假手术组(A)、模型组(B)、100mg/kg尼达尼布组(C)、50mg/kg实施例71低剂量组(D)、250mg/kg实施例71高剂量组(E)左肺肺纤维化病灶内细支气管和肺细小动脉的组织学变化(×200，H&E染色)；其中图A可见正常肺细支气管和伴行的肺细小动脉；图B可见细支气管和肺小动脉损伤；图C-E可见细支气管和肺细小动脉损伤程度均有所减轻。图中a为肺小动脉；b为细支气管。图B-E中的箭头指示炎细胞浸润。Figure 4 shows the histological changes of bronchioles and pulmonary arterioles in the left lung pulmonary fibrosis lesions in the sham operation group (A), model group (B), 100 mg/kg nintedanib group (C), 50 mg/kg Example 71 low-dose group (D), and 250 mg/kg Example 71 high-dose group (E) (×200, H&E staining); Figure A shows normal pulmonary bronchioles and accompanying pulmonary arterioles; Figure B shows damage to bronchioles and pulmonary arterioles; Figures C-E show that the degree of damage to bronchioles and pulmonary arterioles is reduced. Figure a is a pulmonary arteriole; b is a bronchiole. The arrows in Figures B-E indicate inflammatory cell infiltration.
图5显示了假手术组、模型组、100mg/kg尼达尼布组、50mg/kg实施例71低剂量组、250mg/kg实施例71高剂量组左肺肺纤维化病灶内终末细支气管和伴随的细小肺动脉的损伤和炎症评分。图中***为p<0.001(与模型组比)。Figure 5 shows the injury and inflammation scores of the terminal bronchioles and the accompanying small pulmonary arteries in the left lung pulmonary fibrosis lesions in the sham operation group, the model group, the 100 mg/kg nintedanib group, the 50 mg/kg low-dose group of Example 71, and the 250 mg/kg high-dose group of Example 71. In the figure, *** is p<0.001 (compared with the model group).
图6显示了假手术组(A)、模型组(B)、100mg/kg尼达尼布组(C)、50mg/kg实施例71低剂量组(D)、250mg/kg实施例71高剂量组(E)左肺肺纤维化病灶边缘细支气管和肺细小动脉的组织学变化(×200，H&E染色)。其中图A可见正常肺细支气管和伴行的肺细小动脉；图B可见细支气管和肺小动脉损伤；图C-E可见细支气管和肺细小动脉损伤程度均有所减轻。图中a为肺小动脉；b为细支气管。图B-E中的箭头指示炎细胞浸润。Figure 6 shows the histological changes of bronchioles and pulmonary arterioles at the edge of the left lung pulmonary fibrosis lesions in the sham operation group (A), model group (B), 100 mg/kg nintedanib group (C), 50 mg/kg Example 71 low-dose group (D), and 250 mg/kg Example 71 high-dose group (E) (×200, H&E staining). In Figure A, normal pulmonary bronchioles and accompanying pulmonary arterioles can be seen; Figure B shows damage to bronchioles and pulmonary arterioles; Figures C-E show that the degree of damage to bronchioles and pulmonary arterioles is reduced. In the figure, a is a pulmonary arteriole; b is a bronchiole. The arrows in Figures B-E indicate inflammatory cell infiltration.
图7显示了假手术组、模型组、100mg/kg尼达尼布组、50mg/kg实施例71低剂量组、250mg/kg实施例71高剂量组左肺肺纤维化病灶边缘终末细支气管和伴随的细小肺动脉的损伤和炎症评分。图中***为p<0.001，**为p<0.01(与模型组比)。Figure 7 shows the injury and inflammation scores of the terminal bronchioles and the accompanying small pulmonary arteries at the edge of the left lung pulmonary fibrosis lesions in the sham operation group, the model group, the 100 mg/kg nintedanib group, the 50 mg/kg low-dose group of Example 71, and the 250 mg/kg high-dose group of Example 71. In the figure, *** is p<0.001, and ** is p<0.01 (compared with the model group).
图8显示了假手术组(A)、模型组(B)、100mg/kg尼达尼布组(C)、50mg/kg实施例71低剂量组(D)、250mg/kg实施例71高剂量组(E)左肺肺纤维化病灶内肺泡组织的组织学变化(×200，H&E染色)。其中图A可见正常肺泡壁；图B可见纤维化病灶内片状肺泡结构损伤、消失，由大量渗出的炎细胞和增生的结缔组织充填。图C-E肺泡结构虽有异常，但明显好于模型组。E组，也即实施例71高剂量组，部分肺泡接近正常的肺泡壁结构。图B-E中的箭头指示纤维化病灶内肺泡结构损伤，残存肺泡壁增厚。Figure 8 shows the histological changes of alveolar tissue in the left lung pulmonary fibrosis lesions in the sham operation group (A), model group (B), 100 mg/kg nintedanib group (C), 50 mg/kg low-dose group of Example 71 (D), and 250 mg/kg high-dose group of Example 71 (E) (×200, H&E staining). In Figure A, normal alveolar walls can be seen; Figure B shows that the sheet-like alveolar structure in the fibrotic lesions is damaged and disappeared, filled with a large number of exuded inflammatory cells and proliferating connective tissue. Although the alveolar structure in Figures C-E is abnormal, it is significantly better than that in the model group. In Group E, that is, the high-dose group of Example 71, some alveoli are close to the normal alveolar wall structure. The arrows in Figures B-E indicate that the alveolar structure in the fibrotic lesions is damaged and the remaining alveolar wall is thickened.
图9显示了假手术组(A)、模型组(B)、100mg/kg尼达尼布组(C)、50mg/kg实施例71低剂量组(D)、250mg/kg实施例71高剂量组(E)左肺肺纤维化病灶内肺泡纤维化病变(×200，Masson染色)。图A可见正常生理性纤维组织，无纤维组织沉积；图B可见肺泡组织出现严重纤维化病变及明显纤维组织沉积。图C-E同样有纤维化病变，但明显好于模型组。图B-E中的箭头指示纤维组织沉积。Figure 9 shows alveolar fibrosis lesions in the left lung pulmonary fibrosis lesions (×200, Masson staining) in the sham operation group (A), model group (B), 100 mg/kg nintedanib group (C), 50 mg/kg Example 71 low-dose group (D), and 250 mg/kg Example 71 high-dose group (E). Normal physiological fibrous tissue without fibrous tissue deposition can be seen in Figure A; severe fibrotic lesions and obvious fibrous tissue deposition can be seen in alveolar tissue in Figure B. Figures C-E also have fibrotic lesions, but are significantly better than the model group. The arrows in Figures B-E indicate fibrous tissue deposition.
图10显示了假手术组、模型组、100mg/kg尼达尼布组、50mg/kg实施例71低剂量组、250mg/kg实施例71高剂量组左肺肺纤维化评分。图中***为p<0.001，*为p<0.05(与模型组比)。Figure 10 shows the left lung pulmonary fibrosis scores of the sham group, model group, 100 mg/kg nintedanib group, 50 mg/kg low-dose group of Example 71, and 250 mg/kg high-dose group of Example 71. In the figure, *** means p<0.001, and * means p<0.05 (compared with the model group).
图11显示假手术组、模型组、100mg/kg尼达尼布组、50mg/kg实施例71低剂量组、250mg/kg实施例71高剂量组左肺肺纤维化不同病理评分的占比。图中***为p<0.001(与模型组比)。 Figure 11 shows the proportion of different pathological scores of left lung pulmonary fibrosis in the sham operation group, model group, 100 mg/kg nintedanib group, 50 mg/kg low-dose group of Example 71, and 250 mg/kg high-dose group of Example 71. In the figure, *** indicates p<0.001 (compared with the model group).
图12显示假手术组(A)、模型组(B)、100mg/kg尼达尼布组(C)、50mg/kg实施例71低剂量组(D)、250mg/kg实施例71高剂量组(E)左肺肺纤维化病灶内胶原沉积程度(×200，Masson染色)。其中图A存在正常生理性纤维组织，胶原面积<5％；图B出现极其严重的胶原沉积现象。图C-E同样出现胶原沉积的增多，但程度低于模型组。E组，也即实施例71高剂量组，胶原沉积最轻，程度明显低于模型组，同样低于尼达尼布组。Figure 12 shows the degree of collagen deposition in the left lung fibrosis lesions in the sham operation group (A), model group (B), 100 mg/kg nintedanib group (C), 50 mg/kg low-dose group of Example 71 (D), and 250 mg/kg high-dose group of Example 71 (E) (×200, Masson staining). In Figure A, normal physiological fibrous tissue exists, and the collagen area is <5%; Figure B shows extremely serious collagen deposition. Figures C-E also show an increase in collagen deposition, but the degree is lower than that of the model group. Group E, that is, the high-dose group of Example 71, has the lightest collagen deposition, which is significantly lower than the model group and also lower than the nintedanib group.
图13显示了假手术组、模型组、100mg/kg尼达尼布组、50mg/kg实施例71低剂量组、250mg/kg实施例71高剂量组胶原沉积面积百分比。图中***为p<0.001，*为p<0.05(与模型组比)。Figure 13 shows the percentage of collagen deposition area in the sham operation group, model group, 100 mg/kg nintedanib group, 50 mg/kg low-dose group of Example 71, and 250 mg/kg high-dose group of Example 71. In the figure, *** means p<0.001, and * means p<0.05 (compared with the model group).
具体实施方式Detailed ways
本发明人经过长期而深入的研究，筛选得到了一类结构新颖的双环衍生物的整合素抑制剂。所述的双环衍生物对整联蛋白αvβ1、αvβ6和αvβ8具有优异活性和选择性，以及显著优于阳性对照PLN-74809的口服药代性质，并且在纤维化疾病的体外和体内药效模型中显示了优于阳性参照吡非尼酮和尼达尼布的活性效果，因此可以用于制备和预防纤维化疾病的药物组合物。基于上述发现，发明人完成了本发明。After long-term and in-depth research, the inventors screened and obtained a class of integrin inhibitors with novel structures of bicyclic derivatives. The bicyclic derivatives have excellent activity and selectivity for integrins αvβ1, αvβ6 and αvβ8, and oral pharmacokinetic properties that are significantly better than the positive control PLN-74809, and show activity effects that are better than the positive references pirfenidone and nintedanib in in vitro and in vivo pharmacodynamic models of fibrotic diseases, so they can be used to prepare and prevent pharmaceutical compositions for fibrotic diseases. Based on the above findings, the inventors completed the present invention.
术语the term
如本文所用，术语“包含”、“包括”、“含有”可互换使用，不仅包括封闭式定义，还包括半封闭、和开放式的定义。换言之，所述术语包括了“由……构成”、“基本上由……构成”。As used herein, the terms "comprise", "include", and "contain" are used interchangeably and include not only closed definitions, but also semi-closed and open definitions. In other words, the terms include "consisting of", "consisting essentially of".
如本文所用，术语“烷基”是指完全饱和的环状或非环状、支链或非支链碳链部分，其具有指定的碳原子数，或者如果没有指定则最多30个碳原子，例如具有1至3个、1至6个碳原子。例如，具有1至8个碳原子的烷基是指诸如甲基、乙基、丙基、丁基、戊基、己基、庚基和辛基的部分，以及作为这些部分的位置异构体的那些部分。10至30个碳原子的烷基包括癸基、十一烷基、十二烷基、十三烷基、十四烷基、十五烷基、十六烷基、十七烷基、十八烷基、十九烷基、二十烷基、二十二烷基、二十二烷基、三十烷基和二十四烷基，烷基基团可以被取代或未取代的。As used herein, the term "alkyl" refers to a fully saturated cyclic or acyclic, branched or unbranched carbon chain moiety having the specified number of carbon atoms, or if not specified, up to 30 carbon atoms, such as 1 to 3, 1 to 6 carbon atoms. For example, alkyl having 1 to 8 carbon atoms refers to moieties such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl and octyl, and those moieties that are positional isomers of these moieties. Alkyl having 10 to 30 carbon atoms includes decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, docosyl, docosyl, triacontyl and tetracosyl, and the alkyl group may be substituted or unsubstituted.
如本文所用，术语“C1-C6烷基”或“C1-C3烷基”指具有1-6或1-3个碳原子的直链或支链烷基，例如甲基、乙基、丙基、异丙基、丁基、异丁基、仲丁基、叔丁基，或类似基团。As used herein, the term "C1-C6 alkyl" or "C1-C3 alkyl" refers to a straight or branched chain alkyl group having 1-6 or 1-3 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, or the like.
如本文所用，术语“亚烷基”是指具有指定碳原子数例如1至12个碳原子的烷基，其在其最长碳链上包含与化合物其余部分的两个连接点。亚烷基的非限制性实例包括C1-C6亚烷基、C1-C3亚烷基，具体包括亚甲基-(CH2)-、亚乙基-(CH2CH2)-、正亚丙基-(CH2CH2CH2)-、异亚丙基-(CH2CH(CH3))-等。亚烷基可以是环状或非环状、支链或未支链的碳链部分，并且可以任选被一个或多个取代基取代。As used herein, the term "alkylene" refers to an alkyl group having a specified number of carbon atoms, such as 1 to 12 carbon atoms, which contains two points of connection to the rest of the compound on its longest carbon chain. Non-limiting examples of alkylene groups include C1-C6 alkylene groups, C1-C3 alkylene groups, specifically including methylene-(CH 2 )-, ethylene-(CH 2 CH 2 )-, n-propylene-(CH 2 CH 2 CH 2 )-, isopropylene-(CH 2 CH(CH 3 ))-, etc. Alkylene groups can be cyclic or acyclic, branched or unbranched carbon chain moieties, and can be optionally substituted with one or more substituents.
如本文所用，术语“C1-C6烷氧基”指具有1-6个碳原子的直链或支链的烷氧基，例如甲氧基、乙氧基、丙氧基、异丙氧基、丁氧基、异丁氧基、仲丁氧基、叔丁氧基、戊氧基、己氧基，或类似基团。As used herein, the term "C1-C6 alkoxy" refers to a straight or branched alkoxy group having 1 to 6 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, hexyloxy, or the like.
如本文所用，术语“C2-C4酯基”指具有C1-C3烷基-OC(O)-结构的基团或者具有-OC(O)-C1-C3烷基结构的基团，其中烷基可以为直链或支链的，例如CH3COO-、C2H5COO-、C3H8COO-、(CH3)2CHCOO-、-COOCH3、-COOC2H5、-COOC3H8，或类似基团。As used herein, the term "C2-C4 ester group" refers to a group having a C1-C3 alkyl-OC(O)- structure or a group having an -OC(O) -C1-C3 alkyl structure, wherein the alkyl group may be linear or branched, for example CH3COO-, C2H5COO- , C3H8COO- , ( CH3 ) 2CHCOO- , -COOCH3 , -COOC2H5 , -COOC3H8 , or the like.
如本文所用，术语“C2-C4酰胺基”指具有C1-C3烷基-CO-NH-结构的基团或者具有- NH-CO-C1-C3烷基结构的基团，其中烷基可以为直链或支链的，例如CH3-CO-NH-、C2H5-CO-NH-、C3H8-CO-NH-、-COOCH3、-CO-NH-C2H5、-CO-NH-C3H8，或类似基团。As used herein, the term "C2-C4 amide group" refers to a group having a C1-C3 alkyl-CO-NH-structure or a group having a A group of the structure NH-CO-C1-C3 alkyl, wherein the alkyl group may be linear or branched, for example CH3 -CO-NH-, C2H5 - CO- NH-, C3H8-CO-NH-, -COOCH3, -CO-NH-C2H5 , -CO - NH - C3H8 , or the like.
如本文所用，术语“C2-C4酰基”指具有C1-C3烷基-CO-结构的基团，其中烷基可以为直链或支链的，例如CH3-CO-、C2H5-CO-、C3H8-CO-，或类似基团。As used herein, the term "C2-C4 acyl" refers to a group having a C1-C3 alkyl-CO- structure, wherein the alkyl group may be linear or branched, such as CH3 -CO-, C2H5 - CO-, C3H8 - CO- , or the like.
如本文所用，术语“C1-C3卤代烷基”是指具有1-3个碳原子的直链或支链的烷基的一个或多个氢原子被卤素基团取代，如一氯甲基、二氯乙基、三氯丙基，或类似基团。As used herein, the term "C1-C3 haloalkyl" refers to a linear or branched alkyl group having 1 to 3 carbon atoms in which one or more hydrogen atoms are substituted by a halogen group, such as monochloromethyl, dichloroethyl, trichloropropyl, or the like.
如本文所用，术语“C1-C4羧基”是指C1-C3烷基-COOH结构的基团，其中烷基可以为直链或支链的，例如CH3COOH、C2H5COOH、C3H8COOH、(CH3)2CHCOOH，或类似基团。As used herein, the term "C1-C4 carboxyl" refers to a group of a C1-C3 alkyl-COOH structure, wherein the alkyl group may be linear or branched, such as CH3COOH , C2H5COOH , C3H8COOH , ( CH3 ) 2CHCOOH , or the like.
如本文所用，术语“C6-C10芳环”、“C6-C10芳基”是指在环组分中具有6至10个碳原子的单环或二环芳族烃基，诸如苯环、萘环，或相似基团。As used herein, the term "C6-C10 aromatic ring", "C6-C10 aryl group" refers to a monocyclic or bicyclic aromatic hydrocarbon group having 6 to 10 carbon atoms in the ring component, such as a benzene ring, a naphthalene ring, or a similar group.
术语“芳基”包括3至12元取代或未取代的单环芳族基团，其中环的每个原子都是碳(即碳环芳基)或其中一个或多个原子是杂原子(即杂芳基)。优选地，芳基包括5-至12-元环，更优选6-至10-元环。术语“芳基”还包括具有两个或更多个环的多环环系统，其中两个或更多个碳为两个相邻环所共有，其中至少一个环是芳族的，例如，其他环可以是环烷基、环烯基、环炔基、芳基、杂芳基和/或杂环基。碳环芳基包括苯、萘、菲、苯酚、苯胺等。The term "aryl" includes 3 to 12 substituted or unsubstituted monocyclic aromatic groups, wherein each atom of the ring is carbon (i.e., carbocyclic aromatic groups) or wherein one or more atoms are heteroatoms (i.e., heteroaryl groups). Preferably, aryl groups include 5- to 12-membered rings, more preferably 6- to 10-membered rings. The term "aryl" also includes polycyclic ring systems having two or more rings, wherein two or more carbons are shared by two adjacent rings, wherein at least one ring is aromatic, for example, the other rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl and/or heterocyclic radicals. Carbocyclic aromatic groups include benzene, naphthalene, phenanthrene, phenol, aniline, etc.
如本文所用，术语“杂芳基”是指任选取代的芳香基，例如其为5至7元单环环系，所述环系具有的环含有至少一个杂原子和至少一个碳原子，例如吡咯基、噻吩基、吡啶基、哒嗪基、嘧啶基、吡嗪基、呋喃、咪唑、噻唑、噁唑、三氮唑，或类似基团。As used herein, the term "heteroaryl" refers to an optionally substituted aromatic group, for example, a 5- to 7-membered monocyclic ring system having a ring containing at least one heteroatom and at least one carbon atom, such as pyrrolyl, thienyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, furan, imidazole, thiazole, oxazole, triazole, or the like.
术语“环烷环”、“环烷基”是指具有3至12个碳原子(例如具有3至8个、4个、5个、或6个碳原子)的非芳香基团(包括饱和的、部分饱和的或不饱和的基团)，具有单环或稠环(包括桥环体系和螺环体系)。因此，所述烷环或烷环基可以为饱和烷环、或不饱和烷环。在饱和烷环的稠环体系中，一个或多个环均为饱和烷环。在不饱和烷环的稠环体系中，一个或多个环可以是饱和烷环或不饱和烷环。The term "cycloalkane ring", "cycloalkyl" refers to a non-aromatic group (including saturated, partially saturated or unsaturated groups) having 3 to 12 carbon atoms (e.g., 3 to 8, 4, 5, or 6 carbon atoms), having a monocyclic or condensed ring (including a bridged ring system and a spirocyclic system). Therefore, the alkane ring or alkane ring group can be a saturated alkane ring or an unsaturated alkane ring. In the condensed ring system of a saturated alkane ring, one or more rings are saturated alkane rings. In the condensed ring system of an unsaturated alkane ring, one or more rings can be a saturated alkane ring or an unsaturated alkane ring.
如本文所用，术语“5-8元杂芳环”指具有一个到多个(优选为1、2或3个)选自N、O和S杂原子的芳族杂环系，且具有5-8个环原子。应当理解的是，当含有多个杂原子时，杂原子可以相同、可以部分相同，也可以完全不同。在本申请中，所述杂芳环优选为含1至2个氮原子的杂芳环。例如5元杂芳环的实例包括(但不限于)：吡咯环、呋喃环、噻吩环、咪唑环、吡唑环、恶唑环、噻唑环，6元杂芳环的实例包括(但不限于)吡啶环、吡嗪环、哒嗪环、嘧啶环，或相似基团。As used herein, the term "5-8 membered heteroaromatic ring" refers to an aromatic heterocyclic ring system having one to multiple (preferably 1, 2 or 3) heteroatoms selected from N, O and S, and having 5-8 ring atoms. It should be understood that when multiple heteroatoms are contained, the heteroatoms may be the same, partially the same, or completely different. In the present application, the heteroaromatic ring is preferably a heteroaromatic ring containing 1 to 2 nitrogen atoms. For example, examples of 5-membered heteroaromatic rings include (but are not limited to): pyrrole ring, furan ring, thiophene ring, imidazole ring, pyrazole ring, oxazole ring, thiazole ring, examples of 6-membered heteroaromatic rings include (but are not limited to) pyridine ring, pyrazine ring, pyridazine ring, pyrimidine ring, or similar groups.
如本文所用，术语“5-8元杂芳基”指具有一个到多个(优选为1、2或3个)选自N、O和S杂原子的的芳香基，且具有5或8个环原子。应当理解的是，当含有多个杂原子时，杂原子可以相同、可以部分相同，也可以完全不同。例如5元杂芳基的实例包括(但不限于)：吡咯基、呋喃基、噻吩基、咪唑基、恶唑基、噻唑基，或相似基团。As used herein, the term "5-8 membered heteroaryl" refers to an aromatic group having one to more (preferably 1, 2 or 3) heteroatoms selected from N, O and S, and having 5 or 8 ring atoms. It should be understood that when containing multiple heteroatoms, the heteroatoms may be the same, partially the same, or completely different. For example, examples of 5-membered heteroaryl include (but are not limited to): pyrrolyl, furanyl, thienyl, imidazolyl, oxazolyl, thiazolyl, or similar groups.
术语“杂芳基”、“杂芳环”、“芳杂环基”指3-12元芳族基团，其包含一个或多个选自氮、氧和硫的杂原子，包括单环或多环体系，所述多环体系可以为稠环、桥环体系和螺环体系。术语“稠合杂芳环”是指两个或更多个芳族基团稠合形成的稠环，其包含一个或多个选自氮、氧和硫的杂原子。其中，所述稠合杂芳环中的一个或多个环中有杂原子。杂芳基包括取代或未取代的芳族3-12元环结构，更优选5-12元环，更优选5-10元环，其环 结构包括1-4个杂原子。杂芳基包括例如吡咯、呋喃、噻吩、咪唑、恶唑、噻唑、三唑、吡唑、吡啶、吡嗪、哒嗪和嘧啶等。芳基和杂芳基可以是单环、双环或多环的。在本文中，C5-C8杂芳基表示环原子数为5-8个。The terms "heteroaryl", "heteroaromatic ring" and "aromatic heterocyclic group" refer to 3-12 membered aromatic groups, which contain one or more heteroatoms selected from nitrogen, oxygen and sulfur, including monocyclic or polycyclic systems, and the polycyclic system can be a fused ring, a bridged ring system and a spirocyclic system. The term "fused heteroaromatic ring" refers to a fused ring formed by the fusion of two or more aromatic groups, which contains one or more heteroatoms selected from nitrogen, oxygen and sulfur. Among them, one or more rings in the fused heteroaromatic ring have heteroatoms. Heteroaryl includes substituted or unsubstituted aromatic 3-12 membered ring structures, more preferably 5-12 membered rings, more preferably 5-10 membered rings, and its rings The structure includes 1-4 heteroatoms. Heteroaryl includes, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine. Aryl and heteroaryl can be monocyclic, bicyclic or polycyclic. In this article, C5-C8 heteroaryl means that the number of ring atoms is 5-8.
术语“杂烷环”、“杂烷环基”是指3-12元非芳族基团(包括饱和的、部分饱和的或不饱和的基团)，其包含一个或多个选自氮、氧和硫的杂原子，包括单环或多环体系，所述多环体系可以为稠环、桥环体系和螺环体系。因此，所述杂烷环或杂烷环基可以为饱和杂烷环、或不饱和杂烷环。在稠环体系中，一个或多个环可以是烷环基、芳基或杂芳基。杂烷环中的环原子数可以为3至8个、4个、5个或6个。在一个实施方式中，杂环基团的氮原子和/或硫原子任选地被氧化，以提供N-氧化物、亚磺酰基和磺酰基部分。在一个实施方式中，杂环基团的碳原子任选地被氧化，形成(C＝O)部分。在本文中，C3-C8表示环原子数为3-8个。The term "heteroalkyl ring", "heteroalkyl ring group" refers to a 3-12 membered non-aromatic group (including saturated, partially saturated or unsaturated groups) containing one or more heteroatoms selected from nitrogen, oxygen and sulfur, including a monocyclic or polycyclic system, and the polycyclic system can be a fused ring, a bridged ring system and a spirocyclic system. Therefore, the heteroalkyl ring or heteroalkyl ring group can be a saturated heteroalkyl ring or an unsaturated heteroalkyl ring. In a fused ring system, one or more rings can be an alkyl ring group, an aryl group or a heteroaryl group. The number of ring atoms in the heteroalkyl ring can be 3 to 8, 4, 5 or 6. In one embodiment, the nitrogen atom and/or sulfur atom of the heterocyclic group is optionally oxidized to provide N-oxide, sulfinyl and sulfonyl moieties. In one embodiment, the carbon atoms of the heterocyclic group are optionally oxidized to form a (C=O) moiety. In this article, C3-C8 means that the number of ring atoms is 3-8.
如本文所用，术语“5-8元杂烷环”为任何稳定的环上含有一个或多个(优选为1、2或3个)选自N、O和S的杂原子，且环原子个数为5-7个的非芳香族杂环系。杂环可以是饱和的、部分不饱和的、不饱和的环，但不能为芳族的环。在本申请中，所述杂烷环优选为含1至2个氮原子的杂烷环，其任选地可还包括1或2个氧原子。应当理解的是，当含有多个杂原子时，杂原子可以相同、可以部分相同，也可以完全不同。在本申请中，5元杂烷环的实例包括(但不限于)吡咯烷环、吡咯啉环、吡唑烷环、吡唑啉环、1,3-氧五环，6元杂烷环的实例包括(但不限于)哌啶环、吗啉环、哌嗪环、1,4-二氧六环，或类似基团。As used herein, the term "5-8 membered heteroalkyl ring" refers to any stable ring containing one or more (preferably 1, 2 or 3) heteroatoms selected from N, O and S, and a non-aromatic heterocyclic ring system with 5-7 ring atoms. The heterocyclic ring may be a saturated, partially unsaturated, or unsaturated ring, but cannot be an aromatic ring. In the present application, the heteroalkyl ring is preferably a heteroalkyl ring containing 1 to 2 nitrogen atoms, which may optionally further include 1 or 2 oxygen atoms. It should be understood that when multiple heteroatoms are contained, the heteroatoms may be the same, partially the same, or completely different. In the present application, examples of 5-membered heteroalkyl rings include (but are not limited to) pyrrolidine rings, pyrroline rings, pyrazolidine rings, pyrazoline rings, 1,3-oxopentacyclic rings, and examples of 6-membered heteroalkyl rings include (but are not limited to) piperidine rings, morpholine rings, piperazine rings, 1,4-dioxane rings, or similar groups.
如本文所用，术语“四元杂烷环”为任何稳定的含有一个或多个(优选为1、2或3个)选自N、O和S的环内杂原子，且环原子个数为4个的非芳香族杂环系。在本申请中，所述四元杂烷环优选为含1至2个氮原子的四元杂烷环。应当理解的是，当含有多个杂原子时，杂原子可以相同、可以部分相同，也可以完全不同。四元杂烷环的实例包括(但不限于)氧杂环丁烷、氮杂环丁烷，或类似基团。As used herein, the term "four-membered heteroalkane ring" is any stable non-aromatic heterocyclic ring system containing one or more (preferably 1, 2 or 3) heteroatoms selected from N, O and S, and the number of ring atoms is 4. In the present application, the four-membered heteroalkane ring is preferably a four-membered heteroalkane ring containing 1 to 2 nitrogen atoms. It should be understood that when containing multiple heteroatoms, the heteroatoms can be the same, partially the same, or completely different. Examples of four-membered heteroalkane rings include (but are not limited to) oxetane, azetidine, or similar groups.
术语“杂环基”或“杂环基团”是指3至12元环结构，更优选5至12元环，更优选5至10元环，其环结构包括1至4个杂原子。杂环可以是单环、双环、螺环或多环。杂环基包括，例如，吡咯、咪唑、吡唑、异噻唑、异恶唑、吡啶、吡嗪、嘧啶、哒嗪、吲嗪、异吲哚、吲哚、吲唑、嘌呤、喹啉、异喹啉、喹啉、酞嗪、萘啶、喹喔啉、喹唑啉、噌啉、蝶啶、咔唑、咔啉、菲啶、吖啶、嘧啶、菲咯啉、吩嗪、吩嗪、吩噻嗪、呋喃、吩恶嗪、吡咯烷、氧戊环、硫戊烷、恶唑、哌啶、哌嗪、吗啉、内酯、内酰胺类如氮杂环丁酮和吡咯烷酮，苏丹,磺内酯等。杂环可以在一个或多个位置被如上所述的取代基取代，例如卤素、烷基、芳烷基、烯基、炔基、环烷基、羟基、氨基、硝基、巯基、亚氨基、酰氨基、磷酸酯、膦酸酯，次膦酸酯、羰基、羧基、甲硅烷基、氨磺酰基、亚磺酰基、醚、烷硫基、磺酰基、酮、醛、酯、杂环基、芳族或杂芳族部分、-CF3、-CN等，本文中，“杂环”包括杂烷环和杂芳环。The term "heterocyclic radical" or "heterocyclic group" refers to a 3 to 12-membered ring structure, more preferably a 5 to 12-membered ring, more preferably a 5 to 10-membered ring, and its ring structure includes 1 to 4 heteroatoms. The heterocycle can be a monocyclic, bicyclic, spirocyclic or polycyclic ring. The heterocyclic radical includes, for example, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinoline, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenazine, phenothiazine, furan, phenoxazine, pyrrolidine, oxolane, thiopentane, oxazole, piperidine, piperazine, morpholine, lactone, lactams such as azetidinone and pyrrolidone, Sudan, sultone etc. The heterocycle may be substituted at one or more positions by substituents as described above, for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxy, amino, nitro, thiol, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, sulfamoyl, sulfinyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocyclic group, aromatic or heteroaromatic moiety, -CF3 , -CN, etc. Herein, "heterocycle" includes heteroalkyl ring and heteroaromatic ring.
如本文所用，术语“卤素”是指包括例如但不限于放射性和非放射性形式的氟、氯、溴、碘等。在优选的实施方案中，卤素选自氟、氯和溴。As used herein, the term "halogen" refers to, for example but not limited to, radioactive and non-radioactive forms of fluorine, chlorine, bromine, iodine, etc. In a preferred embodiment, the halogen is selected from fluorine, chlorine and bromine.
如本文所用，术语“卤代”是指卤素取代的。As used herein, the term "halo" refers to halogen substituted.
本文所用，术语“自身免疫疾病”是指重症肌无力、多发性肌炎、自身免疫性心肌炎、风湿性多肌痛、银屑病关节炎、类风湿性关节炎、干燥综合征、强直性脊柱炎、复发性多软骨炎、炎症性肠病(如克罗恩病、溃疡性结肠炎)、乳糜泻、自身免疫性肝炎、原 发性胆汁性肝硬化(PBC)、原发性硬化性胆管炎(PSC)、免疫介导的肾病、间质性膀胱炎、阿狄森氏病、自身免疫性甲状腺疾病(如桥本氏甲状腺炎、格雷夫斯病)、糖尿病、皮肌炎、银屑病、斑秃、自身免疫或免疫介导的皮肤病、大疱性类天疱疮、结节性红斑、疱疹样皮炎、化脓性汗腺炎、自身免疫性荨麻疹、多发性硬化症、慢性炎症性脱髓鞘多发性神经病变(CIDP)、中枢或外周神经系统脱髓鞘疾病、特发性脱髓鞘性多发性神经病、格林-巴利综合征、周围神经病变、系统性红斑狼疮、系统性血管炎、自身免疫性溶血性贫血、自身免疫血小板减少症、过敏性疾病(如过敏性紫癜)、结节病、纤维性肺泡炎疾病。As used herein, the term "autoimmune disease" refers to myasthenia gravis, polymyositis, autoimmune myocarditis, polymyalgia rheumatica, psoriatic arthritis, rheumatoid arthritis, Sjögren's syndrome, ankylosing spondylitis, relapsing polychondritis, inflammatory bowel disease (such as Crohn's disease, ulcerative colitis), celiac disease, autoimmune hepatitis, Primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), immune-mediated kidney disease, interstitial cystitis, Addison's disease, autoimmune thyroid disease (e.g., Hashimoto's thyroiditis, Graves' disease), diabetes mellitus, dermatomyositis, psoriasis, alopecia areata, autoimmune or immune-mediated skin diseases, bullous pemphigoid, erythema nodosum, dermatitis herpetiformis, hidradenitis suppurativa, autoimmune urticaria, multiple sclerosis, chronic inflammatory demyelinating polyneuropathy (CIDP), demyelinating diseases of the central or peripheral nervous system, idiopathic demyelinating polyneuropathy, Guillain-Barré syndrome, peripheral neuropathy, systemic lupus erythematosus, systemic vasculitis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, allergic diseases (e.g., Henoch-Schönlein purpura), sarcoidosis, fibrosing alveolitis disease.
药物组合物和施用方法Pharmaceutical compositions and methods of administration
由于本发明化合物具有对整合素αvβ1、αvβ6和αvβ8的抑制活性和选择性，因此本发明化合物及其各种晶型，药学上可接受的无机或有机盐，水合物或溶剂合物，以及含有本发明化合物为主要活性成分的药物组合物可用于治疗、预防以及缓解肺纤维化疾病。Since the compounds of the present invention have inhibitory activity and selectivity against integrins αvβ1, αvβ6 and αvβ8, the compounds of the present invention and their various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and pharmaceutical compositions containing the compounds of the present invention as the main active ingredient can be used to treat, prevent and alleviate pulmonary fibrosis.
本发明的药物组合物包含安全有效量范围内的本发明化合物或其药理上可接受的盐及药理上可以接受的赋形剂或载体。其中“安全有效量”指的是：化合物的量足以明显改善病情，而不至于产生严重的副作用。通常，药物组合物含有0.1-1000mg本发明化合物/剂，更佳地，含有0.5-500mg本发明化合物/剂。较佳地，所述的“一剂”为一个胶囊或药片。The pharmaceutical composition of the present invention comprises a safe and effective amount of the compound of the present invention or a pharmacologically acceptable salt thereof and a pharmacologically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Usually, the pharmaceutical composition contains 0.1-1000 mg of the compound of the present invention per dose, and more preferably, contains 0.5-500 mg of the compound of the present invention per dose. Preferably, the "one dose" is a capsule or tablet.
“药学上可以接受的载体”指的是：一种或多种相容性固体或液体填料或凝胶物质，它们适合于人使用，而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组份能和本发明的化合物以及它们之间相互掺和，而不明显降低化合物的药效。药学上可以接受的载体部分例子有纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如吐温)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。"Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as Tween), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.
本发明化合物或药物组合物的施用方式没有特别限制，代表性的施用方式包括(但并不限于)：口服、直肠、肠胃外(静脉内、肌肉内或皮下)、吸入给药和局部给药。特别优选的施用方式是口服。There is no particular limitation on the administration of the compound or pharmaceutical composition of the present invention. Representative administrations include, but are not limited to, oral, rectal, parenteral (intravenous, intramuscular or subcutaneous), inhalation and topical administration. A particularly preferred administration is oral.
用于口服给药的固体剂型包括胶囊剂、片剂、丸剂、散剂和颗粒剂。在这些固体剂型中，活性化合物与至少一种常规惰性赋形剂(或载体)混合，如柠檬酸钠或磷酸二钙，或与下述成分混合：(a)填料或增容剂，例如，淀粉、乳糖、蔗糖、葡萄糖、甘露醇和硅酸；(b)粘合剂，例如，羟甲基纤维素、藻酸盐、明胶、聚乙烯基吡咯烷酮、蔗糖和阿拉伯胶；(c)保湿剂，例如，甘油；(d)崩解剂，例如，琼脂、碳酸钙、马铃薯淀粉或木薯淀粉、藻酸、某些复合硅酸盐、和碳酸钠；(e)缓溶剂，例如石蜡；(f)吸收加速剂，例如，季胺化合物；(g)润湿剂，例如鲸蜡醇和单硬脂酸甘油酯；(h)吸附剂，例如，高岭土；和(i)润滑剂，例如，滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠，或其混合物。胶囊剂、片剂和丸剂中，剂型也可包含缓冲剂。Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and acacia; (c) humectants, for example, glycerol; (d) disintegrators, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may also contain a buffer.
固体剂型如片剂、糖丸、胶囊剂、丸剂和颗粒剂可采用包衣和壳材制备，如肠衣和其它本领域公知的材料。它们可包含不透明剂，并且，这种组合物中活性化合物或化合 物的释放可以延迟的方式在消化道内的某一部分中释放。可采用的包埋组分的实例是聚合物质和蜡类物质。必要时，活性化合物也可与上述赋形剂中的一种或多种形成微胶囊形式。Solid dosage forms such as tablets, dragees, capsules, pills and granules may be prepared with coatings and shells, such as enteric coatings and other materials known in the art. They may contain opacifying agents and the active compound or compounds in such compositions may be The release of the active compound can be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active compound can also be formed into microcapsules with one or more of the above-mentioned excipients.
用于口服给药的液体剂型包括药学上可接受的乳液、溶液、悬浮液、糖浆或酊剂。除了活性化合物外，液体剂型可包含本领域中常规采用的惰性稀释剂，如水或其它溶剂，增溶剂和乳化剂，例知，乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1,3-丁二醇、二甲基甲酰胺以及油，特别是棉籽油、花生油、玉米胚油、橄榄油、蓖麻油和芝麻油或这些物质的混合物等。Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.
除了这些惰性稀释剂外，组合物也可包含助剂，如润湿剂、乳化剂和悬浮剂、甜味剂、矫味剂和香料。Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
除了活性化合物外，悬浮液可包含悬浮剂，例如，乙氧基化异十八烷醇、聚氧乙烯山梨醇和脱水山梨醇酯、微晶纤维素、甲醇铝和琼脂或这些物质的混合物等。Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances, and the like.
用于肠胃外注射的组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。Compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.
用于局部给药的本发明化合物的剂型包括软膏剂、散剂、贴剂、喷射剂和吸入剂。活性成分在无菌条件下与生理上可接受的载体及任何防腐剂、缓冲剂，或必要时可能需要的推进剂一起混合。Dosage forms for topical administration of the compounds of the invention include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required.
本发明化合物可以单独给药，或者与其他药学上可接受的化合物联合给药。The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.
使用药物组合物时，是将安全有效量的本发明化合物适用于需要治疗的哺乳动物(如人)，其中施用时剂量为药学上认为的有效给药剂量，对于60kg体重的人而言，日给药剂量通常为0.2～1000mg，优选0.5～500mg。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage during administration is a pharmaceutically effective dosage, and for a person weighing 60 kg, the daily dosage is usually 0.2 to 1000 mg, preferably 0.5 to 500 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the health status of the patient, which are all within the skill of a skilled physician.
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.
中间体A：叔-丁基7-(溴甲基)-3,4-二氢-1,8-二氮杂萘-1(2H)-羧酸酯的制备
Intermediate A: Preparation of tert-butyl 7-(bromomethyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylate
步骤1：将2-氨基尼古丁醛(1g，8.2mmol)，1,1-二甲氧基丙烷-2-酮(1.3g，10.6mmol)溶于10/1的乙醇/水(22ml)溶液中，在氮气氛围0℃下向溶液中缓慢滴加氢氧化钠溶液(3M，3.6ml)，混合液室温下搅拌3小时。将反应混合液浓缩，残余物溶于乙酸乙酯中(50ml)，有 机相经饱和食盐水洗(25ml*2)，干燥，过滤，浓缩得到黄色固体2-(二甲氧基甲基)-1,8-二氮杂萘(A-1)2g。LC-MS:ESI m/z:205.2[M+H]+；1H NMR(400MHz,CDCl3)δ＝9.13(dd,J＝2.0,8.0Hz,1H),8.28(d,J＝13.2Hz,1H)8.23(dd,J＝8.0Hz,10.0Hz,1H),7.79(d,J＝8.0Hz,1H),7.51(dd,J＝4.4,8.0Hz,1H),5.48(s,1H),3.52(s,6H)。Step 1: Dissolve 2-aminonicotinaldehyde (1 g, 8.2 mmol) and 1,1-dimethoxypropane-2-one (1.3 g, 10.6 mmol) in a 10/1 ethanol/water (22 ml) solution, slowly add sodium hydroxide solution (3 M, 3.6 ml) to the solution at 0°C under a nitrogen atmosphere, and stir the mixture at room temperature for 3 hours. Concentrate the reaction mixture, dissolve the residue in ethyl acetate (50 ml), and The organic phase was washed with saturated brine (25 ml*2), dried, filtered and concentrated to obtain 2 g of yellow solid 2-(dimethoxymethyl)-1,8-naphthyridine (A-1). LC-MS: ESI m/z: 205.2 [M+H] + ; 1 H NMR (400 MHz, CDCl3) δ = 9.13 (dd, J = 2.0, 8.0 Hz, 1H), 8.28 (d, J = 13.2 Hz, 1H) 8.23 (dd, J = 8.0 Hz, 10.0 Hz, 1H), 7.79 (d, J = 8.0 Hz, 1H), 7.51 (dd, J = 4.4, 8.0 Hz, 1H), 5.48 (s, 1H), 3.52 (s, 6H).
步骤2：将A-1(1.6g，7.8mmol)溶于乙醇(20ml)中，在氮气氛围下向溶液中缓慢加入二氧化铂(50mg，0.2mmol)，混合液在氢气保护下(15psi)室温搅拌16小时。将反应混合液过滤，浓缩滤液得到淡黄色固体7-(二甲氧基甲基)-1,2,3,4-四氢-1,8-二氮杂萘(A-2)1g。LC-MS:ESI m/z:209.2[M+H]+；1H NMR(400MHz,CDCl3)δ＝7.09(d,J＝7.6Hz,1H),6.63(d,J＝7.6Hz,1H),5.07(s,1H),4.85(br s,1H),3.41-3.24(m,2H),3.33(s,6H)2.65(t,J＝6.0Hz,2H),1.84(quin,J＝6.0Hz,2H)。Step 2: A-1 (1.6 g, 7.8 mmol) was dissolved in ethanol (20 ml), platinum dioxide (50 mg, 0.2 mmol) was slowly added to the solution under nitrogen atmosphere, and the mixture was stirred at room temperature for 16 hours under hydrogen protection (15 psi). The reaction mixture was filtered, and the filtrate was concentrated to obtain 1 g of light yellow solid 7-(dimethoxymethyl)-1,2,3,4-tetrahydro-1,8-naphthyridine (A-2). LC-MS: ESI m/z: 209.2 [M+H] + ; 1 H NMR (400 MHz, CDCl 3 ) δ=7.09 (d, J=7.6 Hz, 1H), 6.63 (d, J=7.6 Hz, 1H), 5.07 (s, 1H), 4.85 (br s, 1H), 3.41-3.24 (m, 2H), 3.33 (s, 6H) 2.65 (t, J=6.0 Hz, 2H), 1.84 (quin, J=6.0 Hz, 2H).
步骤3：将A-2(1.2g，5.8mmol)溶于水(10ml)中，在-5℃下向溶液中加入12mmol/ml的浓盐酸(1.1ml，13mmol)，混合液在85℃下搅拌2小时。将混合液冷却至室温，通过3M的氢氧化钠溶液调节pH＝11，用乙酸乙酯萃取水相(30ml*2)，有机相经Na2SO4干燥，过滤并真空浓缩得到粗产物。粗品经正相硅胶柱色谱法纯化(DCM/MeOH＝20/1)得到黄色油状产品5,6,7,8-四氢-1,8-二氮杂萘-2-甲醛(A-3)850mg。1H NMR(400MHz,CDCl3)δ＝9.82(s,1H),7.30(d,J＝7.2Hz,1H),7.16(d,J＝7.2Hz,1H),5.23(brs,1H),3.47(t,J＝5.6Hz,2H),2.81(t,J＝6.4Hz,2H),2.03-1.87(m,2H)。Step 3: A-2 (1.2 g, 5.8 mmol) was dissolved in water (10 ml), and 12 mmol/ml concentrated hydrochloric acid (1.1 ml, 13 mmol) was added to the solution at -5°C, and the mixture was stirred at 85°C for 2 hours. The mixture was cooled to room temperature, and the pH was adjusted to 11 by 3M sodium hydroxide solution. The aqueous phase was extracted with ethyl acetate (30 ml*2), and the organic phase was dried over Na 2 SO 4 , filtered and concentrated in vacuo to obtain a crude product. The crude product was purified by normal phase silica gel column chromatography (DCM/MeOH=20/1) to obtain 850 mg of a yellow oily product 5,6,7,8-tetrahydro-1,8-naphthyridine-2-carboxaldehyde (A-3). 1 H NMR (400 MHz, CDCl 3 ) δ＝9.82 (s, 1H), 7.30 (d, J＝7.2 Hz, 1H), 7.16 (d, J＝7.2 Hz, 1H), 5.23 (brs, 1H), 3.47 (t, J＝5.6 Hz, 2H), 2.81 (t, J＝6.4 Hz, 2H), 2.03-1.87 (m, 2H).
步骤4：将A-3(3.1g，19.1mmol)溶于THF(60ml)中，室温下向溶液中依次加入(Boc)2O(8.3g，38.2mmol)和N，N-二甲胺基吡啶(4.7g，38.2mmol),混合液在氮气保护下80℃搅拌16小时。混合液用饱和氯化铵水溶液洗(20ml)，乙酸乙酯萃取(20ml*3)，有机相经Na2SO4干燥，过滤并真空浓缩得到粗产物。粗品经正相硅胶柱色谱法纯化(PE/EA＝3/1)得到淡黄色油状产品叔-丁基7-甲酰基-3,4-二氢-1,8-二氮杂萘-1(2H)-羧酸酯(A-4)1g。LC-MS:ESI m/z:263.3[M+H]+.1H NMR(400MHz,CDCl3)δ9.97(s,1H)7.65-7.60(m,1H)7.57-7.52(m,1H)3.85-3.81(m,2H)2.85(t,J＝6.50Hz,2H)2.02-1.96(m,2H)1.55(s,9H)。Step 4: A-3 (3.1 g, 19.1 mmol) was dissolved in THF (60 ml), (Boc) 2 O (8.3 g, 38.2 mmol) and N, N-dimethylaminopyridine (4.7 g, 38.2 mmol) were added to the solution in sequence at room temperature, and the mixture was stirred at 80°C for 16 hours under nitrogen protection. The mixture was washed with saturated aqueous ammonium chloride solution (20 ml), extracted with ethyl acetate (20 ml*3), and the organic phase was dried over Na 2 SO 4 , filtered and concentrated in vacuo to obtain a crude product. The crude product was purified by normal phase silica gel column chromatography (PE/EA=3/1) to obtain 1 g of a light yellow oily product, tert-butyl 7-formyl-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylate (A-4). LC-MS: ESI m/z: 263.3 [M+H] + . 1 H NMR (400 MHz, CDCl 3 ) δ 9.97 (s, 1H) 7.65-7.60 (m, 1H) 7.57-7.52 (m, 1H) 3.85-3.81 (m, 2H) 2.85 (t, J=6.50 Hz, 2H) 2.02-1.96 (m, 2H) 1.55 (s, 9H).
步骤5：将A-4(1g，3.8mmol)溶于THF(20ml)中，0℃下加入硼氢化钠(173.1mg，4.6mmol),混合液氮气保护下0℃搅拌1小时。向混合液中加水(5ml)猝灭，乙酸乙酯萃取(10ml*3)，有机相经饱和食盐水洗，Na2SO4干燥，过滤并真空浓缩得到粗产物。粗产品经正相硅胶柱色谱法纯化(PE/EA＝1/0到1/1)得到淡黄色油状产品叔-丁基7-(羟甲基)-3,4-二氢-1,8-二氮杂萘-1(2H)-羧酸酯(A-5)750mg。1H NMR(500MHz,CDCl3)δ7.38(d,J＝8.0Hz,1H)6.83(d,J＝8.0Hz,1H)4.66(s,2H)4.10(brs,1H)3.81-3.77(m,2H)2.78-2.75(m,2H)1.98-1.93(m,2H)1.53(s,9H)。Step 5: A-4 (1 g, 3.8 mmol) was dissolved in THF (20 ml), sodium borohydride (173.1 mg, 4.6 mmol) was added at 0°C, and the mixture was stirred at 0°C for 1 hour under nitrogen protection. Water (5 ml) was added to the mixture to quench it, and ethyl acetate (10 ml*3) was extracted. The organic phase was washed with saturated brine, dried over Na 2 SO 4 , filtered and concentrated in vacuo to obtain a crude product. The crude product was purified by normal phase silica gel column chromatography (PE/EA=1/0 to 1/1) to obtain tert-butyl 7-(hydroxymethyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylate (A-5) 750 mg as a light yellow oily product. 1 H NMR (500 MHz, CDCl3) δ 7.38 (d, J = 8.0 Hz, 1H) 6.83 (d, J = 8.0 Hz, 1H) 4.66 (s, 2H) 4.10 (brs, 1H) 3.81-3.77 (m, 2H) 2.78-2.75 (m, 2H) 1.98-1.93 (m, 2H) 1.53 (s, 9H).
步骤6：将A-5(600mg，2.3mmol)溶于DCM(10ml)中，室温下向溶液中加入三苯基膦(893.1mg，3.4mmol)和四溴化碳(978.6mg，3mmol),混合液氮气保护下室温搅拌3小时。浓缩反应液，粗品经正相硅胶柱色谱法纯化(PE/EA＝1/1)得到淡黄色油状产品叔-丁基7-(溴甲基)-3,4-二氢-1,8-二氮杂萘-1(2H)-羧酸酯(A)450mg。1H NMR(400MHz,CDCl3)δ7.40(d,J＝7.6Hz,1H)7.12(d,J＝7.6Hz,1H)4.51(s,2H)3.82-3.74(m,2H)2.78(t,J＝6.65Hz,2H)1.95(m,2H)1.56(s,9H)。Step 6: A-5 (600 mg, 2.3 mmol) was dissolved in DCM (10 ml), triphenylphosphine (893.1 mg, 3.4 mmol) and carbon tetrabromide (978.6 mg, 3 mmol) were added to the solution at room temperature, and the mixed liquid was stirred at room temperature for 3 hours under nitrogen protection. The reaction solution was concentrated, and the crude product was purified by normal phase silica gel column chromatography (PE/EA=1/1) to obtain 450 mg of light yellow oily product tert-butyl 7-(bromomethyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylate (A). 1 H NMR (400 MHz, CDCl3) δ 7.40 (d, J = 7.6 Hz, 1H) 7.12 (d, J = 7.6 Hz, 1H) 4.51 (s, 2H) 3.82-3.74 (m, 2H) 2.78 (t, J = 6.65 Hz, 2H) 1.95 (m, 2H) 1.56 (s, 9H).
中间体B：N-(7-(溴甲基)喹啉-2-基)-1,1-二苯基甲亚胺的制备
Intermediate B: Preparation of N-(7-(bromomethyl)quinolin-2-yl)-1,1-diphenylmethanimine
步骤1：将3-苯基丙烯酸(10g，67.5mmol)溶于二氯亚砜(50ml，689.3mmol)中，混合液90℃搅拌2小时，真空浓缩得到残余品，将残余品溶于DCM(200ml)中，室温下向溶液中加入碳酸氢钠(10g，119.mmol)中和过多的二氯亚砜，然后向反应液中依次加入4-二甲氨基吡啶(1g，8.2mmol)和m-甲苯胺(7.2g，67.5mmol)并搅拌14小时。反应液用盐酸溶液(5％，150ml)和碳酸氢钠水溶液(150ml)各洗一次，有机相用Na2SO4干燥，过滤，浓缩得到粗产物。粗产品经正相硅胶柱色谱法纯化(PE/EA＝4/1)得到白色固体产品3-苯基-N-(间-苯甲基)丙烯酰基酰胺(B-1)16g。LC-MS:ESI m/z:238.3[M+H]+.1H NMR(400MHz,CDCl3)δ＝7.75(d,J＝15.2Hz,1H),7.54-7.46(m,3H),7.45-7.38(m,1H),7.38-7.32(m,3H),7.20-7.22(m,1H),6.99-6.90(m,1H),6.59(d,J＝15.2Hz,1H),2.33(s,3H).Step 1: 3-phenylacrylic acid (10 g, 67.5 mmol) was dissolved in thionyl chloride (50 ml, 689.3 mmol), the mixture was stirred at 90°C for 2 hours, and vacuum concentrated to obtain a residue. The residue was dissolved in DCM (200 ml), and sodium bicarbonate (10 g, 119. mmol) was added to the solution at room temperature to neutralize the excessive thionyl chloride. Then 4-dimethylaminopyridine (1 g, 8.2 mmol) and m-toluidine (7.2 g, 67.5 mmol) were added to the reaction solution in sequence and stirred for 14 hours. The reaction solution was washed once with hydrochloric acid solution (5%, 150 ml) and sodium bicarbonate aqueous solution (150 ml), and the organic phase was dried over Na2SO4, filtered, and concentrated to obtain a crude product. The crude product was purified by normal phase silica gel column chromatography (PE/EA=4/1) to obtain 16 g of a white solid product 3-phenyl-N-(m-phenylmethyl)acrylamide (B-1). LC-MS: ESI m/z: 238.3 [M+H] + . 1 H NMR (400 MHz, CDCl3) δ = 7.75 (d, J = 15.2 Hz, 1H), 7.54-7.46 (m, 3H), 7.45-7.38 (m, 1H), 7.38-7.32 (m, 3H), 7.20-7.22 (m, 1H), 6.99-6.90 (m, 1H), 6.59 (d, J = 15.2 Hz, 1H), 2.33 (s, 3H).
步骤2：将B-1(15.7g，66mmol)溶于氯苯(200ml)中，氮气氛围下缓慢加入三氯化铝(44g，330mmol)，反应液在氮气保护下90℃搅拌2小时。将反应液冷却后倒入冰水中(300ml)，用乙酸乙酯萃取(200ml*2)，有机相用饱和食盐水洗(100ml)，Na2SO4干燥，过滤并真空浓缩得到粗产物。粗产品经正相硅胶柱色谱法纯化(PE/EA＝1/1)得到棕色固体产品7-甲基喹啉-2(1H)-酮(B-2)10g。1H NMR(400MHz,CDCl3)δ＝12.51(s,1H),8.05(d,J＝9.8Hz,1H),7.79(d,J＝9.3Hz,1H),7.45(d,J＝8.0Hz,1H),7.43-7.37(m,1H),7.34-7.30(m,1H),7.26(s,1H),7.05(d,J＝8.3Hz,2H),6.76(d,J＝9.8Hz,1H),6.68(d,J＝9.5Hz,1H),2.57(s,2H),2.46(s,3H)。Step 2: Dissolve B-1 (15.7 g, 66 mmol) in chlorobenzene (200 ml), slowly add aluminum chloride (44 g, 330 mmol) under nitrogen atmosphere, and stir the reaction solution at 90°C for 2 hours under nitrogen protection. After cooling the reaction solution, pour it into ice water (300 ml), extract it with ethyl acetate (200 ml*2), wash the organic phase with saturated brine (100 ml), dry it with Na 2 SO 4 , filter it and concentrate it in vacuo to obtain a crude product. The crude product is purified by normal phase silica gel column chromatography (PE/EA=1/1) to obtain 10 g of a brown solid product 7-methylquinolin-2(1H)-one (B-2). 1H NMR (400 MHz, CDCl3) δ = 12.51 (s, 1H), 8.05 (d, J = 9.8 Hz, 1H), 7.79 (d, J = 9.3 Hz, 1H), 7.45 (d, J = 8.0 Hz, 1H), 7.43-7.37 (m, 1H), 7.34-7.30 (m, 1H), 7.26 (s, 1H), 7.05 (d, J = 8.3 Hz, 2H), 6.76 (d, J = 9.8 Hz, 1H), 6.68 (d, J = 9.5 Hz, 1H), 2.57 (s, 2H), 2.46 (s, 3H).
步骤3：将B-2(9.8g，61.4mmol)溶于三氯氧磷(130ml，1.4摩尔)中，混合液100℃搅拌2小时。将混合液浓缩，缓慢倒入冰水中(300ml)并保持冰浴，加入氢氧化钠固体调节pH＝7。混合液经乙酸乙酯萃取(200ml)，有机相经饱和食盐水洗，Na2SO4干燥，真空过滤浓缩得到粗产物。粗产品经正相硅胶柱色谱法纯化(PE/EA＝10/1)得到白色固体2-氯-7-甲基喹啉(B-3)3g。1H NMR(400MHz,CDCl3)δ＝8.05(d,J＝8.5Hz,1H),7.80(s,1H),7.70(d,J＝8.2Hz,1H),7.39(d,J＝8.1Hz,1H),7.31(d,J＝8.4Hz,1H),2.56(s,3H)。Step 3: Dissolve B-2 (9.8 g, 61.4 mmol) in phosphorus oxychloride (130 ml, 1.4 mol), and stir the mixture at 100°C for 2 hours. Concentrate the mixture, slowly pour it into ice water (300 ml) and keep it in an ice bath, and add solid sodium hydroxide to adjust the pH to 7. The mixture is extracted with ethyl acetate (200 ml), and the organic phase is washed with saturated brine, dried over Na 2 SO 4 , and vacuum filtered and concentrated to obtain a crude product. The crude product is purified by normal phase silica gel column chromatography (PE/EA=10/1) to obtain 3 g of 2-chloro-7-methylquinoline (B-3) as a white solid. 1 H NMR (400 MHz, CDCl3) δ＝8.05 (d, J＝8.5 Hz, 1H), 7.80 (s, 1H), 7.70 (d, J＝8.2 Hz, 1H), 7.39 (d, J＝8.1 Hz, 1H), 7.31 (d, J＝8.4 Hz, 1H), 2.56 (s, 3H).
步骤4：依次将B-3(300mg，1.7mmol)，二苯基甲亚胺(459.1mg，2.5mmol)，钯催化剂(77.3mg，0.1mmol)，配体(105.2mg，0.2mmol)和碳酸铯(1.1g，3.4mmol)溶于1,4-二氧六环(5ml)中，混合液氮气保护下90℃搅拌16小时。浓缩混合液，粗品经正相硅胶柱色谱法纯化(PE/EA＝19/1)得到淡黄色油状产品N-(7-甲基喹啉-2-基)-1,1-二苯基甲亚胺(B-4)570mg。LC-MS:ESI m/z:323.4[M+H]+.1H NMR(400MHz,DMSO-d6)δ＝8.04(d,J＝8.4Hz,1H),7.75-7.69(m,3H),7.64-7.52(m,5H),7.25-7.20(m,5H),6.83(d,J＝8.4Hz,1H),2.45(s,3H)。Step 4: Dissolve B-3 (300 mg, 1.7 mmol), diphenylmethamine (459.1 mg, 2.5 mmol), palladium catalyst (77.3 mg, 0.1 mmol), ligand (105.2 mg, 0.2 mmol) and cesium carbonate (1.1 g, 3.4 mmol) in 1,4-dioxane (5 ml) in sequence, and stir the mixed solution at 90 ° C for 16 hours under nitrogen protection. Concentrate the mixed solution, and purify the crude product by normal phase silica gel column chromatography (PE/EA=19/1) to obtain 570 mg of light yellow oily product N-(7-methylquinolin-2-yl)-1,1-diphenylmethamine (B-4). LC-MS: ESI m/z: 323.4 [M+H] + . 1 H NMR (400 MHz, DMSO-d 6 ) δ=8.04 (d, J=8.4 Hz, 1H), 7.75-7.69 (m, 3H), 7.64-7.52 (m, 5H), 7.25-7.20 (m, 5H), 6.83 (d, J=8.4 Hz, 1H), 2.45 (s, 3H).
步骤5：将B-4(570mg，1.3mmol)溶于四氯化碳(28ml)中，室温下向溶液中依次加入N-溴代琥珀酰亚胺(258.3mg，1.5mmol)和过氧化二苯甲酰(63.9mg，0.3mmol)，混合液80℃搅拌12小时。反应液用DCM(100ml)稀释后，用氢氧化钠(0.04mol/ml)和饱和食盐水(40ml)各洗一次，有机相用Na2SO4干燥，过滤并真空浓缩得到粗产物，粗产品经正相硅胶柱色谱法纯化(PE/EA＝10/1)得到黄色油状产品N-(7-(溴甲基)喹啉-2-基)-1,1-二苯基甲亚胺(B)300mg。LCMS:ESI-MS m/z＝401.4[M+1]。 Step 5: Dissolve B-4 (570 mg, 1.3 mmol) in carbon tetrachloride (28 ml), add N-bromosuccinimide (258.3 mg, 1.5 mmol) and dibenzoyl peroxide (63.9 mg, 0.3 mmol) to the solution at room temperature, and stir the mixture at 80°C for 12 hours. The reaction solution was diluted with DCM (100 ml), washed once with sodium hydroxide (0.04 mol/ml) and saturated brine (40 ml), and the organic phase was dried over Na 2 SO 4 , filtered and concentrated in vacuo to obtain a crude product, which was purified by normal phase silica gel column chromatography (PE/EA=10/1) to obtain 300 mg of a yellow oily product, N-(7-(bromomethyl)quinolin-2-yl)-1,1-diphenylmethanimine (B). LCMS: ESI-MS m/z=401.4[M+1].
中间体C：(6-((叔-丁氧基羰基)氨基)吡啶-2-基)甲基4-甲基苯磺酸酯的制备
Intermediate C: Preparation of (6-((tert-butoxycarbonyl)amino)pyridin-2-yl)methyl 4-methylbenzenesulfonate
步骤1：参见中间体A-5的制备方法，过滤并真空浓缩后得到白色固体(6-(羟甲基)吡啶-2-基)氨基甲酸叔丁酯(C-1)100mg。Step 1: Refer to the preparation method of intermediate A-5, filter and concentrate in vacuo to obtain 100 mg of white solid tert-butyl (6-(hydroxymethyl)pyridin-2-yl)carbamate (C-1).
步骤2：将C-1(100mg,445.9μmol)和二异丙基乙胺(172.9mg,1.3mmol)溶于乙腈(4ml)中，在0℃下加入对甲苯磺酰氯(47.2mg,668.9μmol)，混合液氮气保护下室温搅拌3小时。反应液经饱和碳酸氢钠水溶液(5ml)淬灭，用乙酸乙酯萃取(10ml*3)，有机相经干燥，过滤并真空浓缩得到粗产物，粗产品经正相硅胶柱色谱法纯化(PE/EA＝1/1)得到淡黄色油状产品(6-((叔-丁氧基羰基)氨基)吡啶-2-基)甲基4-甲基苯磺酸酯(C)120mg。1H NMR(400MHz,CDCl3)δ7.89-7.76(m,3H)7.62(t,J＝8.0Hz,1H)7.33(d,J＝8.0Hz,2H)7.07(s,1H)7.00(d,J＝8.0Hz,1H)5.00(s,2H)2.45(s,3H)1.51(s,9H)。Step 2: C-1 (100 mg, 445.9 μmol) and diisopropylethylamine (172.9 mg, 1.3 mmol) were dissolved in acetonitrile (4 ml), p-toluenesulfonyl chloride (47.2 mg, 668.9 μmol) was added at 0°C, and the mixture was stirred at room temperature for 3 hours under nitrogen protection. The reaction solution was quenched with saturated sodium bicarbonate aqueous solution (5 ml), extracted with ethyl acetate (10 ml*3), and the organic phase was dried, filtered and concentrated in vacuo to obtain a crude product, which was purified by normal phase silica gel column chromatography (PE/EA=1/1) to obtain 120 mg of a light yellow oily product (6-((tert-butoxycarbonyl)amino)pyridin-2-yl)methyl 4-methylbenzenesulfonate (C). 1 H NMR (400 MHz, CDCl 3 ) δ 7.89-7.76 (m, 3H) 7.62 (t, J=8.0 Hz, 1H) 7.33 (d, J=8.0 Hz, 2H) 7.07 (s, 1H) 7.00 (d, J=8.0 Hz, 1H) 5.00 (s, 2H) 2.45 (s, 3H) 1.51 (s, 9H).
中间体D：叔-丁基6-(溴甲基)-2,3-二氢-4H-吡啶并[3,2-b][1,4]噁嗪-4-羧酸酯的制备
Intermediate D: Preparation of tert-butyl 6-(bromomethyl)-2,3-dihydro-4H-pyrido[3,2-b][1,4]oxazine-4-carboxylate
步骤1：依次将6-溴-2-硝基吡啶-3-酚(9g，41.1mmol)，溴乙酸乙酯(8.2g，49.3mmol)和碳酸钾(11.4g，82.2mmol)溶于丙酮(90ml)中，氮气保护下60℃搅拌2小时。反应液用水洗(200ml)，乙酸乙酯萃取(200ml)，有机相用饱和食盐水洗，Na2SO4干燥，过滤并真空浓缩得到棕色油状粗产品乙基2-((6-溴-2-硝基吡啶-3-基)氧代)乙酸酯(D-1)12g。Step 1: Dissolve 6-bromo-2-nitropyridine-3-ol (9 g, 41.1 mmol), ethyl bromoacetate (8.2 g, 49.3 mmol) and potassium carbonate (11.4 g, 82.2 mmol) in acetone (90 ml) in sequence and stir at 60°C for 2 hours under nitrogen protection. Wash the reaction solution with water (200 ml), extract with ethyl acetate (200 ml), wash the organic phase with saturated brine, dry over Na 2 SO 4 , filter and concentrate in vacuo to obtain 12 g of a brown oily crude product, ethyl 2-((6-bromo-2-nitropyridine-3-yl)oxy)acetate (D-1).
步骤2：依次将D-1(12g，39.3mmol)，铁粉(11g，196.7mmol)和氯化铵(10.5g，196.7mmol)溶于10/1的甲醇/水(110ml)中，氮气保护下60℃搅拌16小时。反应液经过滤浓缩后，加入水稀释(300ml)，乙酸乙酯萃取(300ml)，有机相用饱和食盐水洗，Na2SO4干燥，过滤并真空浓缩得到残余物，将其溶于乙酸(100ml)中，90℃搅拌3小时，之后浓缩过滤得到棕色固体产品6-溴-2H-吡啶并[3,2-b][1,4]噁嗪-3(4H)-酮(D-2)7g。LC-MS:ESI m/z:229.1[M+H]+。Step 2: Dissolve D-1 (12 g, 39.3 mmol), iron powder (11 g, 196.7 mmol) and ammonium chloride (10.5 g, 196.7 mmol) in 10/1 methanol/water (110 ml) in sequence, and stir at 60°C for 16 hours under nitrogen protection. After filtering and concentrating, the reaction solution was diluted with water (300 ml), extracted with ethyl acetate (300 ml), and the organic phase was washed with saturated brine, dried over Na 2 SO 4 , filtered and concentrated in vacuo to obtain a residue, which was dissolved in acetic acid (100 ml), stirred at 90°C for 3 hours, and then concentrated and filtered to obtain 7 g of a brown solid product 6-bromo-2H-pyrido[3,2-b][1,4]oxazine-3(4H)-one (D-2). LC-MS: ESI m/z: 229.1[M+H] + .
步骤3：将D-2(1g，4.4mmol)溶于THF(10ml)中，室温下加入硼烷二甲硫醚(10M，1.1ml)，90℃搅拌1.5小时后再加入甲醇(1ml)，90℃搅拌0.5小时。将反应液浓缩，饱和碳酸氢钠洗(50ml)，乙酸乙酯(50ml)萃取，有机相经Na2SO4干燥，过滤并真空浓缩得到白色固体6-溴-3,4-二氢-2H-吡啶并[3,2-b][1,4]噁嗪(D-3)757mg。LC-MS:ESI m/z:214.7[M+H]+。Step 3: Dissolve D-2 (1 g, 4.4 mmol) in THF (10 ml), add borane dimethyl sulfide (10 M, 1.1 ml) at room temperature, stir at 90°C for 1.5 hours, then add methanol (1 ml), stir at 90°C for 0.5 hours. Concentrate the reaction solution, wash with saturated sodium bicarbonate (50 ml), extract with ethyl acetate (50 ml), dry the organic phase over Na 2 SO 4 , filter and concentrate in vacuo to obtain 757 mg of 6-bromo-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazine (D-3) as a white solid. LC-MS: ESI m/z: 214.7 [M+H] + .
步骤4：依次将D-3(700mg，3.3mmol)，乙烯基三氟硼酸钾(654mg，4.9mmol)，钯催化剂(119mg，0.2mmol)和碳酸铯(2.7g，8.1mmol)溶于1，4-二氧六环(10ml)，氮气保护下100℃搅拌2小时。反应液直接浓缩，正相硅胶柱色谱法纯化(PE/EA＝3/1)得到无色油状产品6-乙烯基-3,4-二氢-2H-吡啶并[3,2-b][1,4]噁嗪(D-4)400mg。LC-MS:ESI m/z:163.3[M+H] +。Step 4: Dissolve D-3 (700 mg, 3.3 mmol), potassium vinyl trifluoroborate (654 mg, 4.9 mmol), palladium catalyst (119 mg, 0.2 mmol) and cesium carbonate (2.7 g, 8.1 mmol) in 1,4-dioxane (10 ml) in sequence and stir at 100 ° C for 2 hours under nitrogen protection. The reaction solution was directly concentrated and purified by normal phase silica gel column chromatography (PE/EA=3/1) to obtain 400 mg of colorless oily product 6-vinyl-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazine (D-4). LC-MS: ESI m/z: 163.3 [M+H] + .
步骤5：参见中间体A-4的制备方法，粗品经正相硅胶柱色谱法纯化(PE/EA＝5/1)得到无色油状产品叔-丁基6-乙烯基-2,3-二氢-4H-吡啶并[3,2-b][1,4]噁嗪-4-羧酸酯(D-5)500mg。1H NMR(400MHz,CDCl3)δ＝7.11(d,J＝8.3Hz,1H),6.99(d,J＝8.2Hz,1H),6.70(dd,J＝10.8,17.2Hz,1H),6.10(d,J＝17.2Hz,1H),5.32(d,J＝10.8Hz,1H),4.25(d,J＝3.3Hz,2H),3.92(br s,2H),1.56(s,9H)。Step 5: Referring to the preparation method of intermediate A-4, the crude product was purified by normal phase silica gel column chromatography (PE/EA=5/1) to obtain 500 mg of colorless oily product tert-butyl 6-vinyl-2,3-dihydro-4H-pyrido[3,2-b][1,4]oxazine-4-carboxylate (D-5). 1 H NMR (400 MHz, CDCl 3 ) δ=7.11 (d, J=8.3 Hz, 1H), 6.99 (d, J=8.2 Hz, 1H), 6.70 (dd, J=10.8, 17.2 Hz, 1H), 6.10 (d, J=17.2 Hz, 1H), 5.32 (d, J=10.8 Hz, 1H), 4.25 (d, J=3.3 Hz, 2H), 3.92 (br s, 2H), 1.56 (s, 9H).
步骤6：将D-5(500mg，1.9mmol)溶于4/1的THF/H2O(12.5ml)中，室温下依次加入高碘酸钠(1.1g，5.3mmol)和二水合锇酸钾(4.2mg，0.01mmol),室温搅拌1小时。反应液经过滤，水洗(30ml)，乙酸乙酯(30ml)萃取，浓缩，粗品经正相硅胶柱色谱法纯化(PE/EA＝3/1)得到淡黄色固体产品叔-丁基6-甲酰基-2,3-二氢-4H-吡啶并[3,2-b][1,4]噁嗪-4-羧酸酯(D-6)428mg。LC-MS:ESI m/z:209.2[M+H]+。Step 6: Dissolve D-5 (500 mg, 1.9 mmol) in 4/1 THF/H 2 O (12.5 ml), add sodium periodate (1.1 g, 5.3 mmol) and potassium osmate dihydrate (4.2 mg, 0.01 mmol) in turn at room temperature, and stir at room temperature for 1 hour. The reaction solution was filtered, washed with water (30 ml), extracted with ethyl acetate (30 ml), and concentrated. The crude product was purified by normal phase silica gel column chromatography (PE/EA=3/1) to obtain 428 mg of light yellow solid product tert-butyl 6-formyl-2,3-dihydro-4H-pyrido[3,2-b][1,4]oxazine-4-carboxylate (D-6). LC-MS: ESI m/z: 209.2[M+H] + .
步骤7：参见中间体A-5的制备方法，粗品经正相硅胶柱色谱法纯化(DCM/MeOH＝20/1)得到无色油状产品叔-丁基6-(羟甲基)-2,3-二氢-4H-吡啶并[3,2-b][1,4]噁嗪-4-羧酸酯(D-7)400mg。LC-MS:ESI m/z:289.2[M+23]+。Step 7: Referring to the preparation method of intermediate A-5, the crude product was purified by normal phase silica gel column chromatography (DCM/MeOH=20/1) to obtain 400 mg of colorless oily product tert-butyl 6-(hydroxymethyl)-2,3-dihydro-4H-pyrido[3,2-b][1,4]oxazine-4-carboxylate (D-7). LC-MS: ESI m/z: 289.2 [M+23] + .
步骤8：将D-7(50mg，0.2mmol)溶于DCM(3ml)中，室温下依次加入四溴化碳(93.4mg，0.3mmol)和三苯基磷(74mg，0.3mmol),室温搅拌2小时。将反应液加水(20ml)淬灭，DCM(20ml)萃取，有机相经干燥，过滤，浓缩，粗品经硅胶板纯化(DCM/MeOH＝20/1)得到无色油状产品叔-丁基6-(溴甲基)-2,3-二氢-4H-吡啶并[3,2-b][1,4]噁嗪-4-羧酸酯(D)30mg。LC-MS:ESI m/z:273.2[M-55]+。Step 8: D-7 (50 mg, 0.2 mmol) was dissolved in DCM (3 ml), and carbon tetrabromide (93.4 mg, 0.3 mmol) and triphenylphosphine (74 mg, 0.3 mmol) were added successively at room temperature, and stirred at room temperature for 2 hours. The reaction solution was quenched by adding water (20 ml), extracted with DCM (20 ml), and the organic phase was dried, filtered, and concentrated. The crude product was purified on a silica gel plate (DCM/MeOH=20/1) to obtain 30 mg of a colorless oily product, tert-butyl 6-(bromomethyl)-2,3-dihydro-4H-pyrido[3,2-b][1,4]oxazine-4-carboxylate (D). LC-MS: ESI m/z: 273.2[M-55] + .
中间体E：6-(溴甲基)-2H-吡啶并[3,2-b][1,4]噁嗪-3(4H)-酮的制备
Intermediate E: Preparation of 6-(bromomethyl)-2H-pyrido[3,2-b][1,4]oxazin-3(4H)-one
步骤1：参见中间体D-4的制备方法，粗品经正相硅胶柱色谱法纯化(PE/EA＝3/1)得到白色固体产品6-乙烯基-2H-吡啶并[3,2-b][1,4]噁嗪-3(4H)-酮(E-1)2g。LC-MS:ESI m/z:177.2[M+H]+。Step 1: Referring to the preparation method of intermediate D-4, the crude product was purified by normal phase silica gel column chromatography (PE/EA=3/1) to obtain 2 g of white solid product 6-vinyl-2H-pyrido[3,2-b][1,4]oxazine-3(4H)-one (E-1). LC-MS: ESI m/z: 177.2 [M+H] + .
步骤2：参见中中间体D-6的制备方法，得到棕色固体粗产品3-羰基-3,4-二氢-2H-吡啶并[3,2-b][1,4]噁嗪-6-甲醛(E-2)900mg。LC-MS:ESI m/z:179.3[M+H]+。Step 2: Refer to the preparation method of intermediate D-6 to obtain 900 mg of brown solid crude product 3-carbonyl-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazine-6-carbaldehyde (E-2). LC-MS: ESI m/z: 179.3 [M+H] + .
步骤3：参见中间体A-5的制备方法，粗品经正相硅胶柱色谱法纯化(DCM/MeOH＝10/1)得到黄色固体产品6-(羟甲基)-2H-吡啶并[3,2-b][1,4]噁嗪-3(4H)-酮(E-3)640mg。LC-MS:ESI m/z:181.3[M+H]+。Step 3: Referring to the preparation method of intermediate A-5, the crude product was purified by normal phase silica gel column chromatography (DCM/MeOH=10/1) to obtain 640 mg of yellow solid product 6-(hydroxymethyl)-2H-pyrido[3,2-b][1,4]oxazine-3(4H)-one (E-3). LC-MS: ESI m/z: 181.3[M+H]+.
步骤4：参见中间体D步骤8的制备方法，粗品经硅胶板纯化(DCM/MeOH＝20/1)得到白色固体产品6-(溴甲基)-2H-吡啶并[3,2-b][1,4]噁嗪-3(4H)-酮(E)34mg。LC-MS:ESI m/z:243.2[M+H]+。Step 4: Referring to the preparation method of intermediate D step 8, the crude product was purified by silica gel plate (DCM/MeOH=20/1) to obtain 34 mg of white solid product 6-(bromomethyl)-2H-pyrido[3,2-b][1,4]oxazin-3(4H)-one (E). LC-MS: ESI m/z: 243.2 [M+H] + .
中间体F：叔-丁基5-甲酰基-3H-咪唑并[4,5-b]吡啶-3-羧酸酯的制备
Intermediate F: Preparation of tert-butyl 5-formyl-3H-imidazo[4,5-b]pyridine-3-carboxylate
步骤1：参见中间体A-4的制备方法，浓缩反应液粗品经正相硅胶柱色谱法纯化(PE/EA＝4/1)得到淡黄色固体产品叔-丁基5-溴-3H-咪唑并[4,5-b]吡啶-3-羧酸酯(F-1)550mg。1H NMR(400MHz,CDCl3)δ8.63(s,1H)8.15(d,J＝8.44Hz,1H)7.52(d,J＝8.44Hz,1H)1.71(s,9H)。Step 1: Referring to the preparation method of intermediate A-4, the concentrated reaction solution was purified by normal phase silica gel column chromatography (PE/EA=4/1) to obtain 550 mg of a light yellow solid product, tert-butyl 5-bromo-3H-imidazo[4,5-b]pyridine-3-carboxylate (F-1). 1 H NMR (400 MHz, CDCl3) δ8.63 (s, 1H) 8.15 (d, J=8.44 Hz, 1H) 7.52 (d, J=8.44 Hz, 1H) 1.71 (s, 9H).
步骤2：参见中间体D-4的制备方法，粗品经正相硅胶柱色谱法纯化(EA)得到白色固体产品5-乙烯基-3H-咪唑并[4,5-b]吡啶(F-2)160mg。1H NMR(400MHz,DMSO-d6)δ12.57-13.20(m,1H)8.41(br s,1H)7.88-8.10(m,1H)7.40(d,J＝8.19Hz,1H)6.89(m,1H)6.20(d,J＝17.36Hz,1H)5.42(br d,J＝10.39Hz,1H)。Step 2: Referring to the preparation method of intermediate D-4, the crude product was purified by normal phase silica gel column chromatography (EA) to obtain 160 mg of white solid product 5-vinyl-3H-imidazo[4,5-b]pyridine (F-2). 1 H NMR (400 MHz, DMSO-d 6 ) δ 12.57-13.20 (m, 1H) 8.41 (br s, 1H) 7.88-8.10 (m, 1H) 7.40 (d, J＝8.19 Hz, 1H) 6.89 (m, 1H) 6.20 (d, J＝17.36 Hz, 1H) 5.42 (br d, J＝10.39 Hz, 1H).
步骤3：参见中间体A-4的制备方法，粗品经正相硅胶柱色谱法纯化(PE/EA＝4/1)得到淡黄色固体产品叔-丁基5-乙烯基-3H-咪唑并[4,5-b]吡啶-3-羧酸酯(F-3)120mg。LC-MS:ESI m/z＝246.4[M+H]+.1H NMR(400MHz,CDCl3)δ8.65(s,1H)8.22(d,J＝8.31Hz,1H)7.42(d,J＝8.44Hz,1H)6.87-7.01(m,1H)6.36(d,J＝17.48Hz,1H)5.53(d,J＝10.88Hz,1H)1.71(s,9H)。Step 3: Referring to the preparation method of intermediate A-4, the crude product was purified by normal phase silica gel column chromatography (PE/EA=4/1) to obtain 120 mg of light yellow solid product tert-butyl 5-vinyl-3H-imidazo[4,5-b]pyridine-3-carboxylate (F-3). LC-MS: ESI m/z=246.4[M+H] + . 1 H NMR (400MHz,CDCl3)δ8.65(s,1H)8.22(d,J=8.31Hz,1H)7.42(d,J=8.44Hz,1H)6.87-7.01(m,1H)6.36(d,J=17.48Hz,1H)5.53(d,J=10.88Hz,1H)1.71(s,9H).
步骤4：参见中间体D-6的制备方法，得到淡黄色固体产品叔-丁基5-甲酰基-3H-咪唑并[4,5-b]吡啶-3-羧酸酯(F)120mg。LCMS:ESI-MS m/z＝246.4[M-t-Bu+]；1H NMR(400MHz,CDCl3)δ10.12(s,1H)8.73(s,1H)8.37(d,J＝8.31Hz,1H)8.03(d,J＝8.31Hz,1H)1.66(s,9H)。Step 4: Referring to the preparation method of intermediate D-6, 120 mg of light yellow solid product tert-butyl 5-formyl-3H-imidazo[4,5-b]pyridine-3-carboxylate (F) was obtained. LCMS: ESI-MS m/z=246.4 [Mt-Bu + ]; 1 H NMR (400 MHz, CDCl3) δ10.12 (s, 1H) 8.73 (s, 1H) 8.37 (d, J=8.31 Hz, 1H) 8.03 (d, J=8.31 Hz, 1H) 1.66 (s, 9H).
中间体G：(3-(3,5-二甲基-1H-吡唑-1-基)苯基)硼酸的制备
Intermediate G: Preparation of (3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)boronic acid
步骤1：(3-溴苯基)肼盐酸(4.4g，19.7mmol)，醋酸钾(2.5g，25.6mmol)和乙酰丙酮(2.1g，20.7mmol)溶于乙醇溶液(20ml)中，氮气保护85℃搅拌3小时。加水猝灭，乙酸乙酯萃取，有机相用饱和盐水洗，Na2SO4干燥，过滤并真空浓缩得到黄色油状产品1-(3-溴苯基)-3,5-二甲基-1H-吡唑(G-1)5g。LC-MS:ESI m/z:251.1[M+H]+.1H NMR(400MHz,CDCl3)δ＝7.65(s,1H),7.48(d,J＝7.9Hz,1H),7.41-7.36(m,1H),7.35-7.28(m,1H),6.01(s,1H),2.31(d,J＝12.6Hz,6H)。Step 1: (3-Bromophenyl)hydrazine hydrochloride (4.4 g, 19.7 mmol), potassium acetate (2.5 g, 25.6 mmol) and acetylacetone (2.1 g, 20.7 mmol) were dissolved in ethanol solution (20 ml), stirred at 85 ° C for 3 hours under nitrogen protection. Water was added to quench, extracted with ethyl acetate, and the organic phase was washed with saturated brine, dried over Na 2 SO 4 , filtered and concentrated in vacuo to obtain 5 g of 1-(3-bromophenyl)-3,5-dimethyl-1H-pyrazole (G-1) as a yellow oily product. LC-MS: ESI m/z: 251.1 [M+H] + .1H NMR (400 MHz, CDCl3) δ = 7.65 (s, 1H), 7.48 (d, J = 7.9 Hz, 1H), 7.41-7.36 (m, 1H), 7.35-7.28 (m, 1H), 6.01 (s, 1H), 2.31 (d, J = 12.6 Hz, 6H).
步骤2：将G-1(4g，15.9mmol)和硼酸三异丙酯(1.5eq.)溶于无水THF(40ml)中，氮气保护下-65℃缓慢滴加入正丁基锂(2.5M，16ml)，混合液-65℃～-60℃搅拌2小时。-65℃下向溶液中滴加稀盐酸(3M，11ml)淬灭反应，经氢氧化钠溶液(1M)调节pH＝7，EA萃取，有机相用饱和盐水洗，Na2SO4干燥，过滤并真空浓缩，粗品正相硅胶柱色谱法(DCM/MeOH＝20/1)纯化得到淡黄色固体产品(3-(3,5-二甲基-1H-吡唑-1-基)苯基)硼酸(G)3g。LC-MS:ESI m/z:217.3[M+H]+。Step 2: G-1 (4 g, 15.9 mmol) and triisopropyl borate (1.5 eq.) were dissolved in anhydrous THF (40 ml), and n-butyl lithium (2.5 M, 16 ml) was slowly added dropwise at -65°C under nitrogen protection, and the mixture was stirred at -65°C to -60°C for 2 hours. Dilute hydrochloric acid (3 M, 11 ml) was added dropwise to the solution at -65°C to quench the reaction, and the pH was adjusted to 7 with sodium hydroxide solution (1 M), and EA was extracted. The organic phase was washed with saturated brine, dried over Na 2 SO 4 , filtered and concentrated in vacuo, and the crude product was purified by normal phase silica gel column chromatography (DCM/MeOH=20/1) to obtain 3 g of a light yellow solid product (3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)boronic acid (G). LC-MS: ESI m/z: 217.3 [M+H] + .
中间体H：(3-溴-5-(3,5-二甲基-1H-吡唑-1-基)苯基)硼酸的制备
Intermediate H: Preparation of (3-bromo-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)boronic acid
步骤1：将3,5-二溴苯胺(5g,19.9mmol)溶于40ml浓盐酸中，氮气氛围0℃下向溶液中加入亚硝酸钠(2.1g，29.9mmol)，0℃下搅拌1小时，然后向溶液滴加氯化亚锡(7.6g，Step 1: Dissolve 3,5-dibromoaniline (5 g, 19.9 mmol) in 40 ml of concentrated hydrochloric acid, add sodium nitrite (2.1 g, 29.9 mmol) to the solution under nitrogen atmosphere at 0°C, stir at 0°C for 1 hour, and then add stannous chloride (7.6 g,
40mmol)的浓盐酸(80ml)溶液，氮气氛围室温搅拌16小时。反应液冷却后，过滤并真空浓缩得到黄色固体产品3-溴-5-肼基苯胺盐酸(H-1)6g。LC-MS:ESI m/z:264.9[M+H]+。40mmol) of concentrated hydrochloric acid (80ml), stirred at room temperature for 16 hours under nitrogen atmosphere. After the reaction solution was cooled, filtered and concentrated in vacuo to obtain 6g of yellow solid product 3-bromo-5-hydrazinoaniline hydrochloride (H-1). LC-MS: ESI m/z: 264.9 [M+H] + .
步骤2：参见中间体G-1的制备方法，粗品经柱层析(PE/EA＝95/5)纯化后得到黄色固体产物1-(3,5-二溴苯基)-3,5-二甲基-1H-吡唑(H-2)3g。LC-MS:ESI m/z:329.3[M+H]+。Step 2: Referring to the preparation method of intermediate G-1, the crude product was purified by column chromatography (PE/EA=95/5) to obtain 3 g of yellow solid product 1-(3,5-dibromophenyl)-3,5-dimethyl-1H-pyrazole (H-2). LC-MS: ESI m/z: 329.3 [M+H] + .
步骤3：将H-2(500mg,1.5mmol)，双联嚬哪醇硼酸酯(462mg,1.8mmol)，乙酸钾(446mg,4.6mmol)和1,1-双(二苯基磷)二茂铁氯化钯(111mg,152μmol)溶于5ml无水Diox中，氮气氛围90℃搅拌2小时。浓缩反应液，残渣用2M稀盐酸稀释，用EA萃取，有机相用饱和盐水洗，Na2SO4干燥，过滤并真空浓缩得到黑棕色油状产品1-(3-溴-5-(4,4,5,5-四甲基-1,3,2-二噁硼戊环-2-基)苯基)-3,5-二甲基-1H-吡唑(H-3)400mg。LC-MS:ESI m/z:376.8[M+H]+。Step 3: H-2 (500 mg, 1.5 mmol), bis-naphthalene boronate (462 mg, 1.8 mmol), potassium acetate (446 mg, 4.6 mmol) and 1,1-bis(diphenylphosphino)ferrocenepalladium chloride (111 mg, 152 μmol) were dissolved in 5 ml of anhydrous Diox and stirred at 90°C for 2 hours under nitrogen atmosphere. The reaction solution was concentrated, the residue was diluted with 2M dilute hydrochloric acid, extracted with EA, the organic phase was washed with saturated brine, dried over Na 2 SO 4 , filtered and concentrated in vacuo to obtain 400 mg of a dark brown oily product 1-(3-bromo-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-3,5-dimethyl-1H-pyrazole (H-3). LC-MS: ESI m/z: 376.8 [M+H] + .
步骤4：将H-3(400mg,1.1mmol)溶于4/1的THF/H2O(5ml)中，向反应液中加入高碘酸钠(681mg，3.2mmol)和1M稀盐酸(955μl)，室温下搅拌12小时。浓缩反应液，粗品经高效液相色谱分离后得到白色固体产品(3-溴-5-(3,5-二甲基-1H-吡唑-1-基)苯基)硼酸(H)70mg。LC-MS:ESI m/z:295.0[M+H]+。Step 4: H-3 (400 mg, 1.1 mmol) was dissolved in 4/1 THF/H 2 O (5 ml), sodium periodate (681 mg, 3.2 mmol) and 1M dilute hydrochloric acid (955 μl) were added to the reaction solution, and stirred at room temperature for 12 hours. The reaction solution was concentrated, and the crude product was separated by HPLC to obtain 70 mg of a white solid product (3-bromo-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)boronic acid (H). LC-MS: ESI m/z: 295.0 [M+H] + .
中间体I：(3-(叔丁基)-5-(3,5-二甲基-1H-吡唑-1-基)苯基)硼酸的制备
Intermediate I: Preparation of (3-(tert-butyl)-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)boronic acid
步骤1：参见中间体B-4的制备方法，粗品经柱层析(PE/EA＝98/2)纯化得到黄色油状产品N-(3-溴-5-(叔丁基)苯基)-1,1-二苯基甲亚胺(I-1)5g。LC-MS:ESI m/z:392.1[M+H]+。Step 1: Referring to the preparation method of intermediate B-4, the crude product was purified by column chromatography (PE/EA=98/2) to obtain 5 g of yellow oily product N-(3-bromo-5-(tert-butyl)phenyl)-1,1-diphenylmethanimine (I-1). LC-MS: ESI m/z: 392.1 [M+H] + .
步骤2：将I-1(4g,10.2mmol)溶于4/1的THF/浓HCl(37.5ml)中，氮气氛围室温搅拌2小时。浓缩反应液，残渣加水30ml稀释，水相加入2M氢氧化钠水溶液调节PH＝7-8，后经EA萃取，有机相用饱和盐水洗，Na2SO4干燥，过滤并真空浓缩，粗品经柱层析(PE/EA＝95/5)纯化得到黄色液体产品3-溴-5-(叔丁基)苯胺(I-2)2g。LC-MS:ESI m/z:227.7[M+H]+。Step 2: I-1 (4 g, 10.2 mmol) was dissolved in 4/1 THF/concentrated HCl (37.5 ml), and stirred at room temperature for 2 hours under nitrogen atmosphere. The reaction solution was concentrated, and the residue was diluted with 30 ml of water. The aqueous phase was added with 2M sodium hydroxide aqueous solution to adjust the pH to 7-8, and then extracted with EA. The organic phase was washed with saturated brine, dried with Na 2 SO 4 , filtered and concentrated in vacuo. The crude product was purified by column chromatography (PE/EA=95/5) to obtain 2 g of yellow liquid product 3-bromo-5-(tert-butyl)aniline (I-2). LC-MS: ESI m/z: 227.7 [M+H] + .
步骤3：参见中间体H-1的制备方法，过滤并真空浓缩反应液后得到黄色固体产品(3-溴-5-(叔丁基)苯基)盐酸肼(I-3)1g。LC-MS:ESI m/z:243.1[M+H]+。Step 3: Referring to the preparation method of intermediate H-1, the reaction solution was filtered and concentrated in vacuo to obtain 1 g of a yellow solid product (3-bromo-5-(tert-butyl)phenyl)hydrazine hydrochloride (I-3). LC-MS: ESI m/z: 243.1 [M+H] + .
步骤4：参见中间体G-1的制备方法，粗品经柱层析(PE/EA＝95/5)后得到黄色油状产品1-(3-溴-5-(叔丁基)苯基)-3,5-二甲基-1H-吡唑(I-4)600mg。LC-MS:ESI m/z:306.9[M+H] +。Step 4: Referring to the preparation method of intermediate G-1, the crude product was purified by column chromatography (PE/EA=95/5) to obtain 600 mg of a yellow oily product 1-(3-bromo-5-(tert-butyl)phenyl)-3,5-dimethyl-1H-pyrazole (I-4). LC-MS: ESI m/z: 306.9 [M+H] + .
步骤5：参见中间体H-3的制备方法，得到黑棕色油状产品1-(3-(叔丁基)-5-(4,,5,5-四甲基-1,3,2-二氧硼杂环戊烷-2-基)苯基)-3,5-二甲基-1H-吡唑(I-5)500mg。LC-MS:ESI m/z:355.4[M+H]+。Step 5: Referring to the preparation method of intermediate H-3, 500 mg of dark brown oily product 1-(3-(tert-butyl)-5-(4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-3,5-dimethyl-1H-pyrazole (I-5) was obtained. LC-MS: ESI m/z: 355.4 [M+H] + .
步骤6：参见中间体H的制备方法，粗品经高效液相色谱分离后得到白色固体品(3-(叔丁基)-5-(3,5-二甲基-1H-吡唑-1-基)苯基)硼酸(I)55mg。LC-MS:ESI m/z:273.2[M+H]+。Step 6: Referring to the preparation method of intermediate H, the crude product was separated by HPLC to obtain 55 mg of white solid (3-(tert-butyl)-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)boronic acid (I). LC-MS: ESI m/z: 273.2 [M+H] + .
中间体J：叔-丁基7-(2-(甲苯磺酰氧代)乙基)-3,4-二氢-1,8-二氮杂萘-1(2H)-羧酸酯的制备
Intermediate J: Preparation of tert-butyl 7-(2-(toluenesulfonyloxy)ethyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylate
步骤1：将2-氨基尼古丁醛(10g，81.9mmol)溶于乙醇(100ml)中，加入丙酮(14.3g，245.7mmol)和L-脯氨酸(10.4g，90.1mmol)，氮气保护80℃搅拌16小时。浓缩反应液，粗品经正相硅胶柱色谱法纯化(PE/EA＝0/1)得到白色固体产品2-甲基-1,8-二氮杂萘(J-1)11g。1H NMR(400MHz,CDCl3)δ9.08(d,J＝2.57Hz,1H)8.15(d,J＝8.07Hz,1H)8.08(d,J＝8.19Hz,1H)7.44(dd,J＝8.07,4.28Hz,1H)7.39(d,J＝8.31Hz,1H)2.82(s,3H)Step 1: Dissolve 2-aminonicotinaldehyde (10 g, 81.9 mmol) in ethanol (100 ml), add acetone (14.3 g, 245.7 mmol) and L-proline (10.4 g, 90.1 mmol), and stir at 80°C for 16 hours under nitrogen protection. Concentrate the reaction solution, and purify the crude product by normal phase silica gel column chromatography (PE/EA=0/1) to obtain 11 g of white solid product 2-methyl-1,8-naphthyridine (J-1). 1 H NMR (400 MHz, CDCl3) δ 9.08 (d, J = 2.57 Hz, 1H) 8.15 (d, J = 8.07 Hz, 1H) 8.08 (d, J = 8.19 Hz, 1H) 7.44 (dd, J = 8.07, 4.28 Hz, 1H) 7.39 (d, J = 8.31 Hz, 1H) 2.82 (s, 3H)
步骤2：将J-1(5g，34.7mmol)溶于乙醇(50ml)和中，加入10％钯碳(1.5g)，氢气氛围室温搅拌16小时。过滤并真空浓缩反应液得到白色固体产品7-甲基-1,2,3,4-四氢-1,8-二氮杂萘(J-2)5g。1H NMR(400MHz,CDCl3)δ7.04(d,J＝7.21Hz,1H)6.35(d,J＝7.34Hz,1H)4.78(m,1H)3.36-3.42(m,2H)2.68(m,2H)2.31(s,3H)1.90(m,2H)Step 2: Dissolve J-1 (5 g, 34.7 mmol) in ethanol (50 ml) and add 10% palladium carbon (1.5 g), stir at room temperature under hydrogen atmosphere for 16 hours. Filter and concentrate the reaction solution in vacuo to obtain 5 g of 7-methyl-1,2,3,4-tetrahydro-1,8-naphthyridine (J-2) as a white solid product. 1 H NMR (400 MHz, CDCl3) δ7.04 (d, J = 7.21 Hz, 1H) 6.35 (d, J = 7.34 Hz, 1H) 4.78 (m, 1H) 3.36-3.42 (m, 2H) 2.68 (m, 2H) 2.31 (s, 3H) 1.90 (m, 2H)
步骤3：参见中间体A-4的制备方法，粗品经正相硅胶柱色谱法纯化(PE/EA＝5/1)得到白色固体产品叔-丁基7-甲基-3,4-二氢-1,8-二氮杂萘-1(2H)-羧酸酯(J-3)4g。LC-MS:ESI m/z＝249.4[M+H]+.Step 3: Referring to the preparation method of intermediate A-4, the crude product was purified by normal phase silica gel column chromatography (PE/EA=5/1) to obtain 4 g of white solid product tert-butyl 7-methyl-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylate (J-3). LC-MS: ESI m/z=249.4[M+H] + .
步骤4：将J-3(3.5g，14.1mmol)和碳酸二甲酯(4.4g，49.3mmol)溶于THF(30ml)中，氮气保护-78℃滴加2M的二异丙基胺锂溶液(10.7ml)，并搅拌1小时。反应液用饱和氯化铵水溶液(50ml)淬灭，EA(50ml*3)萃取，有机相经干燥，过滤浓缩，粗品经正相硅胶柱色谱法纯化(PE/EA＝4/1)得到淡黄色固体产品叔-丁基7-(2-甲氧基-2-羰基乙基)-3,4-二氢-1,8-二氮杂萘-1(2H)-羧酸酯(J-4)2.9g。1H NMR(400MHz,CDCl3)δ7.29(d,J＝7.70Hz,1H)6.89(d,J＝7.58Hz,1H)3.71(s,2H)3.66-3.70(m,2H)3.63(s,3H)2.67(t,J＝6.60Hz,2H)1.85(m,2H)1.44(s,9H)Step 4: J-3 (3.5 g, 14.1 mmol) and dimethyl carbonate (4.4 g, 49.3 mmol) were dissolved in THF (30 ml), and 2 M lithium diisopropylamine solution (10.7 ml) was added dropwise at -78 °C under nitrogen protection, and stirred for 1 hour. The reaction solution was quenched with saturated aqueous ammonium chloride solution (50 ml), extracted with EA (50 ml*3), the organic phase was dried, filtered and concentrated, and the crude product was purified by normal phase silica gel column chromatography (PE/EA=4/1) to obtain 2.9 g of light yellow solid product tert-butyl 7-(2-methoxy-2-carbonylethyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylate (J-4). 1 H NMR (400 MHz, CDCl3) δ7.29 (d, J = 7.70 Hz, 1H) 6.89 (d, J = 7.58 Hz, 1H) 3.71 (s, 2H) 3.66-3.70 (m, 2H) 3.63 (s, 3H) 2.67 (t, J = 6.60 Hz, 2H) 1.85 (m, 2H) 1.44 (s, 9H)
步骤5：参见中间体A-4的制备方法，粗品经正相硅胶柱色谱法纯化(PE/EA＝4/1)得到淡黄色固体产品叔-丁基7-(2-羟基乙基)-3,4-二氢-1,8-二氮杂萘-1(2H)-羧酸酯(J-5)800mg。1H NMR(400MHz,CDCl3)δ7.31(d,J＝7.58Hz,1H)6.77(d,J＝7.70Hz,1H)5.58(br s,1H)3.98(br s,2H)3.76-3.82(m,2H)2.92(t,J＝5.14Hz,2H)2.72(t,J＝6.48Hz,2H)1.92(m,2H)1.54(s,9H)。Step 5: Referring to the preparation method of intermediate A-4, the crude product was purified by normal phase silica gel column chromatography (PE/EA=4/1) to obtain 800 mg of a light yellow solid product, tert-butyl 7-(2-hydroxyethyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylate (J-5). 1 H NMR (400 MHz, CDCl3) δ7.31 (d, J=7.58 Hz, 1H) 6.77 (d, J=7.70 Hz, 1H) 5.58 (br s, 1H) 3.98 (br s, 2H) 3.76-3.82 (m, 2H) 2.92 (t, J=5.14 Hz, 2H) 2.72 (t, J=6.48 Hz, 2H) 1.92 (m, 2H) 1.54 (s, 9H).
步骤6：参见中间体C的制备方法，粗品经正相硅胶柱色谱法纯化(PE/EA＝4/1)得到白 色固体产品260mg。1H NMR(400MHz,CDCl3)δ7.74(d,J＝8.19Hz,2H)7.27-7.33(m,3H)6.80(d,J＝7.58Hz,1H)4.42(t,J＝7.03Hz,2H)3.70-3.76(m,2H)3.05(t,J＝6.97Hz,2H)2.72(t,J＝6.60Hz,2H)2.44(s,3H)1.91(m,2H)1.45(s,9H)。Step 6: Referring to the preparation method of intermediate C, the crude product was purified by normal phase silica gel column chromatography (PE/EA=4/1) to obtain white 260 mg of a white solid product. 1 H NMR (400 MHz, CDCl3) δ7.74 (d, J = 8.19 Hz, 2H) 7.27-7.33 (m, 3H) 6.80 (d, J = 7.58 Hz, 1H) 4.42 (t, J = 7.03 Hz, 2H) 3.70-3.76 (m, 2H) 3.05 (t, J = 6.97 Hz, 2H) 2.72 (t, J = 6.60 Hz, 2H) 2.44 (s, 3H) 1.91 (m, 2H) 1.45 (s, 9H).
中间体K：(3-(1-(叔-丁氧基羰基)-1H-吡咯-2-基)苯基)硼酸的制备
Intermediate K: Preparation of (3-(1-(tert-butoxycarbonyl)-1H-pyrrol-2-yl)phenyl)boronic acid
步骤1：将1,3-二溴苯(3g,12.7mmol)，硼酸酯(4.1g，14mmol)，钯催化剂(4.7g)和碳酸钾(5.3g，38.2mmol)溶于Diox/H2O(3/1，40ml)中，氮气保护85℃搅拌过夜。浓缩反应液，粗品经正相硅胶柱色谱法纯化(PE/EA＝20/1)得到无色油状产品叔-丁基2-(3-溴苯基)-1H-吡咯-1-羧酸酯(K-1)4g。1H NMR(400MHz,CDCl3)δ＝7.49(s,1H),7.43(br d,J＝7.9Hz,1H),7.39-7.34(m,1H),7.28(br d,J＝7.5Hz,1H),7.25-7.18(m,1H),6.27-6.15(m,2H),1.37(s,9H)。Step 1: Dissolve 1,3-dibromobenzene (3 g, 12.7 mmol), borate (4.1 g, 14 mmol), palladium catalyst (4.7 g) and potassium carbonate (5.3 g, 38.2 mmol) in Diox/H 2 O (3/1, 40 ml), and stir overnight at 85°C under nitrogen protection. Concentrate the reaction solution, and purify the crude product by normal phase silica gel column chromatography (PE/EA=20/1) to obtain 4 g of colorless oily product tert-butyl 2-(3-bromophenyl)-1H-pyrrole-1-carboxylate (K-1). 1 H NMR (400 MHz, CDCl 3 ) δ＝7.49 (s, 1H), 7.43 (br d, J＝7.9 Hz, 1H), 7.39-7.34 (m, 1H), 7.28 (br d, J＝7.5 Hz, 1H), 7.25-7.18 (m, 1H), 6.27-6.15 (m, 2H), 1.37 (s, 9H).
步骤2：参见中间体G的制备方法啊，粗品经正相硅胶柱色谱法纯化(DCM/MeOH＝20/1)得到紫色固体产品1.8g。LC-MS:ESI m/z＝188.0[M-Boc+1]+。Step 2: Referring to the preparation method of intermediate G, the crude product was purified by normal phase silica gel column chromatography (DCM/MeOH=20/1) to obtain 1.8 g of a purple solid product. LC-MS: ESI m/z=188.0 [M-Boc+1] + .
中间体L：(3-(5-甲基-1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-咪唑-2-基)苯基)硼酸的制备
Intermediate L: Preparation of (3-(5-methyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)phenyl)boronic acid
步骤1：将3-溴苯(甲)醛(1g，5.4mmol)，2-羰基丙醛(14.3g，79.3mmol)和氨水(5.5g，43.6mmol)溶于甲醇(15ml)中，75℃下搅拌3小时。浓缩反应液，粗品经正相硅胶柱色谱法纯化(DCM/MeOH＝10/1)得到黄色固体产品2-(3-溴苯基)-5-甲基-1H-咪唑(L-1)1.5g。LC-MS:ESI m/z＝239.1[M+H]+。Step 1: Dissolve 3-bromobenzaldehyde (1 g, 5.4 mmol), 2-carbonylpropanal (14.3 g, 79.3 mmol) and aqueous ammonia (5.5 g, 43.6 mmol) in methanol (15 ml) and stir at 75°C for 3 hours. Concentrate the reaction solution and purify the crude product by normal phase silica gel column chromatography (DCM/MeOH=10/1) to obtain 1.5 g of yellow solid product 2-(3-bromophenyl)-5-methyl-1H-imidazole (L-1). LC-MS: ESI m/z=239.1[M+H] + .
步骤2：将L-1(1.5g，6.3mmol)和氢化钠(506.1mg，12.7mmol)溶于THF(20ml)中，25℃搅拌0.5小时，0℃下向溶液中缓慢滴加2-(三甲硅烷基)乙氧甲基氯(1.6g，9.5mmol)，0℃下继续搅拌2.5小时。0℃下加15ml饱和NH4Cl溶液淬灭反应，加20ml水稀释，用乙酸乙酯萃取，有机相用饱和盐水洗，Na2SO4干燥，过滤并真空浓缩，粗品经正相硅胶柱色谱法纯化(PE/EA＝1/1)得到黄色油状产品2-(3-溴苯基)-5-甲基-1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-咪唑(L-2)1.3g。LC-MS:ESI m/z＝368.3[M+H]+。Step 2: L-1 (1.5 g, 6.3 mmol) and sodium hydride (506.1 mg, 12.7 mmol) were dissolved in THF (20 ml), stirred at 25°C for 0.5 hours, 2-(trimethylsilyl)ethoxymethyl chloride (1.6 g, 9.5 mmol) was slowly added dropwise to the solution at 0°C, and stirring was continued at 0°C for 2.5 hours. 15 ml of saturated NH 4 Cl solution was added at 0°C to quench the reaction, 20 ml of water was added to dilute, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over Na 2 SO 4 , filtered and concentrated in vacuo. The crude product was purified by normal phase silica gel column chromatography (PE/EA=1/1) to obtain 1.3 g of a yellow oily product 2-(3-bromophenyl)-5-methyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazole (L-2). LC-MS: ESI m/z=368.3[M+H] + .
步骤3：将L-2(1.2g，3.3mmol)，四羟基二硼烷(585.7mg，6.5mmol)，乙酸钾(961.8mg，9.8mmol)，2-二环己基膦-2’4’6-三异丙基联苯(311.5mg，0.7mmol)和甲烷磺酸(2-二环己基膦-2’4’6-三异丙基-1,1’-联苯基)(2’-氨基-1,1’-联苯-2-基)钯(Ⅱ)(276.5mg，0.3mmol)溶于甲醇(20ml)中，氮气保护下80℃搅拌3小时。浓缩反应液，粗品经高效液相色谱纯化得到黄色固体产品670mg。LC-MS:ESI m/z＝334.6[M+H]+。 Step 3: Dissolve L-2 (1.2 g, 3.3 mmol), tetrahydroxydiborane (585.7 mg, 6.5 mmol), potassium acetate (961.8 mg, 9.8 mmol), 2-dicyclohexylphosphino-2'4'6-triisopropylbiphenyl (311.5 mg, 0.7 mmol) and methanesulfonic acid (2-dicyclohexylphosphino-2'4'6-triisopropyl-1,1'-biphenyl) (2'-amino-1,1'-biphenyl-2-yl) palladium (II) (276.5 mg, 0.3 mmol) in methanol (20 ml) and stir at 80°C for 3 hours under nitrogen protection. Concentrate the reaction solution and purify the crude product by HPLC to obtain 670 mg of a yellow solid product. LC-MS: ESI m/z = 334.6 [M+H] + .
中间体M：(3-(1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-咪唑-2-基)苯基)硼酸的制备
Intermediate M: Preparation of (3-(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)phenyl)boronic acid
参见中间体L的制备方法，粗品经高效液相色谱纯化得到白色固体产品(M)1.5g。LC-MS:ESI m/z＝321.0[M+H]+。Referring to the preparation method of intermediate L, the crude product was purified by high performance liquid chromatography to obtain 1.5 g of a white solid product (M). LC-MS: ESI m/z = 321.0 [M+H] + .
中间体N：(5-(叔-丁基)-2-氟苯基)硼酸的制备
Preparation of Intermediate N: (5-(tert-butyl)-2-fluorophenyl)boronic acid
步骤1：参见中间体G的制备方法，粗品经正相硅胶柱色谱法纯化(PE/EA＝100/0～3/1)得到淡黄色固体产品760mg。1H NMR(400MHz,DMSO-d6)δ＝8.17(s,2H),7.57(dd,J＝2.5,5.7Hz,1H),7.43(ddd,J＝2.7,5.4,8.4Hz,1H),7.00(t,J＝8.8Hz,1H),1.29(s,9H)。Step 1: Referring to the preparation method of intermediate G, the crude product was purified by normal phase silica gel column chromatography (PE/EA=100/0-3/1) to obtain 760 mg of a light yellow solid product. 1 H NMR (400 MHz, DMSO-d 6 ) δ=8.17 (s, 2H), 7.57 (dd, J=2.5, 5.7 Hz, 1H), 7.43 (ddd, J=2.7, 5.4, 8.4 Hz, 1H), 7.00 (t, J=8.8 Hz, 1H), 1.29 (s, 9H).
中间体P1(3-环丙基-5-(3,5-二甲基-1H-吡唑-1-基)苯基)硼酸、P2(3-环丁基-5-(3,5-二甲基-1H-吡唑-1-基)苯基)硼酸、P3(3-环戊基-5-(3,5-二甲基-1H-吡唑-1-基)苯基)硼酸、P4(3-环己基-5-(3,5-二甲基-1H-吡唑-1-基)苯基)硼酸的一般制备方法：
General preparation method of intermediates P1 (3-cyclopropyl-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)boric acid, P2 (3-cyclobutyl-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)boric acid, P3 (3-cyclopentyl-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)boric acid, P4 (3-cyclohexyl-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)boric acid:
步骤1：将H-2(1.2g,3.5mmol)、不同的环烷基硼酸(1当量)、Pd(dppf)Cl2(0.1当量)、碳酸钾(2当量)、二氧六环/水溶液(10:1，7ml)，氮气保护，93℃加热反应3h；LCMS检测反应完毕，加水淬灭后加入乙酸乙酯，抽滤除去黄色固体，乙酸乙酯萃取，合并有机相，Na2SO4干燥，过滤，50℃减压蒸馏得黄色油状物，粗品经柱层析纯化(PE/EA≈20/1)后得到相应的黄色油状产品。Step 1: H-2 (1.2 g, 3.5 mmol), different cycloalkylboronic acids (1 equivalent), Pd(dppf)Cl 2 (0.1 equivalent), potassium carbonate (2 equivalents), dioxane/water solution (10:1, 7 ml), under nitrogen protection, heated at 93°C for 3 h; LCMS detected that the reaction was complete, quenched with water, and then ethyl acetate was added, and the yellow solid was removed by suction, extracted with ethyl acetate, and the organic phases were combined, dried over Na2SO4, filtered, and distilled under reduced pressure at 50°C to obtain a yellow oil. The crude product was purified by column chromatography (PE/EA≈20/1) to obtain the corresponding yellow oily product.
步骤2：参见中间体G的制备方法，粗品经反相柱层析纯化(水/乙腈＝7/3)后得到相应的白色固体产品。Step 2: Referring to the preparation method of intermediate G, the crude product was purified by reverse phase column chromatography (water/acetonitrile = 7/3) to obtain the corresponding white solid product.
中间体Q：(5-(叔-丁基)-[1,1'-联苯基]-3-基)硼酸的制备
Intermediate Q: Preparation of (5-(tert-butyl)-[1,1'-biphenyl]-3-yl)boronic acid
参见中间体P1等的制备方法，粗品经硅胶柱层析纯化(PE/EA＝1/1)得到黄色固体产品450mg。LC-MS:ESI m/z:255.1[M+H]+。Referring to the preparation method of intermediate P1, etc., the crude product was purified by silica gel column chromatography (PE/EA=1/1) to obtain 450 mg of a yellow solid product. LC-MS: ESI m/z: 255.1 [M+H] + .
中间体R：(3,5-二-叔-丁基苯基)硼酸的制备
Preparation of Intermediate R: (3,5-di-tert-butylphenyl)boronic acid
参见中间体P1步骤2的制备方法。See the preparation method of intermediate P1 step 2.
中间体1-INTB：甲基(S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(2-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-6-基)丁酸酯的制备
Intermediate 1-INTB: Preparation of methyl (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(2-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-6-yl)butanoate
步骤1：将叔-丁基2,6-二氮杂螺[3.4]辛烷-2-羧酸酯(0.5g，2.4mmol)溶于N，N-二甲基甲酰胺(5ml)中，室温下加入二异丙基乙胺(1.ml，5.9mmol)和甲基(E)-4-溴丁-2-烯酸酯(506mg，2.8mmol)搅拌2小时。反应液加水稀释，乙酸乙酯萃取(30ml*2)，有机相用饱和食盐水洗，Na2SO4干燥，过滤并真空浓缩，粗品经正相硅胶柱色谱法纯化(DCM/MeOH＝10/1)得到白色固体产品叔-丁基(E)-6-(4-甲氧基-4-羰基丁-2-烯-1-基)-2,6-二氮杂螺[3.4]辛烷-2-羧酸酯580mg。LC-MS:ESI m/z:311.3[M+H]+；1H NMR(400MHz,CDCl3)δ＝6.96(td,J＝6.0,15.6Hz,1H),5.99(d,J＝15.6Hz,1H),3.93-3.79(m,4H),3.75(s,3H),3.23(dd,J＝1.6,6.0Hz,2H),2.73(s,2H),2.61(t,J＝7.0Hz,2H),2.05(t,J＝7.0Hz,2H),1.49(s,9H)。Step 1: Dissolve tert-butyl 2,6-diazaspiro[3.4]octane-2-carboxylate (0.5 g, 2.4 mmol) in N,N-dimethylformamide (5 ml), add diisopropylethylamine (1. ml, 5.9 mmol) and methyl (E)-4-bromobut-2-enoate (506 mg, 2.8 mmol) at room temperature and stir for 2 hours. Dilute the reaction solution with water, extract with ethyl acetate (30 ml*2), wash the organic phase with saturated brine, dry over Na 2 SO 4 , filter and concentrate in vacuo, and purify the crude product by normal phase silica gel column chromatography (DCM/MeOH=10/1) to obtain 580 mg of white solid product tert-butyl (E)-6-(4-methoxy-4-carbonylbut-2-en-1-yl)-2,6-diazaspiro[3.4]octane-2-carboxylate. LC-MS: ESI m/z: 311.3 [M+H] + ; 1 H NMR (400 MHz, CDCl3) δ = 6.96 (td, J = 6.0, 15.6 Hz, 1H), 5.99 (d, J = 15.6 Hz, 1H), 3.93-3.79 (m, 4H), 3.75 (s, 3H), 3.23 (dd, J = 1.6, 6.0 Hz, 2H), 2.73 (s, 2H), 2.61 (t, J = 7.0 Hz, 2H), 2.05 (t, J = 7.0 Hz, 2H), 1.49 (s, 9H).
步骤2：将叔-丁基(E)-6-(4-甲氧基-4-羰基丁-2-烯-1-基)-2,6-二氮杂螺[3.4]辛烷-2-羧酸酯(0.6g，1.8mmol)溶于DCM(5ml)中，加入三氟乙酸(2ml)，室温下搅拌2小时。反应液直接旋干得到黄色油状产品甲基(E)-4-(2,6-二氮杂螺[3.4]辛烷-6-基)丁-2-烯酸酯550mg。LC-MS:ESI m/z:211.3[M+H]+。Step 2: Dissolve tert-butyl (E)-6-(4-methoxy-4-carbonylbut-2-ene-1-yl)-2,6-diazaspiro[3.4]octane-2-carboxylate (0.6 g, 1.8 mmol) in DCM (5 ml), add trifluoroacetic acid (2 ml), and stir at room temperature for 2 hours. The reaction solution is directly spin-dried to obtain 550 mg of methyl (E)-4-(2,6-diazaspiro[3.4]octane-6-yl)but-2-enoate as a yellow oily product. LC-MS: ESI m/z: 211.3 [M+H] + .
步骤3：依次将甲基(E)-4-(2,6-二氮杂螺[3.4]辛烷-6-基)丁-2-烯酸酯(0.6g，1.7mmol)，中间体A-3(330mg，2mmol)和二异丙基乙胺(657mg，5.1mmol)溶于1，2-二氯乙烷(5ml) 中，室温下搅拌30分钟。然后加入氰基硼氢化钠(160mg，2.5mmol)并继续反应1.5小时。反应液用水洗(30ml)，乙酸乙酯萃取(30ml*2)。有机相用饱和食盐水洗，Na2SO4干燥，过滤并真空浓缩，粗产品经正相硅胶柱色谱法纯化(DCM/MeOH＝10/1)得到黄色油状产品甲基(E)-4-(2-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-6-基)丁-2-烯酸酯(1-INTA)300mg。LC-MS:ESI m/z:357.4[M+H]+。Step 3: Dissolve methyl (E)-4-(2,6-diazaspiro[3.4]octan-6-yl)but-2-enoate (0.6 g, 1.7 mmol), intermediate A-3 (330 mg, 2 mmol) and diisopropylethylamine (657 mg, 5.1 mmol) in 1,2-dichloroethane (5 ml) in sequence. and stirred at room temperature for 30 minutes. Then sodium cyanoborohydride (160 mg, 2.5 mmol) was added and the reaction was continued for 1.5 hours. The reaction solution was washed with water (30 ml) and extracted with ethyl acetate (30 ml*2). The organic phase was washed with saturated brine, dried with Na2SO4, filtered and concentrated in vacuo. The crude product was purified by normal phase silica gel column chromatography (DCM/MeOH=10/1) to obtain 300 mg of yellow oily product methyl (E)-4-(2-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-6-yl)but-2-enoate (1-INTA). LC-MS:ESI m/z:357.4[M+H] + .
步骤4：依次将甲基(E)-4-(2-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-6-基)丁-2-烯酸酯(90mg，0.3mmol)，中间体G(191mg，0.9mmol)，(1,5-环辛二烯)氯铑(I)二聚体([Rh(COD)Cl]2，6.2mg，0.01mmol)，(R)-(+)-2,2′-双(二苯基膦)-1,1′-联萘((R)-(+)-BINAP，15.7mg，0.03mmol)和碳酸铯(247mg，0.8mmol)溶于二氧六环(2ml)和水(0.2ml)中，氮气保护下加热到95℃并搅拌2小时。反应液直接旋干，高效液相色谱分离得到棕色油状产品50mg。LC-MS:ESI m/z:529.4[M+H]+；1H NMR(400MHz,METHANOL-d4)δ＝7.78-7.20(m,5H),6.77(br s,1H),6.09(s,1H),4.57-4.06(m,6H),3.90-3.33(m,12H),2.93-2.64(m,4H),2.48-2.43(m,2H),2.27(s,3H)2.24(s,3H),1.95-1.90(m,2H)。Step 4: Methyl (E)-4-(2-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-6-yl)but-2-enoate (90 mg, 0.3 mmol), intermediate G (191 mg, 0.9 mmol), (1,5-cyclooctadiene)chlororhodium(I) dimer ([Rh(COD)Cl] 2 , 6.2 mg, 0.01 mmol), (R)-(+)-2,2′-bis(diphenylphosphino)-1,1′-binaphthyl ((R)-(+)-BINAP, 15.7 mg, 0.03 mmol) and cesium carbonate (247 mg, 0.8 mmol) were dissolved in dioxane (2 ml) and water (0.2 ml) in sequence, heated to 95° C. under nitrogen protection and stirred for 2 hours. The reaction solution was directly spin-dried and separated by high performance liquid chromatography to obtain 50 mg of a brown oily product. LC-MS: ESI m/z: 529.4 [M+H] + ; 1 H NMR (400 MHz, METHANOL-d 4 ) δ = 7.78-7.20 (m, 5H), 6.77 (br s, 1H), 6.09 (s, 1H), 4.57-4.06 (m, 6H), 3.90-3.33 (m, 12H), 2.93-2.64 (m, 4H), 2.48-2.43 (m, 2H), 2.27 (s, 3H) 2.24 (s, 3H), 1.95-1.90 (m, 2H).
中间体2-INTB：叔-丁基(S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯的制备
Intermediate 2-INTB: Preparation of tert-butyl (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-2-yl)butyrate
步骤1：在氮气保护,0℃下，将叔-丁基2,6-二氮杂螺[3.4]辛烷-2-羧酸酯(500mg,2.36mmol,1.0eq)和TEA(595.7mg,5.9mmol,2.5eq)溶于DCM(5mL)中，滴加CbzCl(803.5mg,4.7mmol,2.0eq)，反应16h。浓缩反应液，粗品过柱。得到透明油状产品6-苯甲基2-(叔-丁基)2,6-二氮杂螺[3.4]辛烷-2,6-二羧酸酯302mg。LC-MS:ESI m/z:369.15[M+Na]+。Step 1: Under nitrogen protection, at 0°C, tert-butyl 2,6-diazaspiro[3.4]octane-2-carboxylate (500 mg, 2.36 mmol, 1.0 eq) and TEA (595.7 mg, 5.9 mmol, 2.5 eq) were dissolved in DCM (5 mL), and CbzCl (803.5 mg, 4.7 mmol, 2.0 eq) was added dropwise. The reaction mixture was reacted for 16 h. The reaction solution was concentrated and the crude product was passed through a column. 302 mg of a transparent oily product, 6-benzyl 2-(tert-butyl) 2,6-diazaspiro[3.4]octane-2,6-dicarboxylate, was obtained. LC-MS: ESI m/z: 369.15 [M+Na] + .
步骤2：在氮气保护,0℃下，将6-苯甲基2-(叔-丁基)2,6-二氮杂螺[3.4]辛烷-2,6-二羧酸酯(295mg,0.9mmol,1.0eq)溶于DCM(6mL)中，滴加TFA(3mL)，反应2h。反应液用氨水中和，浓缩反应液，得到黄色油状粗品苯甲基2,6-二氮杂螺[3.4]辛烷-6-羧酸酯209mg。LC-MS:ESI m/z:247.1[M+H]+。Step 2: Under nitrogen protection, at 0°C, 6-benzyl 2-(tert-butyl) 2,6-diazaspiro[3.4]octane-2,6-dicarboxylate (295 mg, 0.9 mmol, 1.0 eq) was dissolved in DCM (6 mL), TFA (3 mL) was added dropwise, and the reaction was allowed to react for 2 h. The reaction solution was neutralized with ammonia water, and the reaction solution was concentrated to obtain 209 mg of crude yellow oily benzyl 2,6-diazaspiro[3.4]octane-6-carboxylate. LC-MS: ESI m/z: 247.1[M+H] + .
步骤3：在氮气保护,室温条件下，将苯甲基2,6-二氮杂螺[3.4]辛烷-6-羧酸酯(209.7mg,0.85mmol,1.0eq)和碳酸钾(352.8mg,2.6mmol,3.0eq)溶于MeCN/DMF(2mL/2mL)中，65℃滴加叔-丁基(E)-4-溴丁-2-烯酸酯(178.7mg,0.8mmol,1eq)，反应2h。浓缩反应液，粗品溶于DCM，过滤，滤液浓缩。Prep-TLC(DCM/MeOH＝15/1)得浅黄色油状产品苯甲基(E)-2-(4-(叔-丁氧基)-4-羰基丁-2-烯-1-基)-2,6-二氮杂螺[3.4]辛烷-6-羧酸酯95mg。LC-MS:ESI m/z:387.52[M+H]+。Step 3: Under nitrogen protection and room temperature, benzyl 2,6-diazaspiro[3.4]octane-6-carboxylate (209.7 mg, 0.85 mmol, 1.0 eq) and potassium carbonate (352.8 mg, 2.6 mmol, 3.0 eq) were dissolved in MeCN/DMF (2 mL/2 mL), and tert-butyl (E)-4-bromobut-2-enoate (178.7 mg, 0.8 mmol, 1 eq) was added dropwise at 65°C for 2 h. The reaction solution was concentrated, the crude product was dissolved in DCM, filtered, and the filtrate was concentrated. Prep-TLC (DCM/MeOH=15/1) gave 95 mg of a light yellow oily product, benzyl (E)-2-(4-(tert-butyloxy)-4-carbonylbut-2-en-1-yl)-2,6-diazaspiro[3.4]octane-6-carboxylate. LC-MS: ESI m/z: 387.52 [M+H] + .
其中，叔-丁基(E)-4-溴丁-2-烯酸酯的制备是通过在氮气保护下，将叔-丁基(E)-丁-2-烯酸酯(500mg,3.5mmol,1.0eq)溶于CCl4(6mL)中，加入NBS(501mg,2.8mmol,0.8eq) 和BPO(26mg,0.11mmol,0.03eq)，85℃反应16h。TLC显示反应完毕，过滤，滤液浓缩。Prep-TLC(PE/EA＝10/1)得无色油状产品210mg。1H NMR(400MHz,CDCl3)δ6.94-6.86(m,1H),5.95(d,J＝15.6Hz,1H),3.99(dd,J＝7.6,1.2Hz,2H),1.48(s,9H).Among them, tert-butyl (E)-4-bromobut-2-enoate was prepared by dissolving tert-butyl (E)-but-2-enoate (500 mg, 3.5 mmol, 1.0 eq) in CCl4 (6 mL) under nitrogen protection, adding NBS (501 mg, 2.8 mmol, 0.8 eq) and BPO (26 mg, 0.11 mmol, 0.03 eq), react at 85°C for 16 h. TLC showed that the reaction was complete, filtered, and the filtrate was concentrated. Prep-TLC (PE/EA = 10/1) gave 210 mg of a colorless oily product. 1 H NMR (400 MHz, CDCl 3 ) δ 6.94-6.86 (m, 1H), 5.95 (d, J = 15.6 Hz, 1H), 3.99 (dd, J = 7.6, 1.2 Hz, 2H), 1.48 (s, 9H).
步骤4：在氮气保护,室温条件下，将苯甲基(E)-2-(4-(叔-丁氧基)-4-羰基丁-2-烯-1-基)-2,6-二氮杂螺[3.4]辛烷-6-羧酸酯(45mg,0.1mmol,1.0eq)，中间体G(75.4mg,0.3mmol,3.0eq)，(1,5-环辛二烯)氯铑(I)二聚体([Rh(COD)Cl]2，5.7mg，0.01mmol，0.1eq)，(R)-(+)-2,2′-双(二苯基膦)-1,1′-联萘((R)-(+)-BINAP，14.4mg，0.023mmol，0.2eq)()和氢氧化钾(19.6mg,0.3mmol,3.0eq)的水溶液溶于二氧六环(2mL/2mL)中，反应16h。过滤真空浓缩，粗品Prep-TLC(DCM/MeOH＝17/1)制备得无色油状产品苯甲基(S)-2-(4-(叔-丁氧基)-2-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-羰基丁基)-2,6-二氮杂螺[3.4]辛烷-6-羧酸酯25mg。LC-MS:ESI m/z:559.3[M+H]+。Step 4: Under nitrogen protection and room temperature, benzyl (E)-2-(4-(tert-butyloxy)-4-carbonylbut-2-en-1-yl)-2,6-diazaspiro[3.4]octane-6-carboxylate (45 mg, 0.1 mmol, 1.0 eq), intermediate G (75.4 mg, 0.3 mmol, 3.0 eq), (1,5-cyclooctadiene)chlororhodium (I) dimer ([Rh(COD)Cl] 2 , 5.7 mg, 0.01 mmol, 0.1 eq), (R)-(+)-2,2′-bis(diphenylphosphino)-1,1′-binaphthyl ((R)-(+)-BINAP, 14.4 mg, 0.023 mmol, 0.2 eq) () and potassium hydroxide (19.6 mg, 0.3 mmol, 3.0 eq) in aqueous solution were dissolved in dioxane (2 mL/2 mL) and reacted for 16 h. Filtered and concentrated in vacuo, the crude product Prep-TLC (DCM/MeOH=17/1) prepared 25 mg of colorless oily product benzyl (S)-2-(4-(tert-butyloxy)-2-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-carbonylbutyl)-2,6-diazaspiro[3.4]octane-6-carboxylate. LC-MS: ESI m/z: 559.3 [M+H] + .
步骤5：在氮气保护,室温条件下，将苯甲基(S)-2-(4-(叔-丁氧基)-2-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-羰基丁基)-2,6-二氮杂螺[3.4]辛烷-6-羧酸酯(150mg,0.27mmol,1.0eq)溶于MeOH(2mL)中，加入Pd/C(5mg)和1滴醋酸，室温反应16h。垫硅藻土助滤，浓缩反应液，粗品经Prep-TLC(DCM/MeOH＝10/1)提纯得到浅黄色油状产品叔-丁基(S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯(2-INTA)90mg。LC-MS:ESI m/z:425.3[M+H]+。Step 5: Under nitrogen protection and room temperature, benzyl (S)-2-(4-(tert-butyloxy)-2-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-carbonylbutyl)-2,6-diazaspiro[3.4]octane-6-carboxylate (150 mg, 0.27 mmol, 1.0 eq) was dissolved in MeOH (2 mL), Pd/C (5 mg) and 1 drop of acetic acid were added, and the mixture was reacted at room temperature for 16 h. Celite was used for filtration, and the reaction solution was concentrated. The crude product was purified by Prep-TLC (DCM/MeOH=10/1) to obtain 90 mg of tert-butyl (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(2,6-diazaspiro[3.4]octan-2-yl)butanoate (2-INTA) as a light yellow oily product. LC-MS: ESI m/z: 425.3 [M+H] + .
步骤6：在氮气保护,室温条件下，将叔-丁基(S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯(60mg,0.1mmol,1.0eq)和碳酸钾(58.4mg,0.4mmol,3.0eq)溶于DMF(6mL)中，65℃滴加叔-丁基7-(溴甲基)-3,4-二氢-1,8-二氮杂萘-1(2H)-羧酸酯(A)(30.5mg,0.13mmol,0.95eq)的DMF溶液，反应2h。浓缩反应液，粗品溶于DCM，过滤，滤液浓缩。Prep-TLC(DCM/MeOH＝9/1)得无色油状产品21mg。LC-MS:ESI m/z:571.4[M+H]+。Step 6: Under nitrogen protection and room temperature, tert-butyl (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(2,6-diazaspiro[3.4]octan-2-yl)butanoate (60 mg, 0.1 mmol, 1.0 eq) and potassium carbonate (58.4 mg, 0.4 mmol, 3.0 eq) were dissolved in DMF (6 mL), and a DMF solution of tert-butyl 7-(bromomethyl)-3,4-dihydro-1,8-naphthyridin-1(2H)-carboxylate (A) (30.5 mg, 0.13 mmol, 0.95 eq) was added dropwise at 65°C and reacted for 2 h. The reaction solution was concentrated, the crude product was dissolved in DCM, filtered, and the filtrate was concentrated. Prep-TLC (DCM/MeOH=9/1) gave 21 mg of a colorless oily product. LC-MS: ESI m/z: 571.4 [M+H] + .
中间体3-INTB：甲基(S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(5-羰基-6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯的制备
Intermediate 3-INTB: Preparation of methyl (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(5-carbonyl-6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-2-yl)butanoate
步骤1：将叔-丁基5-羰基-2,6-二氮杂螺[3.4]辛烷-2-羧酸酯(220mg，972.3μmol)溶于THF(10ml)中，0℃下加入氢化钠(110.mg，2.8mmol)和中间体A(450mg，1.4mmol),氮气保护下60℃搅拌3小时。反应液加入饱和氯化铵水溶液(5ml)，乙酸乙酯萃取(15ml*3)。有机相用饱和食盐水洗，Na2SO4干燥，过滤并真空浓缩得到粗产物。粗产品经正相硅胶柱色谱法纯化(PE/EA＝1/0到0/1)得到淡黄色油状产品叔-丁基7-((2-(叔-丁 氧基羰基)-5-羰基-2,6-二氮杂螺[3.4]辛烷-6-基)甲基)-3,4-二氢-1,8-二氮杂萘-1(2H)-羧酸酯220mg。LC-MS:ESI m/z:373.5[M-100]+。Step 1: Dissolve tert-butyl 5-carbonyl-2,6-diazaspiro[3.4]octane-2-carboxylate (220 mg, 972.3 μmol) in THF (10 ml), add sodium hydride (110. mg, 2.8 mmol) and intermediate A (450 mg, 1.4 mmol) at 0°C, and stir at 60°C for 3 hours under nitrogen protection. Add saturated aqueous ammonium chloride solution (5 ml) to the reaction solution, and extract with ethyl acetate (15 ml*3). The organic phase is washed with saturated brine, dried over Na 2 SO 4 , filtered and concentrated in vacuo to obtain a crude product. The crude product is purified by normal phase silica gel column chromatography (PE/EA=1/0 to 0/1) to obtain a light yellow oily product tert-butyl 7-((2-(tert-butyl 220 mg of 5-carbonyl-2,6-diazaspiro[3.4]octan-6-yl)-3,4-dihydro-1,8-naphthyridin-1(2H)-carboxylate. LC-MS: ESI m/z: 373.5 [M-100] + .
步骤2：将叔-丁基7-((2-(叔-丁氧基羰基)-5-羰基-2,6-二氮杂螺[3.4]辛烷-6-基)甲基)-3,4-二氢-1,8-二氮杂萘-1(2H)-羧酸酯(220mg,465.5μmol)溶于甲醇(2ml)中，室温下加入4M盐酸二氧六环(5ml),然后氮气保护下室温搅拌1小时。LCMS检测原料反应完全。将反应液浓缩，得到淡黄色固体产品6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-5-酮200mg。1H NMR(400MHz,DMSO-d6)δ9.32(brs,1H)8.90(brs,1H)7.61(d,J＝7.2Hz,1H)6.63(d,J＝7.2Hz,1H)4.48(s,2H)4.15-4.05(m,2H)3.92-3.82(m,2H)3.50(s,2H)3.34-3.28(m,2H)2.80-2.75(m,2H)2.45(t,J＝6.8Hz,2H)1.79-1.85(m,2H)。Step 2: Dissolve tert-butyl 7-((2-(tert-butoxycarbonyl)-5-carbonyl-2,6-diazaspiro[3.4]octan-6-yl)methyl)-3,4-dihydro-1,8-naphthyridin-1(2H)-carboxylate (220 mg, 465.5 μmol) in methanol (2 ml), add 4M hydrochloric acid dioxane (5 ml) at room temperature, and then stir at room temperature for 1 hour under nitrogen protection. LCMS detected that the raw material reacted completely. The reaction solution was concentrated to obtain 200 mg of a light yellow solid product 6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-5-one. 1 H NMR (400 MHz, DMSO-d 6 ) δ 9.32 (brs, 1H) 8.90 (brs, 1H) 7.61 (d, J=7.2 Hz, 1H) 6.63 (d, J=7.2 Hz, 1H) 4.48 (s, 2H) 4.15-4.05 (m, 2H) 3.92-3.82 (m, 2H) 3.50 (s, 2H) 3.34-3.28 (m, 2H) 2.80-2.75 (m, 2H) 2.45 (t, J=6.8 Hz, 2H) 1.79-1.85 (m, 2H).
步骤3：将6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-5-酮(150mg，485.7μmol)溶于乙腈(5ml)中，室温下依次加入甲基(E)-4-溴丁-2-烯酸酯(34.78mg，194.3μmol)和二异丙基乙胺(188.3mg，1.5mmol),氮气保护下80℃搅拌16小时。反应液浓缩得到粗产品。经高效液相色谱纯化得到淡黄色油状产品甲基(E)-4-(5-羰基-6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-2-基)丁-2-烯酸酯(3-INTA)50mg。LC-MS:ESI m/z:371.2[M+H]+。Step 3: 6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-5-one (150 mg, 485.7 μmol) was dissolved in acetonitrile (5 ml), and methyl (E)-4-bromobut-2-enoate (34.78 mg, 194.3 μmol) and diisopropylethylamine (188.3 mg, 1.5 mmol) were added in sequence at room temperature, and stirred at 80°C for 16 hours under nitrogen protection. The reaction solution was concentrated to obtain a crude product. Purification by high performance liquid chromatography gave 50 mg of methyl (E)-4-(5-carbonyl-6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-2-yl)but-2-enoate (3-INTA) as a pale yellow oily product. LC-MS: ESI m/z: 371.2 [M+H] + .
步骤4：参见中间体1-INTB步骤4的制备方法。粗产品经正相硅胶柱色谱法纯化(PE/EA＝1/0到0/1，然后DCM/MeOH＝1/0到70/30)得到淡黄色油状产品45mg。LC-MS:ESI m/z:543.7[M+H]+。Step 4: See the preparation method of intermediate 1-INTB step 4. The crude product was purified by normal phase silica gel column chromatography (PE/EA=1/0 to 0/1, then DCM/MeOH=1/0 to 70/30) to obtain 45 mg of a light yellow oily product. LC-MS: ESI m/z: 543.7 [M+H] + .
中间体4-INTB：甲基(S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(7-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,7-二氮杂螺[3.5]壬烷-2-基)丁酸酯的制备
Intermediate 4-INTB: Preparation of methyl (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(7-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,7-diazaspiro[3.5]nonan-2-yl)butanoate
步骤1：参见中间体1-INTB步骤3的制备方法。粗品经正相硅胶柱色谱法纯化(DCM/MeOH＝10/1)得到黄色油状产品叔-丁基7-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,7-二氮杂螺[3.5]壬烷-2-羧酸酯2g。LC-MS:ESI m/z:373.2[M+H]+；1H NMR(400MHz,CDCl3)δppm 7.15(d,J＝7.2Hz,1H)6.51(d,J＝7.2Hz,1H)3.75-3.57(m,6H)3.47-3.34(m,4H)2.72(br t,J＝6.2Hz,2H)2.41(brs,2H)1.93-1.86(m,2H)1.81(t,J＝5.6Hz,4H)1.44(s,9H)。Step 1: Refer to the preparation method of intermediate 1-INTB step 3. The crude product was purified by normal phase silica gel column chromatography (DCM/MeOH=10/1) to obtain 2 g of yellow oily product tert-butyl 7-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,7-diazaspiro[3.5]nonane-2-carboxylate. LC-MS: ESI m/z: 373.2 [M+H] + ; 1 H NMR (400 MHz, CDCl3) δ ppm 7.15 (d, J=7.2 Hz, 1H) 6.51 (d, J=7.2 Hz, 1H) 3.75-3.57 (m, 6H) 3.47-3.34 (m, 4H) 2.72 (br t, J=6.2 Hz, 2H) 2.41 (br s, 2H) 1.93-1.86 (m, 2H) 1.81 (t, J=5.6 Hz, 4H) 1.44 (s, 9H).
步骤2：参见中间体3-INTB步骤2的制备方法。反应液浓缩得到黄色固体产品7-((2,7-二氮杂螺[3.5]壬烷-7-基)甲基)-1,2,3,4-四氢-1,8-二氮杂萘1g。LC-MS:ESI m/z:273.2[M+H]+。Step 2: See the preparation method of intermediate 3-INTB step 2. The reaction solution was concentrated to obtain 1 g of a yellow solid product 7-((2,7-diazaspiro[3.5]nonan-7-yl)methyl)-1,2,3,4-tetrahydro-1,8-naphthyridine. LC-MS: ESI m/z: 273.2 [M+H] + .
步骤3：参见中间体1-INT步骤1的制备方法。粗品经正相硅胶柱色谱法纯化(DCM/MeOH＝10/1)得到红棕色油状产品甲基(E)-4-(7-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,7-二氮杂螺[3.5]壬烷-2-基)丁-2-烯酸酯(4-INTA)600mg。LC-MS:ESI m/z:371.1[M+H]+。Step 3: Refer to the preparation method of intermediate 1-INT step 1. The crude product was purified by normal phase silica gel column chromatography (DCM/MeOH=10/1) to obtain 600 mg of methyl (E)-4-(7-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,7-diazaspiro[3.5]nonane-2-yl)but-2-enoate (4-INTA) as a reddish brown oily product. LC-MS: ESI m/z: 371.1 [M+H] + .
步骤4：参见中间体1-INTB步骤4的制备方法。反应液浓缩，经高效液相色谱纯化得到 红棕色油状产品45mg。LC-MS:ESI m/z:543.5[M+H]+；1H NMR(400MHz,METHANOL-d4)δppm 7.57-7.41(m,2H)7.39-7.26(m,3H)6.72(d,J＝7.2Hz,1H)6.04(s,1H)4.11(s,2H)3.98-3.78(m,2H)3.65-3.58(m,2H)3.55-3.50(m,3H)3.48-3.40(m,3H)3.35-3.28(m,3H)3.15-3.08(m,3H)2.83-2.73(m,3H)2.72-2.64(m,1H)2.22(s,3H)2.20(s,3H)2.12-1.95(m,4H)1.92-1.87(m,2H)。Step 4: See the preparation method of intermediate 1-INTB step 4. The reaction solution was concentrated and purified by high performance liquid chromatography to obtain 45 mg of reddish brown oily product. LC-MS: ESI m/z: 543.5 [M+H] + ; 1 H NMR (400 MHz, METHANOL-d4) δ ppm 7.57-7.41 (m, 2H) 7.39-7.26 (m, 3H) 6.72 (d, J=7.2 Hz, 1H) 6.04 (s, 1H) 4.11 (s, 2H) 3.98-3.78 (m, 2H) 3.65-3.58 (m, 2H) 3.55-3.50 (m, 3H) 3.48-3.40 (m, 3H) 3.35-3.28 (m, 3H) 3.15-3.08 (m, 3H) 2.83-2.73 (m, 3H) 2.72-2.64 (m, 1H) 2.22 (s, 3H) 2.20 (s, 3H) 2.12-1.95 (m, 4H) 1.92-1.87 (m, 2H).
中间体5-INTB：甲基(S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(2-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,7-二氮杂螺[3.5]壬烷-7-基)丁酸酯的制备
Intermediate 5-INTB: Preparation of methyl (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(2-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,7-diazaspiro[3.5]nonan-7-yl)butanoate
步骤1：参见中间体1-INT步骤1的制备方法。粗产品经正相硅胶柱色谱法纯化(PE/EA＝1/0到0/1)得到淡黄色油状产品叔-丁基(E)-7-(4-甲氧基-4-羰基丁-2-烯-1-基)-2,7-二氮杂螺[3.5]壬烷-2-羧酸酯1g。1H NMR(400MHz,CDCl3)δ6.96(dt,J＝6.0,15.2Hz,1H)5.99(d,J＝15.2Hz,1H)3.72-3.80(s,3H)3.62(s,4H)3.11(dd,J＝1.6,6.0Hz,2H)2.37(s,4H)1.78(t,J＝5.2Hz,4H)1.46(s,9H)。Step 1: See the preparation method of intermediate 1-INT step 1. The crude product was purified by normal phase silica gel column chromatography (PE/EA=1/0 to 0/1) to obtain 1 g of pale yellow oily product tert-butyl (E)-7-(4-methoxy-4-carbonylbut-2-en-1-yl)-2,7-diazaspiro[3.5]nonane-2-carboxylate. 1 H NMR (400 MHz, CDCl 3 ) δ 6.96 (dt, J=6.0,15.2 Hz,1H) 5.99 (d, J=15.2 Hz,1H) 3.72-3.80 (s,3H) 3.62 (s,4H) 3.11 (dd, J=1.6,6.0 Hz,2H) 2.37 (s,4H) 1.78 (t, J=5.2 Hz,4H) 1.46 (s,9H).
步骤2：参见将中间体3-INTB步骤2的制备方法。得到淡黄色固体产品甲基(E)-4-(2,7-二氮杂螺[3.5]壬烷-7-基)丁-2-烯酸酯810mg。1H NMR(400MHz,D2O)δ6.81(dt,J＝7.2,15.2Hz,1H)6.25(d,J＝15.2Hz,1H)4.05-3.95(m,4H)3.87(d,J＝7.2Hz,2H)3.72(s,3H)3.55-3.45(m,2H)3.02-2.92(m,2H)2.44-2.32(m,2H)2.05-1.95(m,2H)。Step 2: Refer to the preparation method of intermediate 3-INTB step 2. Obtain 810 mg of methyl (E)-4-(2,7-diazaspiro[3.5]nonan-7-yl)but-2-enoate as a pale yellow solid product. 1 H NMR (400 MHz, D 2 O) δ 6.81 (dt, J = 7.2, 15.2 Hz, 1H) 6.25 (d, J = 15.2 Hz, 1H) 4.05-3.95 (m, 4H) 3.87 (d, J = 7.2 Hz, 2H) 3.72 (s, 3H) 3.55-3.45 (m, 2H) 3.02-2.92 (m, 2H) 2.44-2.32 (m, 2H) 2.05-1.95 (m, 2H).
步骤3：将中间体A-3(604.6mg,3.0mmol)和TEA(628.7mg,6.2mmol)溶于DCM(20ml)，氮气保护下加入醋酸硼氢化钠(987.5mg，4.7mmol)和醋酸(18.7mg，0.3mmol)，在室温下搅拌过夜。反应液加入20ml水淬灭，直接旋干得到粗产品，经高效液相色谱纯化得到棕色产品甲基(E)-4-(2-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,7-二氮杂螺[3.5]壬烷-7-基)丁-2-烯酸酯(5-INTA)600mg。LC-MS:ESI m/z:371.5[M+H]+.1H NMR(400MHz,DMSO-d6)δ7.15(d,J＝7.2Hz,1H)6.81(dt,J＝6.0,15.6Hz,1H)6.51-6.41(m,2H)6.00(d,J＝15.6Hz,1H)4.02(s,2H)3.66(s,6H)3.30-3.25(m,2H)3.12-3.08(m,2H)2.64(t,J＝6.0Hz,2H)2.42-2.20(m,4H)1.85-1.67(m,6H)。Step 3: Dissolve intermediate A-3 (604.6 mg, 3.0 mmol) and TEA (628.7 mg, 6.2 mmol) in DCM (20 ml), add sodium acetate borohydride (987.5 mg, 4.7 mmol) and acetic acid (18.7 mg, 0.3 mmol) under nitrogen protection, and stir at room temperature overnight. Add 20 ml of water to the reaction solution for quenching, spin dry directly to obtain a crude product, and HPLC purification to obtain a brown product methyl (E)-4-(2-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,7-diazaspiro[3.5]nonane-7-yl)but-2-enoate (5-INTA) 600 mg. LC-MS: ESI m/z: 371.5 [M+H] + . 1 H NMR (400 MHz, DMSO-d6) δ 7.15 (d, J = 7.2 Hz, 1H) 6.81 (dt, J = 6.0, 15.6 Hz, 1H) 6.51-6.41 (m, 2H) 6.00 (d, J = 15.6 Hz, 1H) 4.02 (s, 2H) 3.66 (s, 6H) 3.30-3.25 (m, 2H) 3.12-3.08 (m, 2H) 2.64 (t, J = 6.0 Hz, 2H) 2.42-2.20 (m, 4H) 1.85-1.67 (m, 6H).
步骤4：参见中间体1-INTB步骤4的制备方法。粗产品经正相硅胶柱色谱法纯化(PE/EA＝1/0到0/1，然后DCM/MeOH＝1/0到70/30)得到浅黄色固体产品100mg。LC-MS:ESI m/z:543.7[M+H]+。Step 4: See the preparation method of intermediate 1-INTB step 4. The crude product was purified by normal phase silica gel column chromatography (PE/EA=1/0 to 0/1, then DCM/MeOH=1/0 to 70/30) to obtain 100 mg of a light yellow solid product. LC-MS: ESI m/z: 543.7 [M+H] + .
中间体6-INTB：甲基(S)-4-(7-((1H-吡咯并[2,3-b]吡啶-6-基)甲基)-2,7-二氮杂螺[3.5]壬烷-2-基)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)丁酸酯的制备
Intermediate 6-INTB: Preparation of methyl (S)-4-(7-((1H-pyrrolo[2,3-b]pyridin-6-yl)methyl)-2,7-diazaspiro[3.5]nonan-2-yl)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)butyrate
步骤1：将叔-丁基2,7-二氮杂螺[3.5]壬烷-7-羧酸酯(500mg，2.2mmol)和甲基(E)-4-溴丁-2-烯酸酯(395.5mg，2.2mmol)溶于10ml乙腈中，然后加入碳酸钾(610.7mg，4.4mmol)。反应液在80℃下搅拌16小时。反应液过滤旋干得到粗品，经正相硅胶柱色谱法纯化(PE/EA从100％到40％)得到黄色油状产品叔-丁基(E)-2-(4-甲氧基-4-羰基丁-2-烯-1-基)-2,7-二氮杂螺[3.5]壬烷-7-羧酸酯610mg。1H NMR(400MHz,CDCl3)δ6.82-6.88(dt,J＝5.6,16Hz,1H),5.95-5.99(d,J＝16Hz,1H),3.73(s,3H),3.28-3.33(m,6H),3.12(s,4H),1.69-1.72(m,4H),1.44(s,9H)。Step 1: Dissolve tert-butyl 2,7-diazaspiro [3.5] nonane-7-carboxylate (500 mg, 2.2 mmol) and methyl (E)-4-bromobut-2-enoate (395.5 mg, 2.2 mmol) in 10 ml of acetonitrile, and then add potassium carbonate (610.7 mg, 4.4 mmol). The reaction solution is stirred at 80 ° C for 16 hours. The reaction solution is filtered and dried to obtain a crude product, which is purified by normal phase silica gel column chromatography (PE/EA from 100% to 40%) to obtain a yellow oily product tert-butyl (E)-2-(4-methoxy-4-carbonylbut-2-ene-1-yl)-2,7-diazaspiro [3.5] nonane-7-carboxylate 610 mg. 1 H NMR (400 MHz, CDCl 3 ) δ 6.82-6.88 (dt, J=5.6, 16 Hz, 1H), 5.95-5.99 (d, J=16 Hz, 1H), 3.73 (s, 3H), 3.28-3.33 (m, 6H), 3.12 (s, 4H), 1.69-1.72 (m, 4H), 1.44 (s, 9H).
步骤2：参见中间体1-INTB步骤4的制备方法。粗品经正相硅胶柱色谱法纯化(PE/EA从100％到20％)得到黄色油状产品叔-丁基(S)-2-(2-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-甲氧基-4-羰基丁基)-2,7-二氮杂螺[3.5]壬烷-7-羧酸酯940mg。LC-MS:ESI m/z:497.7[M+H]+。Step 2: Refer to the preparation method of intermediate 1-INTB step 4. The crude product was purified by normal phase silica gel column chromatography (PE/EA from 100% to 20%) to obtain 940 mg of yellow oily product tert-butyl (S)-2-(2-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-methoxy-4-carbonylbutyl)-2,7-diazaspiro[3.5]nonane-7-carboxylate. LC-MS: ESI m/z: 497.7 [M+H] + .
步骤3：参见中间体3-INTB步骤2的制备方法。反应液浓缩旋干后得到粗品甲基(S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(2,7-二氮杂螺[3.5]壬烷-2-基)丁酸酯(6-INTA)1g。LC-MS:ESI m/z:397.5[M+H]+。Step 3: Refer to the preparation method of intermediate 3-INTB in step 2. The reaction solution was concentrated and dried to obtain 1 g of crude methyl (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(2,7-diazaspiro[3.5]nonane-2-yl)butanoate (6-INTA). LC-MS: ESI m/z: 397.5 [M+H] + .
步骤3：参见中间体5-INTB步骤3的制备方法。粗品经正相硅胶柱色谱法纯化(DCM/MeOH从100％到90％)得到黄色油状产品30mg。LC-MS:ESI m/z:527.7[M+H]+。Step 3: Refer to the preparation method of intermediate 5-INTB in step 3. The crude product was purified by normal phase silica gel column chromatography (DCM/MeOH from 100% to 90%) to obtain 30 mg of a yellow oily product. LC-MS: ESI m/z: 527.7 [M+H] + .
中间体7-INTB：甲基(3S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(7-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,7-二氮杂螺[4.4]壬烷-2-基)丁酸酯的制备
Intermediate 7-INTB: Preparation of methyl (3S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(7-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,7-diazaspiro[4.4]nonan-2-yl)butanoate
步骤1：参见中间体3-INTB步骤3的制备方法。粗品经正相硅胶柱色谱法纯化(PE/EA从100％到0％)得到黄色油状产品叔-丁基(E)-7-(4-甲氧基-4-羰基丁-2-烯-1-基)-2,7-二氮杂螺[4.4]壬烷-2-羧酸酯1g。LC-MS:ESI m/z:325.5[M+H]+。Step 1: Refer to the preparation method of intermediate 3-INTB step 3. The crude product was purified by normal phase silica gel column chromatography (PE/EA from 100% to 0%) to obtain 1 g of yellow oily product tert-butyl (E)-7-(4-methoxy-4-carbonylbut-2-en-1-yl)-2,7-diazaspiro[4.4]nonane-2-carboxylate. LC-MS: ESI m/z: 325.5 [M+H] + .
步骤2：参见中间体3-INTB步骤2的制备方法。反应液直接浓缩得到白色固体甲基(E)-4-(2,7-二氮杂螺[4.4]壬烷-2-基)丁-2-烯酸酯1g。Step 2: Refer to the preparation method of intermediate 3-INTB step 2. The reaction solution was directly concentrated to obtain 1 g of white solid methyl (E)-4-(2,7-diazaspiro[4.4]nonane-2-yl)but-2-enoate.
步骤3：参见中间体5-INTB步骤3的制备方法。粗品经高效液相色谱分离得到黄色油状产品甲基(E)-4-(7-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,7-二氮杂螺[4.4]壬烷-2-基)丁-2-烯 酸酯(7-INTA)740mg。LC-MS:ESI m/z:371.5[M+H]+。Step 3: See the preparation method of intermediate 5-INTB step 3. The crude product was separated by high performance liquid chromatography to obtain a yellow oily product methyl (E)-4-(7-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,7-diazaspiro[4.4]nonane-2-yl)but-2-ene Acid ester (7-INTA) 740mg. LC-MS: ESI m/z: 371.5 [M+H] + .
步骤4：参见中间体1-INTB步骤4的制备方法。粗品经正相硅胶柱色谱法纯化(DCM/MeOH从100％到70％)得到黄色油状产品77mg。LC-MS:ESI m/z:542.73[M+H]+。Step 4: Refer to the preparation method of intermediate 1-INTB step 4. The crude product was purified by normal phase silica gel column chromatography (DCM/MeOH from 100% to 70%) to obtain 77 mg of a yellow oily product. LC-MS: ESI m/z: 542.73 [M+H] + .
中间体8-INTB：甲基(S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(9-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-3,9-二氮杂螺[5.5]十一烷-3-基)丁酸酯的制备
Intermediate 8-INTB: Preparation of methyl (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(9-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-3,9-diazaspiro[5.5]undec-3-yl)butanoate
步骤1：参见中间体1-INTB步骤1的制备方法。粗品经正相硅胶柱色谱法纯化(PE/EA从100％到30％)得到黄色油状产品叔-丁基(E)-9-(4-甲氧基-4-羰基丁-2-烯-1-基)-3,9-二氮杂螺[5.5]十一烷-3-羧酸酯1g。LC-MS:ESI m/z:352.9[M+H]+。Step 1: Refer to the preparation method of intermediate 1-INTB step 1. The crude product was purified by normal phase silica gel column chromatography (PE/EA from 100% to 30%) to obtain 1 g of yellow oily product tert-butyl (E)-9-(4-methoxy-4-carbonylbut-2-en-1-yl)-3,9-diazaspiro[5.5]undecane-3-carboxylate. LC-MS: ESI m/z: 352.9 [M+H] + .
步骤2：参见中间体3-INTB步骤2的制备方法。得到白色固体甲基(E)-4-(3,9-二氮杂螺[5.5]十一烷-3-基)丁-2-烯酸酯500mg。Step 2: Refer to the preparation method of intermediate 3-INTB step 2. 500 mg of white solid methyl (E)-4-(3,9-diazaspiro[5.5]undec-3-yl)but-2-enoate was obtained.
步骤3：参见中间体5-INTB步骤3的制备方法。粗品经高效液相色谱(甲酸)分离得到黄色油状产品甲基(E)-4-(9-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-3,9-二氮杂螺[5.5]十一烷-3-基)丁-2-烯酸酯(8-INTA)60mg。LC-MS:ESI m/z:399.6[M+H]+。Step 3: See the preparation method of intermediate 5-INTB step 3. The crude product was separated by high performance liquid chromatography (formic acid) to obtain 60 mg of yellow oily product methyl (E)-4-(9-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-3,9-diazaspiro[5.5]undecane-3-yl)but-2-enoate (8-INTA). LC-MS: ESI m/z: 399.6 [M+H] + .
步骤4：参见中间体1-INTB步骤4的制备方法。粗品经正相硅胶柱色谱法纯化(DCM/MeOH从100％到70％)得到黄色油状产品50mg。LC-MS:ESI m/z:571.7[M+H]+。Step 4: Refer to the preparation method of intermediate 1-INTB step 4. The crude product was purified by normal phase silica gel column chromatography (DCM/MeOH from 100% to 70%) to obtain 50 mg of a yellow oily product. LC-MS: ESI m/z: 571.7 [M+H] + .
中间体9-INTB：甲基-1H-吡唑-1-基)苯基)-4-(8-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,8-二氮杂螺[4.5]癸烷-2-基)丁酸酯的制备
Intermediate 9-INTB: Preparation of methyl-1H-pyrazol-1-yl)phenyl)-4-(8-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,8-diazaspiro[4.5]decane-2-yl)butanoate
步骤1：参见中间体3-INTB步骤3的制备方法。粗产品经正相硅胶柱色谱法纯化(PE/EA＝1/0到0/1)得到淡黄色油状产品叔-丁基(E)-2-(4-甲氧基-4-羰基丁-2-烯-1-基)-2,8-二氮杂螺[4.5]癸烷-8-羧酸酯120mg。1H NMR(400MHz,CDCl3)δ6.93-6.90(m,1H)5.92(d,J＝17.2Hz,1H)3.68(s,3H)3.29-3.38(m,2H)3.20-3.28(m,2H)3.09-3.18(m,2H)2.56(s,2H)2.36(s,2H)1.57-1.60(m,2H)1.43-1.48(m,4H)1.38(s,9H)。 Step 1: Refer to the preparation method of intermediate 3-INTB step 3. The crude product was purified by normal phase silica gel column chromatography (PE/EA=1/0 to 0/1) to obtain 120 mg of light yellow oily product tert-butyl (E)-2-(4-methoxy-4-carbonylbut-2-en-1-yl)-2,8-diazaspiro[4.5]decane-8-carboxylate. 1 H NMR (400 MHz, CDCl 3 ) δ 6.93-6.90 (m, 1H) 5.92 (d, J=17.2 Hz, 1H) 3.68 (s, 3H) 3.29-3.38 (m, 2H) 3.20-3.28 (m, 2H) 3.09-3.18 (m, 2H) 2.56 (s, 2H) 2.36 (s, 2H) 1.57-1.60 (m, 2H) 1.43-1.48 (m, 4H) 1.38 (s, 9H).
步骤2：参见中间体3-INTB步骤2的制备方法。得到淡黄色固体产品甲基(E)-4-(2,8-二氮杂螺[4.5]癸烷-2-基)丁-2-烯酸酯100mg。1H NMR(400MHz,DMSO-d6)δ8.82(s,2H)6.86-7.02(m,1H)6.29(d,J＝15.6Hz,1H)3.88-4.07(m,2H)3.71(s,3H)3.41-3.62(m,3H)3.06-3.21(m,4H)2.91(m,1H)1.70-2.05(m,6H)。Step 2: Refer to the preparation method of intermediate 3-INTB step 2. Obtain 100 mg of light yellow solid product methyl (E)-4-(2,8-diazaspiro[4.5]decane-2-yl)but-2-enoate. 1 H NMR (400 MHz, DMSO-d 6 )δ8.82(s,2H)6.86-7.02(m,1H)6.29(d,J＝15.6Hz,1H)3.88-4.07(m,2H)3.71(s,3H)3.41-3.62(m,3H)3.06-3.21(m,4H)2.91(m,1H)1.70-2.05(m,6H).
步骤3：参见中间体5-INTB步骤3的制备方法。粗产品经高效液相色谱纯化得到棕色产品甲基(E)-4-(8-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,8-二氮杂螺[4.5]癸烷-2-基)丁-2-烯酸酯(9-INTA)70mg。1H NMR(400MHz,CDCl3)δ9.57(s,1H)7.27-7.29(m,1H)6.87-7.02(m,1H)6.59(d,J＝7.34Hz,1H)6.01(d,J＝15.2Hz,1H)3.75(s,3H)3.62(s,2H)3.46(s,2H)3.33(d,J＝5.8Hz,2H)2.52-2.67(m,8H)1.91(s,2H)1.64-1.78(m,6H)1.26-1.23(m,2H)。Step 3: See the preparation method of intermediate 5-INTB step 3. The crude product was purified by HPLC to obtain 70 mg of a brown product, methyl (E)-4-(8-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,8-diazaspiro[4.5]decane-2-yl)but-2-enoate (9-INTA). 1 H NMR (400 MHz, CDCl 3 ) δ 9.57 (s, 1H) 7.27-7.29 (m, 1H) 6.87-7.02 (m, 1H) 6.59 (d, J=7.34 Hz, 1H) 6.01 (d, J=15.2 Hz, 1H) 3.75 (s, 3H) 3.62 (s, 2H) 3.46 (s, 2H) 3.33 (d, J=5.8 Hz, 2H) 2.52-2.67 (m, 8H) 1.91 (s, 2H) 1.64-1.78 (m, 6H) 1.26-1.23 (m, 2H).
步骤4：参见中间体1-INTB步骤4的制备方法。粗产品经正相硅胶柱色谱法纯化(PE/EA＝1/0到0/1，然后DCM/MeOH＝1/0到70/30)得到浅黄色油状产品40mg。LC-MS:ESI m/z:557.7[M+H]+。Step 4: See the preparation method of intermediate 1-INTB step 4. The crude product was purified by normal phase silica gel column chromatography (PE/EA=1/0 to 0/1, then DCM/MeOH=1/0 to 70/30) to obtain 40 mg of a light yellow oily product. LC-MS: ESI m/z: 557.7 [M+H] + .
中间体10-INTB：甲基(S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(2-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,8-二氮杂螺[4.5]癸烷-8-基)丁酸酯的制备
Intermediate 10-INTB: Preparation of methyl (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(2-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,8-diazaspiro[4.5]decane-8-yl)butanoate
步骤1：参见中间体3-INTB步骤3的制备方法。粗产品经正相硅胶柱色谱法纯化(PE/EA＝1/1)得到黄色油状产品叔-丁基(E)-8-(4-甲氧基-4-羰基丁-2-烯-1-基)-2,8-二氮杂螺[4.5]癸烷-2-羧酸酯78mg。1H NMR(400MHz,CDCl3)δ＝7.01-6.91(m,1H),5.98(d,J＝15.6Hz,1H),3.74(s,3H),3.43-3.30(m,2H),3.19-3.08(m,4H),2.56-2.24(m,4H),1.72-1.65(m,2H),1.45(s,9H),1.34-1.23(m,3H),0.91-0.78(m,1H)。Step 1: Refer to the preparation method of intermediate 3-INTB step 3. The crude product was purified by normal phase silica gel column chromatography (PE/EA=1/1) to obtain 78 mg of yellow oily product tert-butyl (E)-8-(4-methoxy-4-carbonylbut-2-en-1-yl)-2,8-diazaspiro[4.5]decane-2-carboxylate. 1 H NMR (400 MHz, CDCl3) δ＝7.01-6.91 (m, 1H), 5.98 (d, J＝15.6 Hz, 1H), 3.74 (s, 3H), 3.43-3.30 (m, 2H), 3.19-3.08 (m, 4H), 2.56-2.24 (m, 4H), 1.72-1.65 (m, 2H), 1.45 (s, 9H), 1.34-1.23 (m, 3H), 0.91-0.78 (m, 1H).
步骤2参见中间体3-INTB步骤2的制备方法。得到白色固体产品甲基(E)-4-(2,8-二氮杂螺[4.5]癸烷-8-基)丁-2-烯酸酯120mg。1H NMR(400MHz,DMSO-d6)δ＝9.53-9.27(m,2H),7.02-6.91(m,1H),6.26(d,J＝15.8Hz,1H),3.92(s,2H),3.71(s,3H),3.28-3.20(m,2H),3.13(s,1H),3.06-2.89(m,3H),1.90(s,4H),1.83-1.75(m,2H),1.24-1.20(m,1H),0.99-0.66(m,1H)。Step 2 refers to the preparation method of intermediate 3-INTB step 2. 120 mg of methyl (E)-4-(2,8-diazaspiro[4.5]decane-8-yl)but-2-enoate was obtained as a white solid product. 1 H NMR (400 MHz, DMSO-d 6 ) δ＝9.53-9.27 (m, 2H), 7.02-6.91 (m, 1H), 6.26 (d, J＝15.8 Hz, 1H), 3.92 (s, 2H), 3.71 (s, 3H), 3.28-3.20 (m, 2H), 3.13 (s, 1H), 3.06-2.89 (m, 3H), 1.90 (s, 4H), 1.83-1.75 (m, 2H), 1.24-1.20 (m, 1H), 0.99-0.66 (m, 1H).
步骤3：参见中间体5-INTB步骤3的制备方法。粗产品经高效液相色谱分离得到黄色油状产品甲基(E)-4-(2-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,8-二氮杂螺[4.5]癸烷-8-基)丁-2-烯酸酯(10-INTA)70mg。Step 3: See the preparation method of intermediate 5-INTB step 3. The crude product was separated by high performance liquid chromatography to obtain 70 mg of yellow oily product methyl (E)-4-(2-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,8-diazaspiro[4.5]decane-8-yl)but-2-enoate (10-INTA).
步骤4：参见中间体1-INTB步骤4的制备方法。粗产品经正相硅胶柱色谱法纯化(DCM/MeOH＝4/1)得到黄色油状产品10mg。LC-MS:ESI m/z:557.7[M+H]+。Step 4: See the preparation method of intermediate 1-INTB step 4. The crude product was purified by normal phase silica gel column chromatography (DCM/MeOH=4/1) to obtain 10 mg of a yellow oily product. LC-MS: ESI m/z: 557.7 [M+H] + .
中间体11-INTB：甲基(S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(1-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)螺[二氢吲哚-3,4'-哌啶]-1'-基)丁酸酯的制备
Intermediate 11-INTB: Preparation of methyl (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(1-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)spiro[dihydroindole-3,4'-piperidin]-1'-yl)butanoate
步骤1：参见中间体5-INTB步骤3的制备方法。粗品经正相硅胶柱色谱法纯化(PE/EA＝5/1)得到白色固体产品叔-丁基1-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)螺[二氢吲哚-3,4'-哌啶]-1'-羧酸酯140mg。LC-MS:ESI m/z:435.0[M+H]+。Step 1: See the preparation method of intermediate 5-INTB in step 3. The crude product was purified by normal phase silica gel column chromatography (PE/EA=5/1) to obtain 140 mg of white solid product tert-butyl 1-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)spiro[dihydroindole-3,4'-piperidine]-1'-carboxylate. LC-MS: ESI m/z: 435.0 [M+H] + .
步骤2：将叔-丁基1-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)螺[二氢吲哚-3,4'-哌啶]-1'-羧酸酯(140mg,0.32mmol)溶于DCM(2ml)，将盐酸/甲醇(2ml，4M)加到反应体系中，室温搅拌1小时。将反应液直接浓缩得到黄色固体产品1-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)螺[二氢吲哚-3,4'-哌啶]80mg。LC-MS:ESI m/z:335.2[M+H]+。Step 2: Dissolve tert-butyl 1-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)spiro[indoline-3,4'-piperidine]-1'-carboxylate (140 mg, 0.32 mmol) in DCM (2 ml), add hydrochloric acid/methanol (2 ml, 4 M) to the reaction system, and stir at room temperature for 1 hour. The reaction solution is directly concentrated to obtain 80 mg of a yellow solid product 1-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)spiro[indoline-3,4'-piperidine]. LC-MS: ESI m/z: 335.2[M+H] + .
步骤3：将1-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)螺[二氢吲哚-3,4'-哌啶](40mg,0.15mmol),甲基(E)-4-溴丁-2-烯酸酯(18.73mg,0.10mmol)和碳酸钾(41mg,0.30mmol)加入到DMF中，85℃搅拌4小时。将反应液加水(10ml)，用乙酸乙酯(10ml)萃取。有机相用饱和食盐水洗三次(10ml*3)，有机相干燥，浓缩，正相硅胶柱色谱法纯化(DCM/MeOH＝10/1)得到黄色油状产品甲基(E)-4-(1-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)螺[二氢吲哚-3,4'-哌啶]-1'-基)丁-2-烯酸酯(11-INTA)45mg。LC-MS:ESI m/z:433.3[M+H]+。Step 3: 1-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)spiro[indoline-3,4'-piperidine] (40 mg, 0.15 mmol), methyl (E)-4-bromobut-2-enoate (18.73 mg, 0.10 mmol) and potassium carbonate (41 mg, 0.30 mmol) were added to DMF and stirred at 85°C for 4 hours. Water (10 ml) was added to the reaction solution and extracted with ethyl acetate (10 ml). The organic phase was washed three times with saturated brine (10 ml*3), dried, concentrated, and purified by normal phase silica gel column chromatography (DCM/MeOH=10/1) to obtain 45 mg of a yellow oily product, methyl (E)-4-(1-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)spiro[dihydroindole-3,4'-piperidin]-1'-yl)but-2-enoate (11-INTA). LC-MS: ESI m/z: 433.3 [M+H] + .
步骤4：参见中间体1-INTB步骤4的制备方法。粗品经硅胶板(DCM/MeOH＝10/1)纯化得到黄色固体产品30mg。LC-MS:ESI m/z:605.2[M+H]+。Step 4: Refer to the preparation method of intermediate 1-INTB step 4. The crude product was purified by silica gel plate (DCM/MeOH=10/1) to obtain 30 mg of yellow solid product. LC-MS: ESI m/z: 605.2 [M+H] + .
中间体12-INTB：甲基(S)-4-(7-((2-氨基喹啉-7-基)甲基)-2,7-二氮杂螺[3.5]壬烷-2-基)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)丁酸酯的制备
Intermediate 12-INTB: Preparation of methyl (S)-4-(7-((2-aminoquinolin-7-yl)methyl)-2,7-diazaspiro[3.5]nonan-2-yl)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)butyrate
步骤1：将中间体B(300mg，0.5mmol)溶于乙腈(5ml)中，室温下依次加入二异丙基乙胺(0.3ml，1.4mmol)和中间体6-INTA(373.5mg，0.9mmol)，反应液加热到80℃并搅拌6小时。反应液直接旋干，粗产品经正相硅胶柱色谱法纯化(DCM/MeOH＝10/1)得到淡黄色油状产品甲基(3S)-3-(3-(3,5-二甲基-2H-吡咯-2-基)苯基)-4-(7-((2-((二苯亚甲基)氨基)喹啉-7-基)甲基)-2,7-二氮杂螺[3.5]壬烷-2-基)丁酸酯(12-INTA)220mg。LC-MS:ESI m/z:717.8[M+H]+。Step 1: Dissolve intermediate B (300 mg, 0.5 mmol) in acetonitrile (5 ml), add diisopropylethylamine (0.3 ml, 1.4 mmol) and intermediate 6-INTA (373.5 mg, 0.9 mmol) in turn at room temperature, heat the reaction solution to 80°C and stir for 6 hours. The reaction solution is directly spin-dried, and the crude product is purified by normal phase silica gel column chromatography (DCM/MeOH=10/1) to obtain a light yellow oily product methyl (3S)-3-(3-(3,5-dimethyl-2H-pyrrol-2-yl)phenyl)-4-(7-((2-((diphenylmethylene)amino)quinolin-7-yl)methyl)-2,7-diazaspiro[3.5]nonane-2-yl)butanoate (12-INTA) 220 mg. LC-MS: ESI m/z: 717.8[M+H] + .
步骤2：参见中间体3-INTB步骤2的制备方法。得到黄色固体产品230mg。LC-MS:ESI m/z:553.4[M+H]+。Step 2: Refer to the preparation method of intermediate 3-INTB step 2. Obtain 230 mg of yellow solid product. LC-MS: ESI m/z: 553.4 [M+H] + .
中间体13-INTB：甲基(S)-4-(7-((6-氨基吡啶-2-基)甲基)-2,7-二氮杂螺[3.5]壬烷-2-基)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)丁酸酯的制备
Intermediate 13-INTB: Preparation of methyl (S)-4-(7-((6-aminopyridin-2-yl)methyl)-2,7-diazaspiro[3.5]nonan-2-yl)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)butyrate
步骤1：参见中间体12-INTB步骤1的制备方法。粗产品经正相硅胶柱色谱法纯化(PE/EA＝1/0到0/1)得到淡黄色油状产品甲基(S)-4-(7-((6-((叔-丁氧基羰基)氨基)吡啶-2-基)甲基)-2,7-二氮杂螺[3.5]壬烷-2-基)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)丁酸酯100mg。LC-MS:ESI m/z:603.7[M+H]+.Step 1: See the preparation method of intermediate 12-INTB step 1. The crude product was purified by normal phase silica gel column chromatography (PE/EA=1/0 to 0/1) to obtain 100 mg of methyl (S)-4-(7-((6-((tert-butoxycarbonyl)amino)pyridin-2-yl)methyl)-2,7-diazaspiro[3.5]nonane-2-yl)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)butanoate as a light yellow oily product. LC-MS: ESI m/z: 603.7 [M+H] + .
步骤2：参见中间体3-INTB步骤2的制备方法。得到淡黄色油状产品100mg。LC-MS:ESI m/z:503.6[M+H]+。Step 2: Refer to the preparation method of intermediate 3-INTB step 2. Obtain 100 mg of light yellow oily product. LC-MS: ESI m/z: 503.6 [M+H] + .
中间体14-INTB：甲基(S)-4-(7-((3,4-二氢-2H-吡啶并[3,2-b][1,4]噁嗪-6-基)甲基)-2,7-二氮杂螺[3.5]壬烷-2-基)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)丁酸酯的制备
Intermediate 14-INTB: Preparation of methyl (S)-4-(7-((3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazin-6-yl)methyl)-2,7-diazaspiro[3.5]nonan-2-yl)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)butyrate
步骤1：依次将中间体D(30mg，0.01mmol)，中间体6-INTA(36.1mg，0.1mmol)和N,N-二异丙基乙胺(35.3mg，0.3mmol)溶于DCM(5ml)中，室温下搅拌2小时。反应液加水(20ml)淬灭，DCM(20ml)萃取。有机相旋干，硅胶板纯化(DCM/MeOH＝20/1)得到无色油状产品叔-丁基(S)-6-((2-(2-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-甲氧基-4-羰基丁基)-2,7-二氮杂螺[3.5]壬烷-7-基)甲基)-2,3-二氢-4H-吡啶并[3,2-b][1,4]噁嗪-4-羧酸酯30mg。LC-MS:ESI m/z:645.8[M+H]+。Step 1: Dissolve intermediate D (30 mg, 0.01 mmol), intermediate 6-INTA (36.1 mg, 0.1 mmol) and N,N-diisopropylethylamine (35.3 mg, 0.3 mmol) in DCM (5 ml) in turn and stir at room temperature for 2 hours. The reaction solution was quenched with water (20 ml) and extracted with DCM (20 ml). The organic phase was spin-dried and purified on a silica gel plate (DCM/MeOH=20/1) to obtain 30 mg of a colorless oily product, tert-butyl (S)-6-((2-(2-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-methoxy-4-carbonylbutyl)-2,7-diazaspiro[3.5]nonan-7-yl)methyl)-2,3-dihydro-4H-pyrido[3,2-b][1,4]oxazine-4-carboxylate. LC-MS: ESI m/z: 645.8 [M+H] + .
步骤2：将叔-丁基(S)-6-((2-(2-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-甲氧基-4-羰基丁基)-2,7-二氮杂螺[3.5]壬烷-7-基)甲基)-2,3-二氢-4H-吡啶并[3,2-b][1,4]噁嗪-4-羧酸酯(30mg，0.05mmol)溶于DCM(5ml)中，室温下加入盐酸甲醇(4M，5ml)，并搅拌1小时。反应液直接旋干得到黄色固体产品25mg。LC-MS:ESI m/z:545.7[M+H]+。Step 2: Dissolve tert-butyl (S)-6-((2-(2-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-methoxy-4-carbonylbutyl)-2,7-diazaspiro[3.5]nonan-7-yl)methyl)-2,3-dihydro-4H-pyrido[3,2-b][1,4]oxazine-4-carboxylate (30 mg, 0.05 mmol) in DCM (5 ml), add methanolic hydrochloric acid (4 M, 5 ml) at room temperature, and stir for 1 hour. The reaction solution was directly spin-dried to obtain 25 mg of a yellow solid product. LC-MS: ESI m/z: 545.7 [M+H] + .
中间体19-INTB：甲基(S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.3]庚烷-2-基)丁酸酯的制备
Intermediate 19-INTB: Preparation of methyl (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.3]heptane-2-yl)butanoate
步骤1：参见中间体1-INTB步骤1的制备方法。粗产品经正相硅胶柱色谱法纯化(PE/EA＝1/0到3/7)得到淡黄色油状产品叔-丁基(E)-6-(4-甲氧基-4-羰基丁-2-烯-1-基)-2,6-二氮杂螺[3.3]庚烷-2-羧酸酯650mg。1H NMR(400MHz,CDCl3-d)δ6.82(m,1H)5.93(m,1H)3.99(s,4H)3.74(s,3H)3.33(s,4H)3.17(m,2H)1.43(s,9H)。Step 1: Refer to the preparation method of intermediate 1-INTB step 1. The crude product was purified by normal phase silica gel column chromatography (PE/EA=1/0 to 3/7) to obtain 650 mg of pale yellow oily product tert-butyl (E)-6-(4-methoxy-4-carbonylbut-2-en-1-yl)-2,6-diazaspiro[3.3]heptane-2-carboxylate. 1 H NMR (400 MHz, CDCl 3 -d) δ 6.82 (m, 1H) 5.93 (m, 1H) 3.99 (s, 4H) 3.74 (s, 3H) 3.33 (s, 4H) 3.17 (m, 2H) 1.43 (s, 9H).
步骤2：参见中间体1-INTB步骤4的制备方法。粗产品经正相硅胶柱色谱法纯化(PE/EA＝1/0到0/1)得到黄色油状产品叔-丁基(S)-6-(2-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-甲氧基-4-羰基丁基)-2,6-二氮杂螺[3.3]庚烷-2-羧酸酯160mg。LC-MS:ESI m/z＝469.4[M+H]+.Step 2: Refer to the preparation method of intermediate 1-INTB step 4. The crude product was purified by normal phase silica gel column chromatography (PE/EA=1/0 to 0/1) to obtain 160 mg of yellow oily product tert-butyl (S)-6-(2-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-methoxy-4-carbonylbutyl)-2,6-diazaspiro[3.3]heptane-2-carboxylate. LC-MS: ESI m/z=469.4[M+H] + .
步骤3：参见中间体14-INTB步骤2的制备方法。得到淡黄色油状产品甲基(S)-3-(3- (3,5-二甲基-1H-吡唑-1-基)苯基)-4-(2,6-二氮杂螺[3.3]庚烷-2-基)丁酸酯(19-INTA)250mg。LC-MS:ESI m/z＝369.6[M+H]+.Step 3: Refer to the preparation method of intermediate 14-INTB step 2. A pale yellow oily product methyl (S)-3-(3- (3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(2,6-diazaspiro[3.3]heptane-2-yl)butanoate (19-INTA) 250 mg. LC-MS: ESI m/z=369.6 [M+H] + .
步骤4：参见中间体5-INTB步骤3的制备方法。粗产品经正相硅胶柱色谱法纯化(DCM/MeOH＝1/0到8/2)得到黄色油状产品80mg。LC-MS:ESI m/z＝515.7[M+H]+。Step 4: Refer to the preparation method of intermediate 5-INTB in step 3. The crude product was purified by normal phase silica gel column chromatography (DCM/MeOH=1/0 to 8/2) to obtain 80 mg of a yellow oily product. LC-MS: ESI m/z=515.7 [M+H] + .
中间体20-INTB:甲基(S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(6-(2-(5,6,7,8-四氢-1,8-二氮杂萘-2-基)乙基)-2,6-二氮杂螺[3.3]庚烷-2-基)丁酸酯的制备
Intermediate 20-INTB: Preparation of methyl (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(6-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)-2,6-diazaspiro[3.3]heptane-2-yl)butanoate
步骤1：将中间体J(45mg，104μmol)和19-INTA(50.5mg，114.4μmol)溶于乙腈(5ml)中，然后加入二异丙基乙胺(40.3mg，312.1μmol)和碘化钠(31.2mg，208.1μmol)。反应液在60℃下搅拌16小时。反应液过滤旋干，粗品经正相硅胶柱色谱法纯化(100％DCM～甲醇＝5/1)得到淡黄色油状产品叔-丁基(S)-7-(2-(6-(2-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-甲氧基-4-羰基丁基)-2,6-二氮杂螺[3.3]庚烷-2-基)乙基)-3,4-二氢-1,8-二氮杂萘-1(2H)-羧酸酯150mg。LC-MS:ESI m/z＝629.7[M+H]+。Step 1: Dissolve intermediate J (45 mg, 104 μmol) and 19-INTA (50.5 mg, 114.4 μmol) in acetonitrile (5 ml), then add diisopropylethylamine (40.3 mg, 312.1 μmol) and sodium iodide (31.2 mg, 208.1 μmol). The reaction solution was stirred at 60 ° C for 16 hours. The reaction solution was filtered and dried, and the crude product was purified by normal phase silica gel column chromatography (100% DCM ~ methanol = 5/1) to obtain a light yellow oily product tert-butyl (S)-7-(2-(6-(2-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-methoxy-4-carbonylbutyl)-2,6-diazaspiro[3.3]heptane-2-yl)ethyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylate 150 mg. LC-MS: ESI m/z=629.7 [M+H] + .
步骤2：参见中间体3-INTB步骤2的制备方法。得到黄色油状产品500mg。LC-MS:ESI m/z＝529.7[M+H]+。Step 2: Refer to the preparation method of intermediate 3-INTB step 2. 500 mg of yellow oily product was obtained. LC-MS: ESI m/z=529.7 [M+H] + .
中间体21-INTB:甲基4-(5-溴-1'-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)螺[二氢吲哚-3,4'-哌啶]-1-基)丁酸酯的制备
Intermediate 21-INTB: Preparation of methyl 4-(5-bromo-1'-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)spiro[dihydroindole-3,4'-piperidin]-1-yl)butyrate
步骤1：将叔-丁基4-甲酰基哌啶-1-羧酸酯(5g，23.4mmol)和4-溴苯肼盐酸盐(5.2g，23.4mmol)溶于氯仿(50ml)和乙醇(0.1ml)中。氮气保护下冷却到0℃，缓慢滴加三氟乙酸(8g，70.3mmol)，滴加完后升温到50℃并搅拌16小时。反应液加水(30ml)和羟胺水溶液(10ml)淬灭，DCM萃取(30ml)。将有机相旋干，正相硅胶柱色谱法纯化(PE/EA＝10/1～3/1)得到棕色油状产品叔-丁基5-溴螺[吲哚-3,4'-哌啶]-1'-羧酸酯2.4g。LC-MS:ESI m/z＝365.4[M+H]+。Step 1: Dissolve tert-butyl 4-formylpiperidine-1-carboxylate (5g, 23.4mmol) and 4-bromophenylhydrazine hydrochloride (5.2g, 23.4mmol) in chloroform (50ml) and ethanol (0.1ml). Cool to 0°C under nitrogen protection, slowly add trifluoroacetic acid (8g, 70.3mmol), and heat to 50°C after the addition is complete and stir for 16 hours. The reaction solution is quenched with water (30ml) and hydroxylamine aqueous solution (10ml), and extracted with DCM (30ml). The organic phase is spin-dried and purified by normal phase silica gel column chromatography (PE/EA=10/1~3/1) to obtain 2.4g of brown oily product tert-butyl 5-bromospiro[indole-3,4'-piperidine]-1'-carboxylate. LC-MS:ESI m/z=365.4[M+H] + .
步骤2：将叔-丁基5-溴螺[吲哚-3,4'-哌啶]-1'-羧酸酯(2.4g，6.6mmol)溶于乙醇(30ml)中，在室温下缓慢加入硼氢化钠(1.2g，30.2mmol)。室温下搅拌16小时。将反应液旋干，用水洗(50ml)，DCM(50ml)萃取。有机相旋干，正相硅胶柱色谱法纯化(PE/EA＝10/1～3/1)得到棕色固体产品叔-丁基5-溴螺[二氢吲哚-3,4'-哌啶]-1'-羧酸酯900mg。LC-MS:ESI m/z＝367.1[M+H]+。 Step 2: Dissolve tert-butyl 5-bromospiro[indole-3,4'-piperidine]-1'-carboxylate (2.4 g, 6.6 mmol) in ethanol (30 ml), and slowly add sodium borohydride (1.2 g, 30.2 mmol) at room temperature. Stir at room temperature for 16 hours. The reaction solution was spin-dried, washed with water (50 ml), and extracted with DCM (50 ml). The organic phase was spin-dried and purified by normal phase silica gel column chromatography (PE/EA=10/1~3/1) to obtain tert-butyl 5-bromospiro[dihydroindole-3,4'-piperidine]-1'-carboxylate as a brown solid product 900 mg. LC-MS: ESI m/z=367.1[M+H] + .
步骤3：将叔-丁基5-溴螺[二氢吲哚-3,4'-哌啶]-1'-羧酸酯(750mg，2mmol)和醛溶于1,2-二氯乙烷(10ml)中，室温下滴加2滴乙酸并搅拌30分钟。加入醋酸硼氢化钠(1.3g，6.1mmol)，反应16小时。反应液直接旋干，正相硅胶柱色谱法纯化(PE/EA＝10/1～3/1)得到棕色油状产品叔-丁基5-溴-1-(4-甲氧基-4-羰基丁基)螺[二氢吲哚-3,4'-哌啶]-1'-羧酸酯350mg。LC-MS:ESI m/z＝467.5[M+H]+。Step 3: Dissolve tert-butyl 5-bromospiro[dihydroindole-3,4'-piperidine]-1'-carboxylate (750 mg, 2 mmol) and aldehyde in 1,2-dichloroethane (10 ml), add 2 drops of acetic acid at room temperature and stir for 30 minutes. Add sodium acetate borohydride (1.3 g, 6.1 mmol) and react for 16 hours. The reaction solution is directly spin-dried and purified by normal phase silica gel column chromatography (PE/EA=10/1~3/1) to obtain tert-butyl 5-bromo-1-(4-methoxy-4-carbonylbutyl)spiro[dihydroindole-3,4'-piperidine]-1'-carboxylate 350 mg as a brown oily product. LC-MS: ESI m/z=467.5[M+H] + .
步骤4：参见中间体14-INTB步骤2的制备方法。得到棕色固体产品甲基4-(5-溴螺[二氢吲哚-3,4'-哌啶]-1-基)丁酸酯40mg。LC-MS:ESI m/z＝367.4[M+H]+。Step 4: Refer to the preparation method of intermediate 14-INTB in step 2. 40 mg of methyl 4-(5-bromospiro[dihydroindole-3,4'-piperidin]-1-yl)butanoate was obtained as a brown solid product. LC-MS: ESI m/z=367.4 [M+H] + .
步骤5：参见中间体5-INTB步骤3的制备方法。粗品经硅胶板纯化(DCM/MeOH＝20/1，Rf＝0.4)得到淡黄色油状产品25mg。LC-MS:ESI m/z＝513.5[M+H]+。1H NMR(400MHz,CDCl3)δ＝7.21-7.02(m,3H),6.58(d,J＝7.2Hz,1H),6.29(d,J＝8.3Hz,1H),5.03(br s,1H),3.74-3.61(m,3H),3.50-3.34(m,4H),3.24(s,2H),3.09(t,J＝7.0Hz,2H),2.94-2.83(m,2H),2.71(t,J＝6.2Hz,2H),2.49-2.26(m,4H),2.20-2.08(m,2H),1.94-1.89(m,4H),1.65(br d,J＝13.0Hz,2H)。Step 5: Refer to the preparation method of intermediate 5-INTB in step 3. The crude product was purified by silica gel plate (DCM/MeOH=20/1, Rf=0.4) to obtain 25 mg of a light yellow oily product. LC-MS: ESI m/z=513.5 [M+H] + . 1 H NMR (400 MHz, CDCl3) δ＝7.21-7.02 (m, 3H), 6.58 (d, J＝7.2 Hz, 1H), 6.29 (d, J＝8.3 Hz, 1H), 5.03 (br s, 1H), 3.74-3.61 (m, 3H), 3.50-3.34 (m, 4H), 3.24 (s, 2H), 3.09 (t, J＝7.0 Hz, 2H), 2.94-2.83 (m, 2H), 2.71 (t, J＝6.2 Hz, 2H), 2.49-2.26 (m, 4H), 2.20-2.08 (m, 2H), 1.94-1.89 (m, 4H), 1.65 (br d, J＝13.0 Hz, 2H).
中间体23-INTB：甲基(S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(7-((3-羰基-3,4-二氢-2H-吡啶并[3,2-b][1,4]噁嗪-6-基)甲基)-2,7-二氮杂螺[3.5]壬烷-2-基)丁酸酯的制备
Intermediate 23-INTB: Preparation of methyl (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(7-((3-carbonyl-3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazin-6-yl)methyl)-2,7-diazaspiro[3.5]nonan-2-yl)butanoate
步骤1：参见中间体14-INTB步骤1的制备方法。粗品经硅胶板纯化(DCM/MeOH＝10/1)得到淡黄色固体产品40mg。LC-MS:ESI m/z:559.3.3[M+H]+.Step 1: See the preparation method of intermediate 14-INTB step 1. The crude product was purified by silica gel plate (DCM/MeOH=10/1) to obtain 40 mg of light yellow solid product. LC-MS: ESI m/z: 559.3.3 [M+H] + .
中间体24-INTB：甲基(S)-3-(3-溴-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(7-((5,6,7,8-四氢-1),8-萘啶-2-基)甲基)-2,7-二氮杂螺[3.5]壬-2-基)丁酸酯的制备
Intermediate 24-INTB: Preparation of methyl (S)-3-(3-bromo-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(7-((5,6,7,8-tetrahydro-1),8-naphthyridin-2-yl)methyl)-2,7-diazaspiro[3.5]nonan-2-yl)butanoate
步骤1：参见中间体1-INTB步骤4的制备方法。粗品经由硅胶板纯化(DCM/MeOH＝20/1)后得到黄色油状产品叔-丁基(S)-2-(2-(3-溴-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-甲氧基-4-羰基丁基)-2,7-二氮杂螺[3.5]壬烷-7-羧酸酯45mg。LC-MS:ESI m/z:575.6[M+H]+。Step 1: Refer to the preparation method of intermediate 1-INTB step 4. The crude product was purified by silica gel plate (DCM/MeOH=20/1) to obtain 45 mg of yellow oily product tert-butyl (S)-2-(2-(3-bromo-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-methoxy-4-carbonylbutyl)-2,7-diazaspiro[3.5]nonane-7-carboxylate. LC-MS: ESI m/z: 575.6 [M+H] + .
步骤2：参见中间体14-INTB步骤2的制备方法。得到黄色油状产品(S)-3-(3-溴-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(2,7-二氮杂螺[3.5]壬酸-2-基)丁酸甲酯(24-INTA)40mg。LC-MS:ESI m/z:475.5[M+H]+。Step 2: Refer to the preparation method of intermediate 14-INTB step 2. Obtain 40 mg of yellow oily product (S)-3-(3-bromo-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(2,7-diazaspiro[3.5]nonanoic acid-2-yl)butanoic acid methyl ester (24-INTA). LC-MS: ESI m/z: 475.5 [M+H] + .
步骤3：参见中间体14-INTB步骤1的制备方法。粗品经由柱层析(展开剂(含0.2％TEA)：100％DCM～DCM/MeOH＝95/5)后得到黄色油状产品40mg。LC-MS:ESI m/z:621.6[M+H]+。Step 3: Refer to the preparation method of intermediate 14-INTB in step 1. The crude product was purified by column chromatography (developing solvent (containing 0.2% TEA): 100% DCM to DCM/MeOH = 95/5) to obtain 40 mg of a yellow oily product. LC-MS: ESI m/z: 621.6 [M+H] + .
中间体25-INTB：甲基(S)-3-(3-(叔丁基)-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(2,7-二氮杂螺[3.5]壬烷-2-基)丁酸酯的制备
Intermediate 25-INTB: Preparation of methyl (S)-3-(3-(tert-butyl)-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(2,7-diazaspiro[3.5]nonan-2-yl)butanoate
步骤1：参见中间体1-INTB步骤4的制备方法。粗品经由硅胶板纯化(DCM:甲醇＝20:1)后得到黄色油状产品叔丁基(S)-2-(2-(3-(叔丁基)-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-甲氧基-4-氧代丁基)-2,7-二氮杂螺[3.5]壬烷-7-羧酸酯30mg。LC-MS:ESI m/z:553.8[M+H]+。Step 1: Refer to the preparation method of intermediate 1-INTB step 4. The crude product was purified by silica gel plate (DCM: methanol = 20: 1) to obtain 30 mg of yellow oily product tert-butyl (S)-2-(2-(3-(tert-butyl)-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-methoxy-4-oxobutyl)-2,7-diazaspiro[3.5]nonane-7-carboxylate. LC-MS: ESI m/z: 553.8 [M+H] + .
步骤2：参见中间体14-INTB步骤2的制备方法。得到黄色油状产品甲基(S)-3-(3-(叔丁基)-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(2,7-二氮杂螺[3.5]壬-2-基)丁酸酯(25-INTA)30mg。LC-MS:ESI m/z:453.7[M+H]+。Step 2: Refer to the preparation method of intermediate 14-INTB step 2. 30 mg of methyl (S)-3-(3-(tert-butyl)-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(2,7-diazaspiro[3.5]non-2-yl)butanoate (25-INTA) was obtained as a yellow oily product. LC-MS: ESI m/z: 453.7 [M+H] + .
步骤3：参见中间体14-INTB步骤1的制备方法。粗品经由硅胶板纯化(DCM(0.1％DIEA):甲醇＝10:1)后得到黄色油状产品20mg。LC-MS:ESI m/z:599.8[M+H]+。Step 3: Refer to the preparation method of intermediate 14-INTB in step 1. The crude product was purified by silica gel plate (DCM (0.1% DIEA): methanol = 10:1) to obtain 20 mg of yellow oily product. LC-MS: ESI m/z: 599.8 [M+H] + .
中间体29-INTB：甲基(3S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(7-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)八氢-2,7-二氮杂萘-2(1H)-基)丁酸酯的制备
Intermediate 29-INTB: Preparation of methyl (3S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(7-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)octahydro-2,7-naphthyridin-2(1H)-yl)butyrate
步骤1：将3-溴-4-甲基吡啶(20g,116.3mmol)，钯催化剂(5.3g,5.8mmol)，氰化锌(8.2g,69.8mmol),锌粉(0.8g,11.6mmol)以及DPPF(6.5g,11.6mmol)加入到DMF(200ml)中，置换氮气三次，于氮气氛围下100℃搅拌16小时。反应液过滤，滤液浓缩，经正相硅胶柱色谱法纯化(PE/EA＝10/1)得到黄色固体产品4-甲基尼古丁腈7.4g。LC-MS:ESI m/z＝119.0[M+H]+。Step 1: Add 3-bromo-4-methylpyridine (20 g, 116.3 mmol), palladium catalyst (5.3 g, 5.8 mmol), zinc cyanide (8.2 g, 69.8 mmol), zinc powder (0.8 g, 11.6 mmol) and DPPF (6.5 g, 11.6 mmol) to DMF (200 ml), replace nitrogen three times, and stir at 100 ° C for 16 hours under nitrogen atmosphere. The reaction solution was filtered, the filtrate was concentrated, and purified by normal phase silica gel column chromatography (PE/EA=10/1) to obtain 7.4 g of yellow solid product 4-methylnicotinonitrile. LC-MS: ESI m/z=119.0[M+H] + .
步骤2：将4-甲基尼古丁腈(7.4g,62.6mmol)，碳酸二乙酯(74g,626.4mmol)溶于THF(200ml)，氮气置换三次，0℃下将NaH(12.5g,313.2mmol)加入到反应体系中，60℃搅拌16小时。将反应液加饱和氯化铵(100ml)淬灭，用乙酸乙酯(100ml)萃取。有机相用饱和食盐水洗三次(50ml*3)，有机相干燥，浓缩，正相硅胶柱色谱法纯化(PE/EA＝20/1)得到黄色固体产品乙基2-(3-氰基吡啶-4-基)乙酸酯11g。LC-MS:ESI m/z＝191.1[M+H]+。Step 2: Dissolve 4-methylnicotinonitrile (7.4 g, 62.6 mmol) and diethyl carbonate (74 g, 626.4 mmol) in THF (200 ml), replace with nitrogen three times, add NaH (12.5 g, 313.2 mmol) to the reaction system at 0°C, and stir at 60°C for 16 hours. Quench the reaction solution with saturated ammonium chloride (100 ml) and extract with ethyl acetate (100 ml). Wash the organic phase with saturated brine three times (50 ml*3), dry the organic phase, concentrate, and purify by normal phase silica gel column chromatography (PE/EA=20/1) to obtain 11 g of yellow solid product ethyl 2-(3-cyanopyridin-4-yl) acetate. LC-MS: ESI m/z=191.1[M+H] + .
步骤3：将乙基2-(3-氰基吡啶-4-基)乙酸酯(5g,26.3mmol)，Raney-Ni(5g,26.3mmol)加入到乙醇(100ml)和水(100ml)中，氢气置换三次，于氢气氛围下升温至50℃搅拌16小时。反应液过滤。滤液浓缩，得到黄色油状产品1,4-二氢-2,7-二氮杂萘-3(2H)-酮3.8g。LC-MS:ESI m/z＝149.1[M+H]+。Step 3: Add ethyl 2-(3-cyanopyridin-4-yl)acetate (5 g, 26.3 mmol) and Raney-Ni (5 g, 26.3 mmol) to ethanol (100 ml) and water (100 ml), replace with hydrogen three times, and heat to 50 ° C under hydrogen atmosphere and stir for 16 hours. The reaction solution is filtered. The filtrate is concentrated to obtain 3.8 g of yellow oily product 1,4-dihydro-2,7-diazinaphan-3(2H)-one. LC-MS: ESI m/z＝149.1[M+H] + .
步骤4：将1,4-二氢-2,7-二氮杂萘-3(2H)-酮(3.8g,25.7mmol)溶于THF(300ml)和 DCM(100ml)中，0℃下将硼烷二甲硫醚(10M,25.7ml)加入到反应体系中，反应升温至80℃搅拌16小时。反应液于-78℃下加甲醇淬灭，并于室温下搅拌半小时。将盐酸甲醇(4M,200ml)加入到反应液中，并室温搅拌16小时。反应液浓缩得到黄色油状产品1,2,3,4-四氢-2,7-二氮杂萘3.4g。LC-MS:ESI m/z＝135.1[M+H]+。Step 4: Dissolve 1,4-dihydro-2,7-naphthyridin-3(2H)-one (3.8 g, 25.7 mmol) in THF (300 ml) and In DCM (100ml), borane dimethyl sulfide (10M, 25.7ml) was added to the reaction system at 0℃, and the reaction temperature was raised to 80℃ and stirred for 16 hours. The reaction solution was quenched by adding methanol at -78℃ and stirred at room temperature for half an hour. Methanol hydrochloride (4M, 200ml) was added to the reaction solution and stirred at room temperature for 16 hours. The reaction solution was concentrated to obtain 3.4g of yellow oily product 1,2,3,4-tetrahydro-2,7-naphthyridine. LC-MS: ESI m/z＝135.1[M+H] + .
步骤5：将1,2,3,4-四氢-2,7-二氮杂萘(3.4g,25.3mmol),(Boc)2O(8.3g，38mmol),TEA(7.7g，76mmol)和DMAP(309.6mg，2.5mmol)溶于DCM(50ml)中，室温搅拌16小时。将反应液加水(20ml)淬灭，用DCM(20ml)萃取。有机相用饱和食盐水洗三次(10ml*3)，有机相干燥，浓缩，正相硅胶柱色谱法纯化(DCM/MeOH＝20/1)得到黄色油状产品叔-丁基3,4-二氢-2,7-二氮杂萘-2(1H)-羧酸酯1.4g。LC-MS:ES m/z＝235.3[M+H]+。Step 5: Dissolve 1,2,3,4-tetrahydro-2,7-naphthyridine (3.4 g, 25.3 mmol), (Boc) 2 O (8.3 g, 38 mmol), TEA (7.7 g, 76 mmol) and DMAP (309.6 mg, 2.5 mmol) in DCM (50 ml) and stir at room temperature for 16 hours. Quench the reaction solution with water (20 ml) and extract with DCM (20 ml). Wash the organic phase with saturated brine three times (10 ml*3), dry the organic phase, concentrate, and purify by normal phase silica gel column chromatography (DCM/MeOH=20/1) to obtain 1.4 g of yellow oily product tert-butyl 3,4-dihydro-2,7-naphthyridine-2(1H)-carboxylate. LC-MS: ES m/z=235.3[M+H] + .
步骤6：将叔-丁基3,4-二氢-2,7-二氮杂萘-2(1H)-羧酸酯(1.4g,6.0mmol),氢氧化钯(557.6mg，0.8mmol)溶于醋酸(100ml)中，氢气置换三次，于氢气氛围下升温至80℃搅拌12小时。将反应液过滤，滤液浓缩后加水(20ml)，用氨水将pH调到9，并用DCM(20ml)萃取。有机相用饱和食盐水洗三次(10ml*3)，有机相干燥，浓缩得到淡黄色油状产品叔-丁基八氢-2,7-二氮杂萘-2(1H)-羧酸酯800mg。LC-MS:ESI m/z＝241.2[M+H]+。Step 6: Dissolve tert-butyl 3,4-dihydro-2,7-naphthyridine-2(1H)-carboxylate (1.4g, 6.0mmol) and palladium hydroxide (557.6mg, 0.8mmol) in acetic acid (100ml), replace with hydrogen three times, heat to 80°C and stir for 12 hours under hydrogen atmosphere. Filter the reaction solution, add water (20ml) after concentrating the filtrate, adjust the pH to 9 with ammonia water, and extract with DCM (20ml). Wash the organic phase with saturated brine three times (10ml*3), dry the organic phase, and concentrate to obtain 800mg of tert-butyl octahydro-2,7-naphthyridine-2(1H)-carboxylate as a light yellow oily product. LC-MS: ESI m/z＝241.2[M+H] + .
步骤7：将叔-丁基八氢-2,7-二氮杂萘-2(1H)-羧酸酯(800mg,3.3mmol)，碳酸钾(1.4g,4.4mmol)和4-溴巴豆酸甲酯(595mg,3.3mmol)溶于乙腈(15ml)中，室温搅拌8小时。将反应液浓缩，正相硅胶柱色谱法纯化(DCM/MeOH＝10/1)得到黄色油状产品叔-丁基(E)-7-(4-甲氧基-4-羰基丁-2-烯-1-基)八氢-2,7-二氮杂萘-2(1H)-羧酸酯1g。LC-MS:ESI m/z＝339.5[M+H]+；1H NMR(400MHz,CDCl3)δppm 6.84-7.03(m,1H)5.97(br d,J＝15.53Hz,1H)3.46-3.80(m,5H)2.90-3.36(m,4H)2.37(br s,2H)1.84(br s,2H)1.49-1.78(m,6H)1.45(br s,9H)。Step 7: Dissolve tert-butyl octahydro-2,7-naphthyridine-2(1H)-carboxylate (800 mg, 3.3 mmol), potassium carbonate (1.4 g, 4.4 mmol) and methyl 4-bromocrotonate (595 mg, 3.3 mmol) in acetonitrile (15 ml) and stir at room temperature for 8 hours. Concentrate the reaction solution and purify by normal phase silica gel column chromatography (DCM/MeOH=10/1) to obtain 1 g of yellow oily product tert-butyl (E)-7-(4-methoxy-4-carbonylbut-2-en-1-yl)octahydro-2,7-naphthyridine-2(1H)-carboxylate. LC-MS: ESI m/z=339.5 [M+H] + ; 1 H NMR (400 MHz, CDCl 3 ) δ ppm 6.84-7.03 (m, 1H) 5.97 (br d, J=15.53 Hz, 1H) 3.46-3.80 (m, 5H) 2.90-3.36 (m, 4H) 2.37 (br s, 2H) 1.84 (br s, 2H) 1.49-1.78 (m, 6H) 1.45 (br s, 9H).
步骤8：参见中间体1-INTB步骤4的制备方法。粗品经正相硅胶柱色谱法纯化(DCM/MeOH＝10/1)纯化得到黄色固体产品叔-丁基7-((S)-2-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-甲氧基-4-羰基丁基)八氢-2,7-二氮杂萘-2(1H)-羧酸酯115mg。LC-MS:ESI m/z＝511.7[M+H]+。Step 8: Refer to the preparation method of intermediate 1-INTB in step 4. The crude product was purified by normal phase silica gel column chromatography (DCM/MeOH=10/1) to obtain 115 mg of yellow solid product tert-butyl 7-((S)-2-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-methoxy-4-carbonylbutyl)octahydro-2,7-naphthyridine-2(1H)-carboxylate. LC-MS: ESI m/z=511.7 [M+H] + .
步骤9：参见中间体14-INTB步骤2的制备方法。得到黄色固体产品甲基(3S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(八氢-2,7-二氮杂萘-2(1H)-基)丁酸酯(29-INTA)80mg。Step 9: Refer to the preparation method of intermediate 14-INTB in step 2. 80 mg of yellow solid product methyl (3S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(octahydro-2,7-naphthyridin-2(1H)-yl)butanoate (29-INTA) was obtained.
步骤10：参见中间体5-INTB步骤3的制备方法。粗品经正相硅胶柱色谱法纯化(DCM/MeOH＝10/1)得到黄色固体产品40mg。LC-MS:ESI m/z＝557.5[M+H]+。Step 10: Refer to the preparation method of intermediate 5-INTB in step 3. The crude product was purified by normal phase silica gel column chromatography (DCM/MeOH=10/1) to obtain 40 mg of a yellow solid product. LC-MS: ESI m/z=557.5 [M+H] + .
中间体30-INTB：甲基(S)-4-(2-((3,4-二氢-2H-吡啶并[3,2-b][1,4]噁嗪-6-基)甲基)-2,6-二氮杂螺[3.4]辛烷-6-基)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)丁酸酯的制备
Intermediate 30-INTB: Preparation of methyl (S)-4-(2-((3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazin-6-yl)methyl)-2,6-diazaspiro[3.4]octan-6-yl)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)butyrate
步骤1：参见中间体3-INTB步骤3的制备方法。粗品经硅胶柱层析纯化(DCM/MeOH＝15/1)得浅黄色油状产品叔-丁基(E)-6-(4-甲氧基-4-羰基丁-2-烯-1-基)-2,6-二氮杂螺[3.4]辛烷- 2-羧酸酯170mg。LC-MS:ESI m/z:311.2[M+H]+。Step 1: Refer to the preparation method of intermediate 3-INTB step 3. The crude product was purified by silica gel column chromatography (DCM/MeOH=15/1) to obtain a light yellow oily product tert-butyl (E)-6-(4-methoxy-4-carbonylbut-2-en-1-yl)-2,6-diazaspiro[3.4]octane- 2-Carboxylate 170 mg. LC-MS: ESI m/z: 311.2 [M+H] + .
步骤2：参见中间体1-INTB步骤4的制备方法。粗品经正相硅胶柱色谱法纯化(DCM/MeOH＝10/1)纯化得到黄色固体产品叔-丁基(S)-6-(2-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-甲氧基-4-羰基丁基)-2,6-二氮杂螺[3.4]辛烷-2-羧酸酯185mg。LC-MS:ESI m/z:483.3[M+H]+。Step 2: Refer to the preparation method of intermediate 1-INTB step 4. The crude product was purified by normal phase silica gel column chromatography (DCM/MeOH=10/1) to obtain 185 mg of yellow solid product tert-butyl (S)-6-(2-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-methoxy-4-carbonylbutyl)-2,6-diazaspiro[3.4]octane-2-carboxylate. LC-MS: ESI m/z: 483.3 [M+H] + .
步骤3：参见中间体14-INTB步骤2的制备方法。反应液直接浓缩得到黄色油状产品甲基(S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(2,6-二氮杂螺[3.4]辛烷-6-基)丁酸酯(30-INTA)150mg。Step 3: Refer to the preparation method of intermediate 14-INTB in step 2. The reaction solution was directly concentrated to obtain 150 mg of yellow oily product methyl (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(2,6-diazaspiro[3.4]octan-6-yl)butanoate (30-INTA).
步骤4：参见中间体5-INTB步骤3的制备方法。粗品经正相硅胶柱色谱法纯化(DCM/MeOH＝10/1)得到黄色油状产品叔-丁基(S)-6-((6-(2-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-甲氧基-4-羰基丁基)-2,6-二氮杂螺[3.4]辛烷-2-基)甲基)-2,3-二氢-4H-吡啶并[3,2-b][1,4]噁嗪-4-羧酸酯154mg。LC-MS:ESI m/z＝631.7[M+H]+。Step 4: Refer to the preparation method of intermediate 5-INTB in step 3. The crude product was purified by normal phase silica gel column chromatography (DCM/MeOH=10/1) to obtain 154 mg of yellow oily product tert-butyl (S)-6-((6-(2-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-methoxy-4-carbonylbutyl)-2,6-diazaspiro[3.4]octan-2-yl)methyl)-2,3-dihydro-4H-pyrido[3,2-b][1,4]oxazine-4-carboxylate. LC-MS: ESI m/z=631.7[M+H] + .
步骤5：参见中间体14-INTB步骤2的制备方法。得到黄色油状产品120mg。LC-MS:ESI m/z＝531.6[M+H]+。Step 5: Refer to the preparation method of intermediate 14-INTB in step 2. 120 mg of yellow oily product was obtained. LC-MS: ESI m/z=531.6 [M+H] + .
中间体31-INTB：甲基(S)-4-(6-((3,4-二氢-2H-吡啶并[3,2-b][1,4]噁嗪-6-基)甲基)-2,6-二氮杂螺[3.4]辛烷-2-基)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)丁酸酯的制备
Intermediate 31-INTB: Preparation of methyl (S)-4-(6-((3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazin-6-yl)methyl)-2,6-diazaspiro[3.4]octan-2-yl)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)butyrate
步骤1：参见中间体12-INTB步骤1的制备方法，其中31-INTA的制备参见30-INTA的制备。粗品经正相硅胶柱色谱法纯化(100％DCM～甲醇＝4/1)得到黄色油状产品叔-丁基(S)-6-((2-(2-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-甲氧基-4-羰基丁基)-2,6-二氮杂螺[3.4]辛烷-6-基)甲基)-2,3-二氢-4H-吡啶并[3,2-b][1,4]噁嗪-4-羧酸酯160mg。LC-MS:ESI m/z＝631.7[M+H]+。Step 1: Refer to the preparation method of step 1 of intermediate 12-INTB, wherein the preparation of 31-INTA refers to the preparation of 30-INTA. The crude product was purified by normal phase silica gel column chromatography (100% DCM-methanol = 4/1) to obtain 160 mg of yellow oily product tert-butyl (S)-6-((2-(2-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-methoxy-4-carbonylbutyl)-2,6-diazaspiro[3.4]octan-6-yl)methyl)-2,3-dihydro-4H-pyrido[3,2-b][1,4]oxazine-4-carboxylate. LC-MS: ESI m/z = 631.7 [M+H] + .
步骤2：参见中间体14-INTB步骤2的制备方法。得到黄色油状产品200mg。LC-MS:ESI m/z＝531.6[M+H]+。Step 2: Refer to the preparation method of intermediate 14-INTB step 2. 200 mg of yellow oily product was obtained. LC-MS: ESI m/z=531.6 [M+H] + .
中间体32-INTB：甲基(3S)-4-(7-((3,4-二氢-2H-吡啶并[3,2-b][1,4]噁嗪-6-基)甲基)-2,7-二氮杂螺[4.4]壬烷-2-基)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)丁酸酯的制备
Intermediate 32-INTB: Preparation of methyl (3S)-4-(7-((3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazin-6-yl)methyl)-2,7-diazaspiro[4.4]nonan-2-yl)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)butyrate
步骤1：参见中间体12-INTB步骤1的制备方法，其中32-INTA的制备参见30-INTA的制备。粗品经正相硅胶柱色谱法纯化(DCM/甲醇＝4/1)得到黄色油状产品叔-丁基6-((7-((S)-2-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-甲氧基-4-羰基丁基)-2,7-二氮杂螺[4.4]壬烷-2-基)甲基)-2,3-二氢-4H-吡啶并[3,2-b][1,4]噁嗪-4-羧酸酯150mg。LC-MS:ESI m/z＝645.7[M+H]+。 Step 1: Refer to the preparation method of step 1 of intermediate 12-INTB, wherein the preparation of 32-INTA refers to the preparation of 30-INTA. The crude product was purified by normal phase silica gel column chromatography (DCM/methanol=4/1) to obtain 150 mg of yellow oily product tert-butyl 6-((7-((S)-2-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-methoxy-4-carbonylbutyl)-2,7-diazaspiro[4.4]nonane-2-yl)methyl)-2,3-dihydro-4H-pyrido[3,2-b][1,4]oxazine-4-carboxylate. LC-MS: ESI m/z=645.7[M+H] + .
步骤2：参见中间体14-INTB步骤2的制备方法。得到黄色油状产品150mg。LC-MS:ESI m/z＝545.7[M+H]+。Step 2: Refer to the preparation method of intermediate 14-INTB step 2. 150 mg of yellow oily product was obtained. LC-MS: ESI m/z=545.7 [M+H] + .
中间体34-INTB：甲基(S)-4-(2-((1H-吡咯并[2,3-b]吡啶-6-基)甲基)-2,6-二氮杂螺[3.4]辛烷-6-基)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)丁酸酯的制备
Intermediate 34-INTB: Preparation of methyl (S)-4-(2-((1H-pyrrolo[2,3-b]pyridin-6-yl)methyl)-2,6-diazaspiro[3.4]octan-6-yl)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)butyrate
步骤1：参见中间体5-INTB步骤3的制备方法。粗品经正相硅胶柱色谱法纯化(DCM/MeOH＝10/1)得到黄色固体产品85mg。LC-MS:ESI m/z＝513.1[M+H]+。Step 1: Refer to the preparation method of intermediate 5-INTB in step 3. The crude product was purified by normal phase silica gel column chromatography (DCM/MeOH=10/1) to obtain 85 mg of a yellow solid product. LC-MS: ESI m/z=513.1 [M+H] + .
中间体35-INTB：甲基(S)-4-(6-((1H-吡咯并[2,3-b]吡啶-6-基)甲基)-2,6-二氮杂螺[3.4]辛烷-2-基)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)丁酸酯的制备
Intermediate 35-INTB: Preparation of methyl (S)-4-(6-((1H-pyrrolo[2,3-b]pyridin-6-yl)methyl)-2,6-diazaspiro[3.4]octan-2-yl)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)butyrate
步骤1：参见中间体5-INTB步骤3的制备方法。粗产品经正相硅胶柱色谱法纯化(DCM/MeOH＝1/0到8/2)得到黄色油状产品90mg。LC-MS:ESI m/z＝513.6[M+H]+。Step 1: Refer to the preparation method of intermediate 5-INTB in step 3. The crude product was purified by normal phase silica gel column chromatography (DCM/MeOH=1/0 to 8/2) to obtain 90 mg of a yellow oily product. LC-MS: ESI m/z=513.6 [M+H] + .
中间体36-INTB：甲基(3S)-4-(7-((1H-吡咯并[2,3-b]吡啶-6-基)甲基)-2,7-二氮杂螺[4.4]壬烷-2-基)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)丁酸酯的制备
Intermediate 36-INTB: Preparation of methyl (3S)-4-(7-((1H-pyrrolo[2,3-b]pyridin-6-yl)methyl)-2,7-diazaspiro[4.4]nonan-2-yl)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)butyrate
步骤1：参见中间体5-INTB步骤3的制备方法。粗产品经正相硅胶柱色谱法纯化(DCM/MeOH＝1/0到8/2)得到黄色油状产品110mg。LC-MS:ESI m/z＝527.7[M+H]+。Step 1: Refer to the preparation method of intermediate 5-INTB in step 3. The crude product was purified by normal phase silica gel column chromatography (DCM/MeOH=1/0 to 8/2) to obtain 110 mg of a yellow oily product. LC-MS: ESI m/z=527.7 [M+H] + .
中间体37-INTB：甲基(S)-4-(2-((1H-吡咯并[2,3-b]吡啶-6-基)甲基)-2,7-二氮杂螺[3.5]壬-7-基)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)丁酸酯的制备
Intermediate 37-INTB: Preparation of methyl (S)-4-(2-((1H-pyrrolo[2,3-b]pyridin-6-yl)methyl)-2,7-diazaspiro[3.5]nonan-7-yl)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)butyrate
步骤1：参见中间体1-INTB步骤1的制备方法。粗品经柱层析(DCM/MeOH＝95/5)后得到黄色液体产品(E)-7-(4-甲氧基-4-氧代丁-2-烯-1-基)-2,7-二氮杂螺[3.5]壬烷-2-羧酸叔丁酯400mg。LC-MS:ESI m/z:324.7[M+H]+。Step 1: Refer to the preparation method of intermediate 1-INTB step 1. The crude product was purified by column chromatography (DCM/MeOH=95/5) to obtain 400 mg of yellow liquid product (E)-7-(4-methoxy-4-oxobut-2-en-1-yl)-2,7-diazaspiro[3.5]nonane-2-carboxylic acid tert-butyl ester. LC-MS: ESI m/z: 324.7 [M+H] + .
步骤2：参见中间体1-INTB步骤4的制备方法。粗品经柱层析(DCM/MeOH＝95/5)后得到黄色油状产品叔丁基(S)-7-(2-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-甲氧基-4-氧丁基)-2,7-二氮杂螺[3.5]壬烷-2-羧酸酯50mg。LC-MS:ESI m/z:497.7[M+H]+。Step 2: Refer to the preparation method of intermediate 1-INTB step 4. The crude product was purified by column chromatography (DCM/MeOH=95/5) to obtain 50 mg of yellow oily product tert-butyl (S)-7-(2-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-methoxy-4-oxobutyl)-2,7-diazaspiro[3.5]nonane-2-carboxylate. LC-MS: ESI m/z: 497.7 [M+H] + .
步骤3：参见中间体14-INTB步骤2的制备方法。反应液旋干得到黄色油状产品(S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(2,7-二氮杂螺[3.5]壬酸-7-基)丁酸甲酯(37-INTA)40mg。LC-MS:ESI m/z:397.6[M+H]+。Step 3: Refer to the preparation method of intermediate 14-INTB in step 2. The reaction solution was spin-dried to obtain 40 mg of a yellow oily product (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(2,7-diazaspiro[3.5]nonanoic acid-7-yl)butanoic acid methyl ester (37-INTA). LC-MS: ESI m/z: 397.6 [M+H] + .
步骤4：参见中间体5-INTB步骤3的制备方法。粗品经柱层析(DCM/MeOH＝95/5)后得到黄色油状产品50mg。LC-MS:ESI m/z:527.7[M+H]+。Step 4: Refer to the preparation method of intermediate 5-INTB in step 3. The crude product was purified by column chromatography (DCM/MeOH=95/5) to obtain 50 mg of a yellow oily product. LC-MS: ESI m/z: 527.7 [M+H] + .
中间体38-INTB：甲基(S)-3-(3-溴-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.3]庚烷-2-基)丁酸酯的制备
Intermediate 38-INTB: Preparation of methyl (S)-3-(3-bromo-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.3]heptane-2-yl)butanoate
步骤1：参见中间体5-INTB步骤3的制备方法，其中38-INTA的制备参见19-INTA的制备。粗产品经正相硅胶柱色谱法纯化(DCM/MeOH＝8/2)得到黄色油状产品80mg。LC-MS:ESI m/z＝593.6[M+H]+。Step 1: Refer to the preparation method of step 3 of intermediate 5-INTB, wherein the preparation of 38-INTA refers to the preparation of 19-INTA. The crude product was purified by normal phase silica gel column chromatography (DCM/MeOH=8/2) to obtain 80 mg of a yellow oily product. LC-MS: ESI m/z=593.6 [M+H] + .
中间体39-INTB：甲基(S)-3-(3-(叔-丁基)-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.3]庚烷-2-基)丁酸酯的制备
Intermediate 39-INTB: Preparation of methyl (S)-3-(3-(tert-butyl)-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.3]heptane-2-yl)butanoate
步骤1：参见中间体5-INTB步骤3的制备方法，其中39-INTA的制备参见19-INTA的制备。粗产品经正相硅胶柱色谱法纯化(DCM/MeOH＝1/0到8/2)得到黄色油状产品50mg。LC-MS:ESI m/z＝570.8[M+H]+。Step 1: Refer to the preparation method of step 3 of intermediate 5-INTB, wherein the preparation of 39-INTA refers to the preparation of 19-INTA. The crude product was purified by normal phase silica gel column chromatography (DCM/MeOH=1/0 to 8/2) to obtain 50 mg of a yellow oily product. LC-MS: ESI m/z=570.8 [M+H] + .
中间体40-INTB：甲基(S)-3-(3-(叔-丁基)-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(6-((3,4-二氢-2H-吡啶并[3,2-b][1,4]噁嗪-6-基)甲基)-2,6-二氮杂螺[3.3]庚烷-2-基)丁酸酯的制备
Intermediate 40-INTB: Preparation of methyl (S)-3-(3-(tert-butyl)-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(6-((3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazin-6-yl)methyl)-2,6-diazaspiro[3.3]heptane-2-yl)butanoate
步骤1：参见中间体5-INTB步骤3的制备方法。粗产品经正相硅胶柱色谱法纯化 (DCM/MeOH＝1/0到8/2)得到黄色油状产品叔-丁基(S)-6-((6-(2-(3-(叔-丁基)-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-甲氧基-4-羰基丁基)-2,6-二氮杂螺[3.3]庚烷-2-基)甲基)-2,3-二氢-4H-吡啶并[3,2-b][1,4]噁嗪-4-羧酸酯80mg。LC-MS:ESI m/z＝672.9[M+H]+。Step 1: See the preparation method of intermediate 5-INTB step 3. The crude product was purified by normal phase silica gel column chromatography (DCM/MeOH=1/0 to 8/2) to obtain 80 mg of a yellow oily product, tert-butyl (S)-6-((6-(2-(3-(tert-butyl)-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-methoxy-4-carbonylbutyl)-2,6-diazaspiro[3.3]heptan-2-yl)methyl)-2,3-dihydro-4H-pyrido[3,2-b][1,4]oxazine-4-carboxylate. LC-MS: ESI m/z=672.9 [M+H] + .
步骤2：参见中间体14-INTB步骤2的制备方法。得到淡黄色油状产品250mg。LC-MS:ESI m/z＝572.8[M+H]+。Step 2: Refer to the preparation method of intermediate 14-INTB step 2. Obtain 250 mg of a light yellow oily product. LC-MS: ESI m/z = 572.8 [M+H] + .
中间体41-INTB：甲基(S)-4-(6-((1H-吡咯并[2,3-b]吡啶-6-基)甲基)-2,6-二氮杂螺[3.3]庚烷-2-基)-3-(3-(叔-丁基)-5-(3,5-二甲基-1H-吡唑-1-基)苯基)丁酸酯的制备
Intermediate 41-INTB: Preparation of methyl (S)-4-(6-((1H-pyrrolo[2,3-b]pyridin-6-yl)methyl)-2,6-diazaspiro[3.3]heptane-2-yl)-3-(3-(tert-butyl)-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)butyrate
步骤1：参见5-INTB步骤3的制备方法。粗品经柱层析(DCM/MeOH＝95/5)后得到黄色油状产品50mg。LC-MS:ESI m/z:554.7[M+H]+。Step 1: Refer to the preparation method of 5-INTB step 3. The crude product was purified by column chromatography (DCM/MeOH=95/5) to obtain 50 mg of a yellow oily product. LC-MS: ESI m/z: 554.7 [M+H] + .
中间体42-INTB：甲基(S)-3-(3-溴-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯的制备
Intermediate 42-INTB: Preparation of methyl (S)-3-(3-bromo-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-2-yl)butanoate
步骤1：参见中间体1-INTB步骤4的制备方法。反应液浓缩，正相硅胶柱色谱法纯化(DCM/MeOH＝10/1)得到黄色油状产品叔-丁基(S)-2-(2-(3-溴-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-甲氧基-4-羰基丁基)-2,6-二氮杂螺[3.4]辛烷-6-羧酸酯60mg。LC-MS:ESI m/z＝563.5[M+2H]+。Step 1: Refer to the preparation method of intermediate 1-INTB step 4. The reaction solution was concentrated and purified by normal phase silica gel column chromatography (DCM/MeOH=10/1) to obtain 60 mg of yellow oily product tert-butyl (S)-2-(2-(3-bromo-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-methoxy-4-carbonylbutyl)-2,6-diazaspiro[3.4]octane-6-carboxylate. LC-MS: ESI m/z=563.5[M+2H] + .
步骤2：参见中间体14-INTB步骤2的制备方法。得到黄色油状产品甲基(S)-3-(3-溴-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯(42-INTA)30mg。LC-MS:ESI m/z＝461.3[M+H]+。Step 2: Refer to the preparation method of intermediate 14-INTB step 2. 30 mg of methyl (S)-3-(3-bromo-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(2,6-diazaspiro[3.4]octan-2-yl)butanoate (42-INTA) was obtained as a yellow oily product. LC-MS: ESI m/z=461.3 [M+H] + .
步骤3：参见中间体5-INTB步骤3的制备方法。粗品经高效液相色谱纯化得到无色油状产品15mg。LC-MS:ESI m/z＝607.5[M+H]+。Step 3: See the preparation method of intermediate 5-INTB in step 3. The crude product was purified by HPLC to obtain 15 mg of a colorless oily product. LC-MS: ESI m/z = 607.5 [M+H] + .
中间体43-INTB：甲基(S)-3-(3-(叔-丁基)-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯的制备
Intermediate 43-INTB: Preparation of methyl (S)-3-(3-(tert-butyl)-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-2-yl)butanoate
步骤1：参见中间体1-INTB步骤4的制备方法。粗产品用正相硅胶柱色谱法纯化 (100％DCM～DCM/MeOH＝10/1)得到黄色油状产品苯甲基(S)-2-(2-(3-(叔-丁基)-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-甲氧基-4-羰基丁基)-2,6-二氮杂螺[3.4]辛烷-6-羧酸酯750mg。LC-MS:ESI m/z＝575.2[M+H]+。Step 1: See the preparation method of intermediate 1-INTB step 4. The crude product was purified by normal phase silica gel column chromatography (100% DCM to DCM/MeOH=10/1) to obtain 750 mg of yellow oily product benzyl (S)-2-(2-(3-(tert-butyl)-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-methoxy-4-carbonylbutyl)-2,6-diazaspiro[3.4]octane-6-carboxylate. LC-MS: ESI m/z=575.2 [M+H] + .
步骤2：参见中间体2-INTB步骤5的制备方法。将反应液过滤浓缩，得到粗产品甲基(S)-3-(3-(叔-丁基)-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯(43-INTA)520mg。LC-MS:ESI m/z＝439.6[M+H]+。Step 2: Refer to the preparation method of intermediate 2-INTB step 5. The reaction solution was filtered and concentrated to obtain 520 mg of crude product methyl (S)-3-(3-(tert-butyl)-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(2,6-diazaspiro[3.4]octan-2-yl)butanoate (43-INTA). LC-MS: ESI m/z=439.6[M+H] + .
步骤3：参见中间体5-INTB步骤3的制备方法。粗品经高效液相色谱纯化得到黄色油状产品210mg。LC-MS:ESI m/z＝585.5[M+H]+。Step 3: See the preparation method of intermediate 5-INTB in step 3. The crude product was purified by HPLC to obtain 210 mg of a yellow oily product. LC-MS: ESI m/z = 585.5 [M+H] + .
中间体44-INTB：甲基(S)-3-(5-(3,5-二甲基-1H-吡唑-1-基)吡啶-3-基)-4-(7-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,7-二氮杂螺[3.5]壬烷-2-基)丁酸酯的制备
Intermediate 44-INTB: Preparation of methyl (S)-3-(5-(3,5-dimethyl-1H-pyrazol-1-yl)pyridin-3-yl)-4-(7-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,7-diazaspiro[3.5]nonan-2-yl)butanoate
步骤1：参见中间体1-INTB步骤4的制备方法。粗品经正相硅胶柱色谱法纯化(DCM/甲醇＝4/1)得到淡黄色油状产品叔-丁基(S)-2-(2-(5-溴吡啶-3-基)-4-甲氧基-4-羰基丁基)-2,7-二氮杂螺[3.5]壬烷-7-羧酸酯150mg。LC-MS:ESI m/z＝484.4[M+H]+。Step 1: Refer to the preparation method of intermediate 1-INTB step 4. The crude product was purified by normal phase silica gel column chromatography (DCM/methanol=4/1) to obtain 150 mg of light yellow oily product tert-butyl (S)-2-(2-(5-bromopyridin-3-yl)-4-methoxy-4-carbonylbutyl)-2,7-diazaspiro[3.5]nonane-7-carboxylate. LC-MS: ESI m/z=484.4[M+H] + .
步骤2：依次将叔-丁基(S)-2-(2-(5-溴吡啶-3-基)-4-甲氧基-4-羰基丁基)-2,7-二氮杂螺[3.5]壬烷-7-羧酸酯(65mg，134.7μmol)，3,5-二甲基-1H-吡唑(19.4mg，202.1μmol)，甲磺酸-2-(二叔丁基膦基)-3,6-二甲氧基-2,4,6-三异丙基-1,1-联苯(2-氨基-1,1-联苯-2-基)钯(II)(11.5mg)和碳酸铯(109.8mg，336.9μmol)溶于1,4-二氧六环(4ml)中，氮气保护下加热到110℃并搅拌16小时。反应液直接过滤，旋干，粗产品经高效液相色谱分离得到淡黄色固体产品叔-丁基(S)-2-(2-(5-(3,5-二甲基-1H-吡唑-1-基)吡啶-3-基)-4-甲氧基-4-羰基丁基)-2,7-二氮杂螺[3.5]壬烷-7-羧酸酯20mg。LC-MS:ESI m/z＝498.5[M+H]+。Step 2: Dissolve tert-butyl (S)-2-(2-(5-bromopyridin-3-yl)-4-methoxy-4-carbonylbutyl)-2,7-diazaspiro[3.5]nonane-7-carboxylate (65 mg, 134.7 μmol), 3,5-dimethyl-1H-pyrazole (19.4 mg, 202.1 μmol), methanesulfonic acid-2-(di-tert-butylphosphino)-3,6-dimethoxy-2,4,6-triisopropyl-1,1-biphenyl(2-amino-1,1-biphenyl-2-yl)palladium(II) (11.5 mg) and cesium carbonate (109.8 mg, 336.9 μmol) in 1,4-dioxane (4 ml) in sequence, heat to 110°C under nitrogen protection and stir for 16 hours. The reaction solution was directly filtered and dried in a rotary oven. The crude product was separated by high performance liquid chromatography to obtain 20 mg of a light yellow solid product, tert-butyl (S)-2-(2-(5-(3,5-dimethyl-1H-pyrazol-1-yl)pyridin-3-yl)-4-methoxy-4-carbonylbutyl)-2,7-diazaspiro[3.5]nonane-7-carboxylate. LC-MS: ESI m/z=498.5[M+H] + .
步骤3：参见中间体14-INTB步骤2的制备方法。得到淡黄色油状产品甲基(S)-3-(5-(3,5-二甲基-1H-吡唑-1-基)吡啶-3-基)-4-(2,7-二氮杂螺[3.5]壬烷-2-基)丁酸酯(44-INTA)20mg。LC-MS:ESI m/z＝398.5[M+H]+。Step 3: Refer to the preparation method of intermediate 14-INTB in step 2. 20 mg of methyl (S)-3-(5-(3,5-dimethyl-1H-pyrazol-1-yl)pyridin-3-yl)-4-(2,7-diazaspiro[3.5]nonane-2-yl)butanoate (44-INTA) was obtained as a light yellow oily product. LC-MS: ESI m/z=398.5 [M+H] + .
步骤4：参见中间体5-INTB步骤3的制备方法。粗产品经正相硅胶柱色谱法纯化(100％DCM～甲醇＝7/3)得到淡黄色油状产品20mg。LC-MS:ESI m/z＝544.3[M+H]+。Step 4: Refer to the preparation method of intermediate 5-INTB in step 3. The crude product was purified by normal phase silica gel column chromatography (100% DCM-methanol = 7/3) to obtain 20 mg of a light yellow oily product. LC-MS: ESI m/z = 544.3 [M+H] + .
中间体45-INTB：甲基(S)-4-(7-((1H-吡咯并[2,3-b]吡啶-6-基)甲基)-2,7-二氮杂螺[3.5]壬烷-2-基)-3-(3-溴-5-(3,5-二甲基-1H-吡唑-1-基)苯基)丁酸酯的制备
Intermediate 45-INTB: Preparation of methyl (S)-4-(7-((1H-pyrrolo[2,3-b]pyridin-6-yl)methyl)-2,7-diazaspiro[3.5]nonan-2-yl)-3-(3-bromo-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)butyrate
步骤1：参见中间体5-INTB步骤3的制备方法，其中24-INTA的制备同6-INTA。粗品经柱层析(DCM/MeOH＝95/5)后得到黄色油状产品50mg。LC-MS:ESI m/z:605.6[M+H]+。Step 1: Refer to the preparation method of step 3 of intermediate 5-INTB, wherein the preparation of 24-INTA is the same as 6-INTA. The crude product was purified by column chromatography (DCM/MeOH=95/5) to obtain 50 mg of a yellow oily product. LC-MS: ESI m/z: 605.6 [M+H] + .
中间体46-INTB：甲基(S)-4-(7-((1H-吡咯并[2,3-b]吡啶-6-基)甲基)-2,7-二氮杂螺[3.5]壬烷-2-基)-3-(3-(叔-丁基)-5-(3,5-二甲基-1H-吡唑-1-基)苯基)丁酸酯的制备
Intermediate 46-INTB: Preparation of methyl (S)-4-(7-((1H-pyrrolo[2,3-b]pyridin-6-yl)methyl)-2,7-diazaspiro[3.5]nonan-2-yl)-3-(3-(tert-butyl)-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)butyrate
步骤1：参见中间体5-INTB步骤3的制备方法，其中25-INTA的制备同6-INTA。粗品经柱层析(DCM/MeOH＝95/5)后得到黄色油状产品50mg。LC-MS:ESI m/z:582.8[M+H]+。Step 1: Refer to the preparation method of step 3 of intermediate 5-INTB, wherein the preparation of 25-INTA is the same as 6-INTA. The crude product was purified by column chromatography (DCM/MeOH=95/5) to obtain 50 mg of a yellow oily product. LC-MS: ESI m/z: 582.8 [M+H] + .
中间体47-INTB：甲基(S)-3-(3-溴-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(7-((3,4-二氢-2H-吡啶并[3,2-b][1,4]噁嗪-6-基)甲基)-2,7-二氮杂螺[3.5]壬烷-2-基)丁酸酯的制备
Intermediate 47-INTB: Preparation of methyl (S)-3-(3-bromo-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(7-((3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazin-6-yl)methyl)-2,7-diazaspiro[3.5]nonan-2-yl)butanoate
步骤1：参见中间体5-INTB步骤3的制备方法。粗产品经正相硅胶柱色谱法纯化(DCM/MeOH＝1/0到8/2)得到黄色油状产品叔-丁基(S)-6-((2-(2-(3-溴-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-甲氧基-4-羰基丁基)-2,7-二氮杂螺[3.5]壬烷-7-基)甲基)-2,3-二氢-4H-吡啶并[3,2-b][1,4]噁嗪-4-羧酸酯80mg。LC-MS:ESI m/z＝723.3[M+H]+。Step 1: Refer to the preparation method of intermediate 5-INTB step 3. The crude product was purified by normal phase silica gel column chromatography (DCM/MeOH=1/0 to 8/2) to obtain 80 mg of yellow oily product tert-butyl (S)-6-((2-(2-(3-bromo-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-methoxy-4-carbonylbutyl)-2,7-diazaspiro[3.5]nonan-7-yl)methyl)-2,3-dihydro-4H-pyrido[3,2-b][1,4]oxazine-4-carboxylate. LC-MS: ESI m/z=723.3[M+H] + .
步骤2：参见中间体3-INTB步骤2的制备方法。得到淡黄色油状产品50mg。LC-MS:ESI m/z＝623.2[M+H]+。Step 2: Refer to the preparation method of intermediate 3-INTB step 2. 50 mg of a light yellow oily product was obtained. LC-MS: ESI m/z = 623.2 [M+H] + .
中间体48-INTB：甲基(S)-3-(3-(叔-丁基)-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(7-((3,4-二氢-2H-吡啶并[3,2-b][1,4]噁嗪-6-基)甲基)-2,7-二氮杂螺[3.5]壬烷-2-基)丁酸酯的制备
Intermediate 48-INTB: Preparation of methyl (S)-3-(3-(tert-butyl)-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(7-((3,4-dihydro-2H-pyrido[3,2-b][1,4]oxazin-6-yl)methyl)-2,7-diazaspiro[3.5]nonan-2-yl)butanoate
步骤1：步骤1：参见中间体5-INTB步骤3的制备方法。粗产品经正相硅胶柱色谱法纯化(DCM/MeOH＝1/0到8/2)得到黄色油状产品叔-丁基(S)-6-((2-(2-(3-(叔-丁基)-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-甲氧基-4-羰基丁基)-2,7-二氮杂螺[3.5]壬烷-7-基)甲基)-2,3-二氢-4H- 吡啶并[3,2-b][1,4]噁嗪-4-羧酸酯100mg。LC-MS:ESI m/z＝701.4[M+H]+。Step 1: Step 1: Refer to the preparation method of intermediate 5-INTB step 3. The crude product was purified by normal phase silica gel column chromatography (DCM/MeOH=1/0 to 8/2) to obtain a yellow oily product tert-butyl (S)-6-((2-(2-(3-(tert-butyl)-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-methoxy-4-carbonylbutyl)-2,7-diazaspiro[3.5]nonan-7-yl)methyl)-2,3-dihydro-4H- Pyrido[3,2-b][1,4]oxazine-4-carboxylate 100 mg. LC-MS: ESI m/z=701.4 [M+H] + .
步骤2：参见中间体3-INTB步骤2的制备方法。得到淡黄色油状产品70mg。LC-MS:ESI m/z＝601.4[M+H]+。Step 2: Refer to the preparation method of intermediate 3-INTB step 2. Obtain 70 mg of light yellow oily product. LC-MS: ESI m/z=601.4 [M+H] + .
中间体49-INTB：甲基(S)-3-(3-溴-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(2-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-6-基)丁酸酯的制备
Intermediate 49-INTB: Preparation of methyl (S)-3-(3-bromo-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(2-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-6-yl)butanoate
步骤1：参见中间体5-INTB步骤3的制备方法，其中49-INTA的制备同30-INTA。粗品经柱层析(DCM/MeOH＝10/1)得到黄色油状产品60mg。LC-MS:ESI m/z:607.2[M+H]+。Step 1: Refer to the preparation method of step 3 of intermediate 5-INTB, wherein the preparation of 49-INTA is the same as 30-INTA. The crude product was purified by column chromatography (DCM/MeOH=10/1) to obtain 60 mg of a yellow oily product. LC-MS: ESI m/z: 607.2 [M+H] + .
中间体50-INTB：甲基(S)-3-(3-(叔-丁基)-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(2-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-6-基)丁酸酯的制备
Intermediate 50-INTB: Preparation of methyl (S)-3-(3-(tert-butyl)-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(2-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-6-yl)butanoate
步骤1：参见中间体5-INTB步骤3的制备方法，其中50-INTA的制备同30-INTA。粗品经柱层析(DCM/MeOH＝10/1)得到黄色油状产品60mg。LC-MS:ESI m/z:585.4[M+H]+。Step 1: Refer to the preparation method of step 3 of intermediate 5-INTB, wherein the preparation of 50-INTA is the same as 30-INTA. The crude product was purified by column chromatography (DCM/MeOH=10/1) to obtain 60 mg of a yellow oily product. LC-MS: ESI m/z: 585.4 [M+H] + .
中间体51-INTB：甲基(3S)-3-(3-溴-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(7-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,7-二氮杂螺[4.4]壬烷-2-基)丁酸酯的制备
Intermediate 51-INTB: Preparation of methyl (3S)-3-(3-bromo-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(7-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,7-diazaspiro[4.4]nonan-2-yl)butanoate
步骤1：参见中间体5-INTB步骤3的制备方法，其中51-INTA的制备同32-INTA。粗品经柱层析(DCM/MeOH＝10/1)得到黄色油状产品50mg。LC-MS:ESI m/z:621.2[M+H]+。Step 1: Refer to the preparation method of step 3 of intermediate 5-INTB, wherein the preparation of 51-INTA is the same as 32-INTA. The crude product was purified by column chromatography (DCM/MeOH=10/1) to obtain 50 mg of a yellow oily product. LC-MS: ESI m/z: 621.2 [M+H] + .
中间体52-INTB：甲基(3S)-3-(3-(叔-丁基)-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(7-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,7-二氮杂螺[4.4]壬烷-2-基)丁酸酯的制备
Intermediate 52-INTB: Preparation of methyl (3S)-3-(3-(tert-butyl)-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(7-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,7-diazaspiro[4.4]nonan-2-yl)butanoate
步骤1：参见中间体5-INTB步骤3的制备方法，其中52-INTA的制备同32-INTA。粗产 品经正相硅胶柱色谱法纯化(DCM/甲醇＝9/1)得到黄色油状产品140mg。LC-MS:ESI m/z＝599.6[M+H]+。Step 1: Refer to the preparation method of intermediate 5-INTB step 3, wherein the preparation of 52-INTA is the same as 32-INTA. The product was purified by normal phase silica gel column chromatography (DCM/methanol=9/1) to obtain 140 mg of a yellow oily product. LC-MS: ESI m/z=599.6 [M+H] + .
中间体53-INTB：甲基(S)-3-(3-溴-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(2-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,7-二氮杂螺[3.5]壬烷-7-基)丁酸酯的制备
Intermediate 53-INTB: Preparation of methyl (S)-3-(3-bromo-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(2-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,7-diazaspiro[3.5]nonan-7-yl)butanoate
步骤1：参见中间体1-INTB步骤4的制备方法。粗产品经正相硅胶柱色谱法纯化(DCM/MeOH＝10/1)得到黄色油状产品50mg。LC-MS:ESI m/z:621.2[M+H]+。Step 1: Refer to the preparation method of intermediate 1-INTB step 4. The crude product was purified by normal phase silica gel column chromatography (DCM/MeOH=10/1) to obtain 50 mg of a yellow oily product. LC-MS: ESI m/z: 621.2 [M+H] + .
中间体54-INTB：甲基(S)-3-(3-(叔-丁基)-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(2-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,7-二氮杂螺[3.5]壬烷-7-基)丁酸酯的制备
Intermediate 54-INTB: Preparation of methyl (S)-3-(3-(tert-butyl)-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(2-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,7-diazaspiro[3.5]nonan-7-yl)butanoate
步骤1：参见中间体1-INTB步骤4的制备方法。粗产品经正相硅胶柱色谱法纯化(DCM/甲醇＝9/1)得到黄色油状产品50mg。。LC-MS:ESI m/z:599.4[M+H]+。Step 1: Refer to the preparation method of intermediate 1-INTB step 4. The crude product was purified by normal phase silica gel column chromatography (DCM/methanol=9/1) to obtain 50 mg of a yellow oily product. LC-MS: ESI m/z: 599.4 [M+H] + .
中间体62-INTB：甲基2-(7-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,7-二氮杂螺[4.4]壬烷-2-基)乙酸酯的制备
Intermediate 62-INTB: Preparation of methyl 2-(7-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,7-diazaspiro[4.4]nonan-2-yl)acetate
步骤1：将叔-丁基2,7-二氮杂螺[4.4]壬烷-2-羧酸酯(100mg，441.9μmol)溶于DCM(5ml)中，然后依次加入TEA(184.5微升，1.3摩尔)，氯乙酸甲酯(62mg，574.4μmol)，室温下反应2小时。将反应液旋干，水洗(10ml)，乙酸乙酯(10ml)萃取，有机相干燥，旋干得到黄色油状产品叔-丁基7-(2-甲氧基-2-羰基乙基)-2,7-二氮杂螺[4.4]壬烷-2-羧酸酯130mg。LC-MS:ESI m/z＝299.0[M+H]+。Step 1: Dissolve tert-butyl 2,7-diazaspiro[4.4]nonane-2-carboxylate (100 mg, 441.9 μmol) in DCM (5 ml), then add TEA (184.5 μl, 1.3 mol) and methyl chloroacetate (62 mg, 574.4 μmol) in sequence, and react at room temperature for 2 hours. The reaction solution was spin-dried, washed with water (10 ml), extracted with ethyl acetate (10 ml), and the organic phase was dried and spin-dried to obtain 130 mg of yellow oily product tert-butyl 7-(2-methoxy-2-carbonylethyl)-2,7-diazaspiro[4.4]nonane-2-carboxylate. LC-MS: ESI m/z=299.0[M+H] + .
步骤2：参见中间体14-INB步骤2的制备方法。得到黄色油状产品甲基2-(2,7-二氮杂螺[4.4]壬烷-2-基)乙酸酯90mg。LC-MS:ESI m/z＝199.2[M+H]+。Step 2: Refer to the preparation method of intermediate 14-INB step 2. 90 mg of methyl 2-(2,7-diazaspiro[4.4]nonan-2-yl)acetate was obtained as a yellow oily product. LC-MS: ESI m/z=199.2 [M+H] + .
步骤3：参见中间体5-INTB步骤3的制备方法。粗品高效液相色谱制备得到白色固体产品100mg。LC-MS:ESI m/z＝345.5[M+H]+。Step 3: Refer to the preparation method of intermediate 5-INTB in step 3. The crude product was purified by HPLC to obtain 100 mg of a white solid product. LC-MS: ESI m/z = 345.5 [M+H] + .
中间体63-INTB：甲基(S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(9-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-1-氧杂-4,9-二氮杂螺[5.5]十一烷-4-基)丁酸酯的制备
Intermediate 63-INTB: Preparation of methyl (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(9-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-1-oxa-4,9-diazaspiro[5.5]undec-4-yl)butanoate
步骤1：参见中间体3-INTB步骤3的制备方法。粗品经正相硅胶柱色谱法纯化(100％石油醚～乙酸乙酯＝2/1)得到黄色油状产品叔-丁基(E)-4-(4-甲氧基-4-羰基丁-2-烯-1-基)-1-氧杂-4,9-二氮杂螺[5.5]十一烷-9-羧酸酯400mg。1H NMR(400MHz,CDCl3)δ6.83(m,1H)5.93(br d,J＝15.77Hz,1H)3.55-3.77(m,8H)3.06(m,2H)2.99(m,2H)2.31-2.40(m,2H)2.16(s,2H)1.87(m,2H)1.30-1.43(m,12H)。Step 1: Refer to the preparation method of intermediate 3-INTB step 3. The crude product was purified by normal phase silica gel column chromatography (100% petroleum ether ~ ethyl acetate = 2/1) to obtain 400 mg of yellow oily product tert-butyl (E)-4-(4-methoxy-4-carbonylbut-2-en-1-yl)-1-oxa-4,9-diazaspiro[5.5]undecane-9-carboxylate. 1 H NMR (400MHz, CDCl3) δ 6.83 (m, 1H) 5.93 (br d, J = 15.77Hz, 1H) 3.55-3.77 (m, 8H) 3.06 (m, 2H) 2.99 (m, 2H) 2.31-2.40 (m, 2H) 2.16 (s, 2H) 1.87 (m, 2H) 1.30-1.43 (m, 12H).
步骤2：参见中间体1-INTB步骤4的制备方法。粗品经正相硅胶柱色谱法纯化(100％石油醚～乙酸乙酯＝1/2)得到黄色油状产品叔-丁基(S)-4-(2-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-甲氧基-4-羰基丁基)-1-氧杂-4,9-二氮杂螺[5.5]十一烷-9-羧酸酯420mg。LC-MS:ESI m/z＝527.7[M+H]+。Step 2: Refer to the preparation method of intermediate 1-INTB step 4. The crude product was purified by normal phase silica gel column chromatography (100% petroleum ether to ethyl acetate = 1/2) to obtain 420 mg of yellow oily product tert-butyl (S)-4-(2-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-methoxy-4-carbonylbutyl)-1-oxa-4,9-diazaspiro[5.5]undecane-9-carboxylate. LC-MS: ESI m/z = 527.7 [M+H] + .
步骤3：参见中间体3-INTB步骤2的制备方法。得到淡黄色油状产品甲基(S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(1-氧杂-4,9-二氮杂螺[5.5]十一烷-4-基)丁酸酯(63-INTA)500mg。LC-MS:ESI m/z＝427.6[M+H]+。Step 3: Refer to the preparation method of intermediate 3-INTB in step 2. 500 mg of methyl (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(1-oxa-4,9-diazaspiro[5.5]undec-4-yl)butanoate (63-INTA) was obtained as a light yellow oily product. LC-MS: ESI m/z=427.6 [M+H] + .
步骤4：参见中间体5-INTB步骤3的制备方法。粗产品经正相硅胶柱色谱法纯化(100％DCM～甲醇＝4/1)得到黄色油状产品200mg。LC-MS:ESI m/z＝573.7[M+H]+。Step 4: Refer to the preparation method of intermediate 5-INTB in step 3. The crude product was purified by normal phase silica gel column chromatography (100% DCM-methanol = 4/1) to obtain 200 mg of a yellow oily product. LC-MS: ESI m/z = 573.7 [M+H] + .
中间体64-INTB：甲基(S)-4-(7-((3H-咪唑并[4,5-b]吡啶-5-基)甲基)-2,7-二氮杂螺[3.5]壬烷-2-基)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)丁酸酯的制备
Intermediate 64-INTB: Preparation of methyl (S)-4-(7-((3H-imidazo[4,5-b]pyridin-5-yl)methyl)-2,7-diazaspiro[3.5]nonan-2-yl)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)butyrate
步骤1：参见中间体5-INTB步骤3的制备方法。粗产品经正相硅胶柱色谱法纯化(100％DCM～甲醇＝4/1)得到黄色油状产品叔-丁基(S)-5-((2-(2-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-甲氧基-4-羰基丁基)-2,7-二氮杂螺[3.5]壬烷-7-基)甲基)-3H-咪唑并[4,5-b]吡啶-3-羧酸酯30mg。LC-MS:ESI m/z:628.7[M+H]+。Step 1: Refer to the preparation method of intermediate 5-INTB step 3. The crude product was purified by normal phase silica gel column chromatography (100% DCM-methanol = 4/1) to obtain tert-butyl (S)-5-((2-(2-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-methoxy-4-carbonylbutyl)-2,7-diazaspiro[3.5]nonan-7-yl)methyl)-3H-imidazo[4,5-b]pyridine-3-carboxylate 30 mg as a yellow oily product. LC-MS: ESI m/z: 628.7 [M+H] + .
步骤2：参见中间体3-INTB步骤2的制备方法。浓缩得到淡黄色油状产品30mg。LC-MS:ESI m/z:528.6[M+H]+。Step 2: Refer to the preparation method of intermediate 3-INTB step 2. Concentration afforded 30 mg of a light yellow oily product. LC-MS: ESI m/z: 528.6 [M+H] + .
中间体65-INTB：甲基(S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(7-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)氧代)-2-氮杂螺[3.5]壬烷-2-基)丁酸酯的制备
Intermediate 65-INTB: Preparation of methyl (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(7-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)oxy)-2-azaspiro[3.5]nonan-2-yl)butanoate
步骤1：依次将7-羟基-2-氮杂螺[3.5]壬烷-2-羧酸叔丁酯(200mg，0.8mmol)，1,8-萘啶-2-醇(1当量)和三苯基膦(1.2当量)溶于干燥的THF(20ml)中。在氮气保护0℃条件下，向溶液中滴加偶氮二甲酸二叔丁酯(1.2当量)，反应液搅拌1小时。浓缩反应液，粗品经正相硅胶柱色谱法纯化(PE/EA＝4/1)得到黄色油状产品叔-丁基7-((1,8-二氮杂萘-2-基)氧代)-2-氮杂螺[3.5]壬烷-2-羧酸酯160mg。LC-MS:ESI m/z:370.2[M+H]+。Step 1: Dissolve tert-butyl 7-hydroxy-2-azaspiro[3.5]nonane-2-carboxylate (200 mg, 0.8 mmol), 1,8-naphthyridin-2-ol (1 equivalent) and triphenylphosphine (1.2 equivalents) in dry THF (20 ml) in sequence. Add di-tert-butyl azodicarboxylate (1.2 equivalents) dropwise to the solution at 0°C under nitrogen protection, and stir the reaction solution for 1 hour. Concentrate the reaction solution, and purify the crude product by normal phase silica gel column chromatography (PE/EA=4/1) to obtain 160 mg of yellow oily product tert-butyl 7-((1,8-naphthyridin-2-yl)oxy)-2-azaspiro[3.5]nonane-2-carboxylate. LC-MS: ESI m/z: 370.2[M+H] + .
步骤2：参见中间体J步骤2的制备方法。得到白色固体产品叔-丁基7-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)氧代)-2-氮杂螺[3.5]壬烷-2-羧酸酯50mg。LC-MS:ESI m/z:374.2[M+H]+。Step 2: Refer to the preparation method of intermediate J step 2. 50 mg of white solid product tert-butyl 7-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)oxy)-2-azaspiro[3.5]nonane-2-carboxylate was obtained. LC-MS: ESI m/z: 374.2 [M+H] + .
步骤3：参见中间体14-INTB步骤2的制备方法。得到白色固体产品7-((2-氮杂螺[3.5]壬烷-7-基)氧代)-1,2,3,4-四氢-1,8-二氮杂萘40mg。LC-MS:ESI m/z:274.2[M+H]+。Step 3: Refer to the preparation method of intermediate 14-INTB in step 2. Obtain 40 mg of white solid product 7-((2-azaspiro[3.5]nonan-7-yl)oxy)-1,2,3,4-tetrahydro-1,8-naphthyridine. LC-MS: ESI m/z: 274.2 [M+H] + .
步骤4：参见中间体3-INTB步骤3的制备方法。粗品经正相硅胶柱色谱法纯化(PE/EA＝2/1)得到黄色油状产品甲基(E)-4-(7-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)氧代)-2-氮杂螺[3.5]壬烷-2-基)丁-2-烯酸酯(65-INTA)50mg。LC-MS:ESI m/z:372.2[M+H]+。Step 4: Refer to the preparation method of intermediate 3-INTB in step 3. The crude product was purified by normal phase silica gel column chromatography (PE/EA=2/1) to obtain 50 mg of methyl (E)-4-(7-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)oxy)-2-azaspiro[3.5]nonane-2-yl)but-2-enoate (65-INTA) as a yellow oily product. LC-MS: ESI m/z: 372.2 [M+H] + .
步骤5：参见中间体1-INTB步骤4的制备方法。粗品经高效液相色谱制备得到黄色固体产品26mg。LC-MS:ESI m/z:544.3[M+H]+。Step 5: Refer to the preparation method of intermediate 1-INTB in step 4. The crude product was purified by HPLC to obtain 26 mg of a yellow solid product. LC-MS: ESI m/z: 544.3 [M+H] + .
中间体66-INTB：甲基(S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-((7-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-7-氮杂螺[3.5]壬烷-2-基)氨基)丁酸酯的制备
Intermediate 66-INTB: Preparation of methyl (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-((7-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-7-azaspiro[3.5]nonan-2-yl)amino)butanoate
步骤1：参见中间体3-INTB步骤3的制备方法。粗品经正相硅胶柱色谱法纯化(DCM/MeOH＝10/1)得到无色油状产品叔-丁基(E)-2-((4-甲氧基-4-羰基丁-2-烯-1-基)氨基)-7-氮杂螺[3.5]壬烷-7-羧酸酯1.9g。LC-MS:ESI m/z＝339.5[M+H]+；1H NMR(400MHz,CDCl3)δ＝6.98(td,J＝5.6,15.7Hz,1H),5.97(br d,J＝15.7Hz,1H),3.74(s,3H),3.37-3.23(m,7H),2.25-2.08(m,2H),1.52-1.42(m,15H)。Step 1: Refer to the preparation method of intermediate 3-INTB step 3. The crude product was purified by normal phase silica gel column chromatography (DCM/MeOH=10/1) to obtain 1.9 g of colorless oily product tert-butyl (E)-2-((4-methoxy-4-carbonylbut-2-en-1-yl)amino)-7-azaspiro[3.5]nonane-7-carboxylate. LC-MS: ESI m/z=339.5[M+H] + ; 1 H NMR (400MHz, CDCl3) δ=6.98 (td, J=5.6,15.7 Hz,1H), 5.97 (br d, J=15.7 Hz,1H), 3.74 (s,3H), 3.37-3.23 (m,7H), 2.25-2.08 (m,2H), 1.52-1.42 (m,15H).
步骤2：参见中间体14-INTB步骤2的制备方法。得到黄色固体产品甲基(E)-4-((7-氮杂螺[3.5]壬烷-2-基)氨基)丁-2-烯酸酯640mg。Step 2: Refer to the preparation method of intermediate 14-INTB step 2. 640 mg of methyl (E)-4-((7-azaspiro[3.5]nonan-2-yl)amino)but-2-enoate was obtained as a yellow solid product.
步骤3：参见中间体5-INTB步骤3的制备方法。粗品经高效液相色谱制备得到棕色固体产品甲基(E)-4-((7-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-7-氮杂螺[3.5]壬烷-2-基)氨基)丁-2- 烯酸酯(66-INTA)250mg。LC-MS:ESI m/z＝385.5[M+H]+。Step 3: See the preparation method of intermediate 5-INTB step 3. The crude product was subjected to high performance liquid chromatography to obtain a brown solid product methyl (E)-4-((7-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-7-azaspiro[3.5]nonane-2-yl)amino)butane-2-yl Enoic acid ester (66-INTA) 250 mg. LC-MS: ESI m/z = 385.5 [M+H] + .
步骤4：参见中间体1-INTB步骤4的制备方法。粗品经高效液相色谱制备得到棕色固体产品50mg。LC-MS:ESI m/z＝557.4[M+H]+；1H NMR(400MHz,METHANOL-d4)δ＝7.58-7.50(m,1H),7.44-7.34(m,3H),7.25(d,J＝7.2Hz,1H),6.53(d,J＝7.2Hz,1H),6.09(s,1H),3.91(s,2H),3.71-3.62(m,1H),3.59(s,3H),3.56-3.48(m,1H),3.40(br t,J＝5.3Hz,2H),3.20(br d,J＝7.3Hz,2H),3.08-2.92(m,4H),2.88(br dd,J＝6.5,16.5Hz,1H),2.79-2.70(m,3H),2.28(br d,J＝14.4Hz,8H),1.98-1.78(m,8H)。Step 4: See the preparation method of intermediate 1-INTB step 4. The crude product was purified by HPLC to obtain 50 mg of a brown solid product. LC-MS: ESI m/z=557.4 [M+H] + ; 1 H NMR (400 MHz, METHANOL-d 4 ) δ=7.58-7.50 (m, 1H), 7.44-7.34 (m, 3H), 7.25 (d, J=7.2 Hz, 1H), 6.53 (d, J=7.2 Hz, 1H), 6.09 (s, 1H), 3.91 (s, 2H), 3.71-3.62 (m, 1H), 3.59 (s, 3H), 3.56-3.48 (m, 1H), 3.40 (br t, J=5.3 Hz, 2H), 3.20 (br d, J=7.3 Hz, 2H), 3.08-2.92 (m, 4H), 2.88 (br dd, J = 6.5, 16.5 Hz, 1H), 2.79-2.70 (m, 3H), 2.28 (br d, J = 14.4 Hz, 8H), 1.98-1.78 (m, 8H).
中间体67-INTB：甲基(S)-4-(8,8-二氟-2-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-6-基)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)丁酸酯的制备
Intermediate 67-INTB: Preparation of methyl (S)-4-(8,8-difluoro-2-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-6-yl)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)butyrate
步骤1：参见中间体3-INTB步骤3的制备方法。粗品经正相硅胶柱色谱法纯化(100％石油醚～乙酸乙酯＝2/1)得到黄色油状产品叔-丁基(E)-8,8-二氟-6-(4-甲氧基-4-羰基丁-2-烯-1-基)-2,6-二氮杂螺[3.4]辛烷-2-羧酸酯100mg。1H NMR(400MHz,CDCl3)δ6.88(m,1H)6.00(d,J＝15.65Hz,1H)4.19(d,J＝9.29Hz,2H)3.76(s,3H)3.70(d,J＝9.17Hz,2H)3.25(d,J＝5.14Hz,2H)2.92-3.09(m,4H)1.45(s,9H)。Step 1: Refer to the preparation method of intermediate 3-INTB step 3. The crude product was purified by normal phase silica gel column chromatography (100% petroleum ether ~ ethyl acetate = 2/1) to obtain 100 mg of yellow oily product tert-butyl (E)-8,8-difluoro-6-(4-methoxy-4-carbonylbut-2-en-1-yl)-2,6-diazaspiro[3.4]octane-2-carboxylate. 1 H NMR (400 MHz, CDCl3) δ 6.88 (m, 1H) 6.00 (d, J = 15.65 Hz, 1H) 4.19 (d, J = 9.29 Hz, 2H) 3.76 (s, 3H) 3.70 (d, J = 9.17 Hz, 2H) 3.25 (d, J = 5.14 Hz, 2H) 2.92-3.09 (m, 4H) 1.45 (s, 9H).
步骤2：参见中间体1-INTB步骤4的制备方法。粗品经正相硅胶柱色谱法纯化(100％石油醚～乙酸乙酯＝1/1)得到淡黄色油状产品叔-丁基(S)-6-(2-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-甲氧基-4-羰基丁基)-8,8-二氟-2,6-二氮杂螺[3.4]辛烷-2-羧酸酯120mg。LC-MS:ESI m/z＝519.5[M+H]+。Step 2: Refer to the preparation method of intermediate 1-INTB step 4. The crude product was purified by normal phase silica gel column chromatography (100% petroleum ether ~ ethyl acetate = 1/1) to obtain 120 mg of light yellow oily product tert-butyl (S)-6-(2-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-methoxy-4-carbonylbutyl)-8,8-difluoro-2,6-diazaspiro[3.4]octane-2-carboxylate. LC-MS: ESI m/z = 519.5 [M+H] + .
步骤3：参见中间体14-INTB步骤2的制备方法。得到淡黄色油状产品甲基(S)-4-(8,8-二氟-2,6-二氮杂螺[3.4]辛烷-6-基)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)丁酸酯(67-INTA)110mg。LC-MS:ESI m/z＝419.4[M+H]+。Step 3: Refer to the preparation method of intermediate 14-INTB in step 2. 110 mg of methyl (S)-4-(8,8-difluoro-2,6-diazaspiro[3.4]octan-6-yl)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)butanoate (67-INTA) was obtained as a light yellow oily product. LC-MS: ESI m/z=419.4 [M+H] + .
步骤4：参见中间体5-INTB步骤3的制备方法。粗品经正相硅胶柱色谱法纯化(100％DCM～甲醇＝4/1)得到黄色油状产品120mg。LC-MS:ESI m/z＝565.5[M+H]+。Step 4: Refer to the preparation method of intermediate 5-INTB in step 3. The crude product was purified by normal phase silica gel column chromatography (100% DCM-methanol = 4/1) to obtain 120 mg of a yellow oily product. LC-MS: ESI m/z = 565.5 [M+H] + .
中间体68-INTB：甲基(3S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(8-氟-2-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-6-基)丁酸酯的制备
Intermediate 68-INTB: Preparation of methyl (3S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(8-fluoro-2-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-6-yl)butanoate
以叔-丁基8-氟-2,6-二氮杂螺[3.4]辛烷-2-羧酸酯为起始原料，制备方法和步骤同67-INTB 的制备。得到淡黄色油状中间体甲基(3S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(8-氟-2,6-二氮杂螺[3.4]辛烷-6-基)丁酸酯(68-INTA)110mg。LC-MS:ESI m/z＝401.2[M+H]+。得到黄色油状产品120mg。LC-MS:ESI m/z＝547.3[M+H]+。Using tert-butyl 8-fluoro-2,6-diazaspiro[3.4]octane-2-carboxylate as the starting material, the preparation method and steps are the same as 67-INTB Preparation. 110 mg of the intermediate methyl (3S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(8-fluoro-2,6-diazaspiro[3.4]octan-6-yl)butanoate (68-INTA) was obtained as a light yellow oil. LC-MS: ESI m/z=401.2[M+H] + . 120 mg of a yellow oily product was obtained. LC-MS: ESI m/z=547.3[M+H] + .
中间体69-INTB：甲基(S)-4-(8,8-二氟-6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-2-基)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)丁酸酯的制备
Intermediate 69-INTB: Preparation of methyl (S)-4-(8,8-difluoro-6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-2-yl)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)butanoate
以叔-丁基8,8-二氟-2,6-二氮杂螺[3.4]辛烷-6-羧酸酯为起始原料，制备方法和步骤同67-INTB的制备。得到淡黄色油状中间体甲基(S)-4-(8,8-二氟-2,6-二氮杂螺[3.4]辛烷-2-基)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)丁酸酯(69-INTA)110mg。LC-MS:ESI m/z＝419.2[M+H]+。得到黄色油状产品120mg。LC-MS:ESI m/z＝565.3[M+H]+。Using tert-butyl 8,8-difluoro-2,6-diazaspiro[3.4]octane-6-carboxylate as the starting material, the preparation method and steps are the same as those of 67-INTB. 110 mg of the pale yellow oily intermediate methyl (S)-4-(8,8-difluoro-2,6-diazaspiro[3.4]octane-2-yl)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)butanoate (69-INTA) was obtained. LC-MS: ESI m/z=419.2[M+H] + . 120 mg of the yellow oily product was obtained. LC-MS: ESI m/z=565.3[M+H] + .
中间体70-INTB：甲基(3S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(8-氟-6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯的制备
Intermediate 70-INTB: Preparation of methyl (3S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(8-fluoro-6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-2-yl)butanoate
以叔-丁基8-氟-2,6-二氮杂螺[3.4]辛烷-6-羧酸酯为起始原料，制备方法和步骤同67-INTB的制备。得到淡黄色油状中间体甲基(3S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(8-氟-2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯(70-INTA)110mg。LC-MS:ESI m/z＝401.2[M+H]+。得到黄色油状产品120mg。LC-MS:ESI m/z＝547.3[M+H]+。Using tert-butyl 8-fluoro-2,6-diazaspiro[3.4]octane-6-carboxylate as the starting material, the preparation method and steps are the same as those for 67-INTB. 110 mg of the pale yellow oily intermediate methyl (3S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(8-fluoro-2,6-diazaspiro[3.4]octane-2-yl)butanoate (70-INTA) was obtained. LC-MS: ESI m/z=401.2[M+H] + . 120 mg of the yellow oily product was obtained. LC-MS: ESI m/z=547.3[M+H] + .
中间体71-INTB：甲基(S)-4-(5,5-二氟-7-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,7-二氮杂螺[3.5]壬烷-2-基)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)丁酸酯的制备
Intermediate 71-INTB: Preparation of methyl (S)-4-(5,5-difluoro-7-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,7-diazaspiro[3.5]nonan-2-yl)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)butyrate
步骤1：参见中间体5-INTB步骤3的制备方法。粗品经正相硅胶柱色谱法纯化(100％DCM～甲醇＝7/3)得到黄色油状产品叔-丁基5,5-二氟-7-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,7-二氮杂螺[3.5]壬烷-2-羧酸酯390mg。LC-MS:ESI m/z＝409.5[M+H]+。Step 1: Refer to the preparation method of intermediate 5-INTB in step 3. The crude product was purified by normal phase silica gel column chromatography (100% DCM-methanol = 7/3) to obtain 390 mg of yellow oily product tert-butyl 5,5-difluoro-7-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,7-diazaspiro[3.5]nonane-2-carboxylate. LC-MS: ESI m/z = 409.5 [M+H] + .
步骤2：参见中间体3-INTB步骤2的制备方法。得到淡黄色油状产品7-((5,5-二氟-2,7-二氮杂螺[3.5]壬烷-7-基)甲基)-1,2,3,4-四氢-1,8-二氮杂萘500mg。 Step 2: Refer to the preparation method of intermediate 3-INTB step 2. 500 mg of a light yellow oily product 7-((5,5-difluoro-2,7-diazaspiro[3.5]nonan-7-yl)methyl)-1,2,3,4-tetrahydro-1,8-naphthyridine was obtained.
步骤3：参见中间体3-INTB步骤3的制备方法。粗品经正相硅胶柱色谱法纯化(100％石油醚～乙酸乙酯＝0/1)得到淡黄色油状产品甲基(E)-4-(5,5-二氟-7-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,7-二氮杂螺[3.5]壬烷-2-基)丁-2-烯酸酯(71-INTA)60mg。1H NMR(400MHz,CDCl3)δ8.41(br s,1H)7.32(br d,J＝7.21Hz,1H)6.83(dt,J＝15.80,5.49Hz,1H)6.61(d,J＝7.21Hz,1H)5.97(br d,J＝15.77Hz,1H)3.75(s,3H)3.64(s,2H)3.49(br s,2H)3.27-3.38(m,6H)2.75(br t,J＝6.17Hz,2H)2.63(br t,J＝10.94Hz,2H)2.55(br s,2H)2.13(br d,J＝4.77Hz,2H)1.89-1.97(m,2H)。Step 3: See the preparation method of intermediate 3-INTB step 3. The crude product was purified by normal phase silica gel column chromatography (100% petroleum ether-ethyl acetate = 0/1) to obtain 60 mg of methyl (E)-4-(5,5-difluoro-7-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,7-diazaspiro[3.5]nonane-2-yl)but-2-enoate (71-INTA) as a light yellow oily product. 1 H NMR (400 MHz, CDCl3) δ 8.41 (br s, 1H) 7.32 (br d, J = 7.21 Hz, 1H) 6.83 (dt, J = 15.80, 5.49 Hz, 1H) 6.61 (d, J = 7.21 Hz, 1H) 5.97 (br d, J = 15.77 Hz, 1H) 3.75 (s, 3H) 3.64 (s, 2H) 3.49 (br s, 2H) 3.27-3.38 (m, 6H) 2.75 (br t, J = 6.17 Hz, 2H) 2.63 (br t, J = 10.94 Hz, 2H) 2.55 (br s, 2H) 2.13 (br d, J = 4.77 Hz, 2H) 1.89-1.97 (m, 2H).
步骤4：参见中间体1-INTB步骤4的制备方法。粗品经正相硅胶柱色谱法纯化(100％DCM～甲醇＝85/15)得到淡黄色油状产品70mg。LC-MS:ESI m/z＝579.6[M+H]+。Step 4: See the preparation method of intermediate 1-INTB step 4. The crude product was purified by normal phase silica gel column chromatography (100% DCM-methanol=85/15) to obtain 70 mg of a light yellow oily product. LC-MS: ESI m/z=579.6 [M+H] + .
中间体72-INTB：甲基(3S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(5-氟-7-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,7-二氮杂螺[3.5]壬烷-2-基)丁酸酯的制备
Intermediate 72-INTB: Preparation of methyl (3S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(5-fluoro-7-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,7-diazaspiro[3.5]nonan-2-yl)butanoate
步骤1：参见中间体1-INTB步骤4的制备方法，其中72-INTA的制备同71-INTA。粗品经正相硅胶柱色谱法纯化(100％DCM～甲醇＝85/15)得到淡黄色油状产品120mg。LC-MS:ESI m/z＝561.3[M+H]+。Step 1: Refer to the preparation method of step 4 of intermediate 1-INTB, wherein the preparation of 72-INTA is the same as 71-INTA. The crude product was purified by normal phase silica gel column chromatography (100% DCM-methanol = 85/15) to obtain 120 mg of a light yellow oily product. LC-MS: ESI m/z = 561.3 [M+H] + .
中间体73-INTB：甲基(S)-4-(5,5-二氟-2-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,7-二氮杂螺[3.5]壬烷-7-基)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)丁酸酯的制备
Intermediate 73-INTB: Preparation of methyl (S)-4-(5,5-difluoro-2-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,7-diazaspiro[3.5]nonan-7-yl)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)butyrate
以叔-丁基5,5-二氟-2,7-二氮杂螺[3.5]壬烷-2-羧酸酯为起始原料，制备方法和步骤同67-INTB的制备。得到淡黄色油状中间体甲基(S)-4-(5,5-二氟-2,7-二氮杂螺[3.5]壬烷-7-基)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)丁酸酯(73-INTA)100mg。LC-MS:ESI m/z＝433.5[M+H]+。得到黄色油状产品85mg。LC-MS:ESI m/z＝579.7[M+H]+。Using tert-butyl 5,5-difluoro-2,7-diazaspiro[3.5]nonane-2-carboxylate as the starting material, the preparation method and steps are the same as those for 67-INTB. 100 mg of the pale yellow oily intermediate methyl (S)-4-(5,5-difluoro-2,7-diazaspiro[3.5]nonane-7-yl)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)butanoate (73-INTA) was obtained. LC-MS: ESI m/z=433.5[M+H] + . 85 mg of the yellow oily product was obtained. LC-MS: ESI m/z=579.7[M+H] + .
中间体74-INTB：甲基(3S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(5-氟-2-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,7-二氮杂螺[3.5]壬烷-7-基)丁酸酯的制备
Intermediate 74-INTB: Preparation of methyl (3S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(5-fluoro-2-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,7-diazaspiro[3.5]nonan-7-yl)butanoate
以叔-丁基5-氟-2,7-二氮杂螺[3.5]壬烷-2-羧酸酯为起始原料，制备方法和步骤同67-INTB的制备。得到淡黄色油状中间体甲基(3S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(5-氟-2,7-二 氮杂螺[3.5]壬烷-7-基)丁酸酯(74-INTA)100mg。LC-MS:ESI m/z＝415.2[M+H]+。得到黄色油状产品85mg。LC-MS:ESI m/z＝561.3[M+H]+。Using tert-butyl 5-fluoro-2,7-diazaspiro[3.5]nonane-2-carboxylate as the starting material, the preparation method and steps were the same as those for 67-INTB. A pale yellow oily intermediate methyl (3S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(5-fluoro-2,7-dihydro-1H-pyrazol-1-yl)phenyl) ... 100 mg of azaspiro[3.5]nonan-7-yl)butanoate (74-INTA). LC-MS: ESI m/z = 415.2 [M+H] + . 85 mg of a yellow oily product was obtained. LC-MS: ESI m/z = 561.3 [M+H] + .
中间体75-INTB：甲基(S)-3-(3-溴苯基)-4-(7-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,7-二氮杂螺[3.5]壬烷-2-基)丁酸酯的制备
Intermediate 75-INTB: Preparation of methyl (S)-3-(3-bromophenyl)-4-(7-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,7-diazaspiro[3.5]nonan-2-yl)butanoate
步骤1：参见中间体1-INTB步骤4的制备方法。粗品经高效液相色谱纯化得到黄色油状产品110mg。LC-MS:ESI m/z＝527.4[M+H]+。Step 1: Refer to the preparation method of intermediate 1-INTB step 4. The crude product was purified by HPLC to obtain 110 mg of a yellow oily product. LC-MS: ESI m/z = 527.4 [M+H] + .
中间体76-INTB：甲基(S)-3-(3-溴-5-(叔-丁基)苯基)-4-(7-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,7-二氮杂螺[3.5]壬烷-2-基)丁酸酯的制备
Intermediate 76-INTB: Preparation of methyl (S)-3-(3-bromo-5-(tert-butyl)phenyl)-4-(7-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,7-diazaspiro[3.5]nonan-2-yl)butanoate
步骤1：参见中间体1-INTB步骤4的制备方法。粗品经高效液相色谱纯化得到无色油状产品叔-丁基(S)-2-(2-(3-溴-5-(叔-丁基)苯基)-4-甲氧基-4-羰基丁基)-2,7-二氮杂螺[3.5]壬烷-7-羧酸酯210mg。LC-MS:ESI m/z＝537.5[M+H]+。Step 1: Refer to the preparation method of intermediate 1-INTB step 4. The crude product was purified by HPLC to obtain 210 mg of colorless oily product tert-butyl (S)-2-(2-(3-bromo-5-(tert-butyl)phenyl)-4-methoxy-4-carbonylbutyl)-2,7-diazaspiro[3.5]nonane-7-carboxylate. LC-MS: ESI m/z=537.5[M+H] + .
步骤2：参见中间体14-INTB步骤2的制备方法。反应液直接浓缩得到黄色油状产品甲基(S)-3-(3-溴-5-(叔-丁基)苯基)-4-(2,7-二氮杂螺[3.5]壬烷-2-基)丁酸酯(76-INTA)75mg。Step 2: Refer to the preparation method of intermediate 14-INTB step 2. The reaction solution was directly concentrated to obtain 75 mg of yellow oily product methyl (S)-3-(3-bromo-5-(tert-butyl)phenyl)-4-(2,7-diazaspiro[3.5]nonan-2-yl)butanoate (76-INTA).
步骤3：参见中间体5-INTB步骤3的制备方法。粗品经正相硅胶柱色谱法纯化(DCM/MeOH＝10/1)得到黄色油状产品70mg。LC-MS:ESI m/z＝583.6[M+H]+。Step 3: See the preparation method of intermediate 5-INTB in step 3. The crude product was purified by normal phase silica gel column chromatography (DCM/MeOH=10/1) to obtain 70 mg of a yellow oily product. LC-MS: ESI m/z=583.6 [M+H] + .
中间体77-INTB：甲基(S)-3-(5-溴-2-氟苯基)-4-(7-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,7-二氮杂螺[3.5]壬烷-2-基)丁酸酯的制备
Intermediate 77-INTB: Preparation of methyl (S)-3-(5-bromo-2-fluorophenyl)-4-(7-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,7-diazaspiro[3.5]nonan-2-yl)butanoate
以叔-丁基(E)-2-(4-甲氧基-4-羰基丁-2-烯-1-基)-2,7-二氮杂螺[3.5]壬烷-7-羧酸酯和(5-溴-2-氟苯基)硼酸为起始原料，制备方法和步骤同76-INTB的制备。得到淡黄色油状中间体甲基(S)-3-(5-溴-2-氟苯基)-4-(2,7-二氮杂螺[3.5]壬烷-2-基)丁酸酯(77-INTA)290mg。LC-MS:ESI m/z＝401.1[M+H]+。得到无色油状产品150mg。LC-MS:ESI m/z＝547.2[M+H]+。Using tert-butyl (E)-2-(4-methoxy-4-carbonylbut-2-en-1-yl)-2,7-diazaspiro[3.5]nonane-7-carboxylate and (5-bromo-2-fluorophenyl)boronic acid as starting materials, the preparation method and steps are the same as the preparation of 76-INTB. The intermediate methyl (S)-3-(5-bromo-2-fluorophenyl)-4-(2,7-diazaspiro[3.5]nonane-2-yl)butanoate (77-INTA) 290 mg was obtained as a pale yellow oil. LC-MS: ESI m/z = 401.1 [M+H] + . The product was obtained as a colorless oil 150 mg. LC-MS: ESI m/z = 547.2 [M+H] + .
中间体78-INTB：甲基(S)-3-(异喹啉-7-基)-4-(7-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,7-二氮杂螺[3.5]壬烷-2-基)丁酸酯的制备
Intermediate 78-INTB: Preparation of methyl (S)-3-(isoquinolin-7-yl)-4-(7-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,7-diazaspiro[3.5]nonan-2-yl)butanoate
步骤1：参见中间体1-INTB步骤4的制备方法。粗品经正相硅胶柱色谱法纯化(DCM/MeOH＝10/1)得到黄色油状产品40mg。LC-MS:ESI m/z＝500.6[M+H]+。Step 1: Refer to the preparation method of intermediate 1-INTB step 4. The crude product was purified by normal phase silica gel column chromatography (DCM/MeOH=10/1) to obtain 40 mg of a yellow oily product. LC-MS: ESI m/z=500.6 [M+H] + .
中间体79-INTB：甲基(S)-3-(3-(2-甲氧基乙氧基)苯基)-4-(7-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,7-二氮杂螺[3.5]壬烷-2-基)丁酸酯的制备
Intermediate 79-INTB: Preparation of methyl (S)-3-(3-(2-methoxyethoxy)phenyl)-4-(7-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,7-diazaspiro[3.5]nonan-2-yl)butanoate
步骤1：参见中间体1-INTB步骤4的制备方法。粗品经高效液相色谱纯化得到黄色固体产品叔-丁基(S)-2-(4-甲氧基-2-(3-(2-甲氧基乙氧基)苯基)-4-羰基丁基)-2,7-二氮杂螺[3.5]壬烷-7-羧酸酯180mg。LC-MS:ESI m/z＝478.7[M+H]+。Step 1: Refer to the preparation method of intermediate 1-INTB step 4. The crude product was purified by HPLC to obtain 180 mg of yellow solid product tert-butyl (S)-2-(4-methoxy-2-(3-(2-methoxyethoxy)phenyl)-4-carbonylbutyl)-2,7-diazaspiro[3.5]nonane-7-carboxylate. LC-MS: ESI m/z=478.7[M+H] + .
步骤2：参见中间体3-INTB步骤2的制备方法。得到粗品甲基(S)-3-(3-(2-甲氧基乙氧基)苯基)-4-(2,7-二氮杂螺[3.5]壬烷-2-基)丁酸酯(79-INTA)180mg。LC-MS:ESI m/z＝378.3[M+H]+。Step 2: Refer to the preparation method of intermediate 3-INTB step 2. Obtain 180 mg of crude methyl (S)-3-(3-(2-methoxyethoxy)phenyl)-4-(2,7-diazaspiro[3.5]nonan-2-yl)butanoate (79-INTA). LC-MS: ESI m/z=378.3 [M+H] + .
步骤3：参见中间体5-INTB步骤3的制备方法。粗品经高效液相色谱纯化得到黄色油状产品90mg。LC-MS:ESI m/z＝523.5[M+H]+；1H NMR(400MHz,METHANOL-d4)8.42(s,2H),7.34-7.24(m,2H),6.94-6.85(m,3H),6.59-6.52(m,1H),5.28-5.05(m,4H),4.17-4.11(m,2H),3.78-3.75(m,2H),3.72(s,2H),3.61(s,3H),3.57-3.53(m,4H),3.44(s,3H),3.44-3.42(m,1H),2.82-2.61(m,8H),1.97-1.87(m,6H)。Step 3: See the preparation method of intermediate 5-INTB step 3. The crude product was purified by HPLC to obtain 90 mg of a yellow oily product. LC-MS: ESI m/z=523.5 [M+H] + ; 1 H NMR (400 MHz, METHANOL-d 4 ) 8.42 (s, 2H), 7.34-7.24 (m, 2H), 6.94-6.85 (m, 3H), 6.59-6.52 (m, 1H), 5.28-5.05 (m, 4H), 4.17-4.11 (m, 2H), 3.78-3.75 (m, 2H), 3.72 (s, 2H), 3.61 (s, 3H), 3.57-3.53 (m, 4H), 3.44 (s, 3H), 3.44-3.42 (m, 1H), 2.82-2.61 (m, 8H), 1.97-1.87 (m, 6H).
中间体80-INTB：甲基(S)-3-(5-(3,5-二甲基-1H-吡唑-1-基)-2-氟苯基)-4-(6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯的制备
Intermediate 80-INTB: Preparation of methyl (S)-3-(5-(3,5-dimethyl-1H-pyrazol-1-yl)-2-fluorophenyl)-4-(6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-2-yl)butanoate
步骤1：参见中间体1-ITNB步骤4的制备方法。粗品经正相硅胶柱色谱法纯化(DCM/MeOH＝10/1)得到黄色油状产品叔-丁基(S)-2-(2-(5-溴-2-氟苯基)-4-甲氧基-4-羰基丁基)-2,6-二氮杂螺[3.4]辛烷-6-羧酸酯480mg。LC-MS:ESI m/z＝485.4[M+H]+。Step 1: Refer to the preparation method of intermediate 1-ITNB step 4. The crude product was purified by normal phase silica gel column chromatography (DCM/MeOH=10/1) to obtain 480 mg of yellow oily product tert-butyl (S)-2-(2-(5-bromo-2-fluorophenyl)-4-methoxy-4-carbonylbutyl)-2,6-diazaspiro[3.4]octane-6-carboxylate. LC-MS: ESI m/z=485.4[M+H] + .
步骤2：将叔-丁基(S)-2-(2-(5-溴-2-氟苯基)-4-甲氧基-4-羰基丁基)-2,6-二氮杂螺[3.4]辛烷-6-羧酸酯(380mg,0.8mmol)，(5-溴-2-氟苯基)硼酸(90.3mg,0.9mmol),钯催化剂(66.9mg,0.1mmol)和碳酸铯(765.2mg,2.4mmol)溶于二氧六环(10ml)中，氮气置换三次，氮气保护下加 热到120℃并搅拌16小时。将反应液浓缩，粗品经正相硅胶柱色谱法纯化(DCM/MeOH＝10/1)得到黄色油状产品叔-丁基(S)-2-(2-(5-(3,5-二甲基-1H-吡唑-1-基)-2-氟苯基)-4-甲氧基-4-羰基丁基)-2,6-二氮杂螺[3.4]辛烷-6-羧酸酯200mg。LC-MS:ESI m/z＝501.5[M+H]+。Step 2: Dissolve tert-butyl (S)-2-(2-(5-bromo-2-fluorophenyl)-4-methoxy-4-carbonylbutyl)-2,6-diazaspiro[3.4]octane-6-carboxylate (380 mg, 0.8 mmol), (5-bromo-2-fluorophenyl)boric acid (90.3 mg, 0.9 mmol), palladium catalyst (66.9 mg, 0.1 mmol) and cesium carbonate (765.2 mg, 2.4 mmol) in dioxane (10 ml), replace with nitrogen three times, and add under nitrogen protection. Heat to 120°C and stir for 16 hours. The reaction solution was concentrated and the crude product was purified by normal phase silica gel column chromatography (DCM/MeOH=10/1) to obtain 200 mg of yellow oily product tert-butyl (S)-2-(2-(5-(3,5-dimethyl-1H-pyrazol-1-yl)-2-fluorophenyl)-4-methoxy-4-carbonylbutyl)-2,6-diazaspiro[3.4]octane-6-carboxylate. LC-MS: ESI m/z=501.5[M+H] + .
步骤3：参见中间体3-INTB步骤2的制备方法。粗品经高效液相色谱纯化得到黄色油状产品甲基(S)-3-(5-(3,5-二甲基-1H-吡唑-1-基)-2-氟苯基)-4-(2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯(80-INTA)60mg。LC-MS:ESI m/z＝401.5[M+H]+。Step 3: Refer to the preparation method of intermediate 3-INTB in step 2. The crude product was purified by HPLC to obtain 60 mg of methyl (S)-3-(5-(3,5-dimethyl-1H-pyrazol-1-yl)-2-fluorophenyl)-4-(2,6-diazaspiro[3.4]octan-2-yl)butanoate (80-INTA) as a yellow oily product. LC-MS: ESI m/z=401.5[M+H] + .
步骤4：参见中间体5-INTB步骤3的制备方法。粗品经高效液相色谱纯化得到黄色油状产品40mg。LC-MS:ESI m/z＝547.4[M+H]+。Step 4: Refer to the preparation method of intermediate 5-INTB in step 3. The crude product was purified by HPLC to obtain 40 mg of a yellow oily product. LC-MS: ESI m/z = 547.4 [M+H] + .
中间体81-INTB：甲基(S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)-5-氟苯基)-4-(6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯的制备
Intermediate 81-INTB: Preparation of methyl (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)-5-fluorophenyl)-4-(6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-2-yl)butanoate
以(3-溴-5-氟苯基)硼酸为起始原料，制备方法和步骤同80-INTB的制备。得到淡黄色油状中间体甲基(S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)-5-氟苯基)-4-(2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯(81-INTA)60mg。LC-MS:ESI m/z＝401.2[M+H]+。得到黄色油状产品30mg。LC-MS:ESI m/z＝547.5[M+H]+。Using (3-bromo-5-fluorophenyl)boronic acid as the starting material, the preparation method and steps are the same as those for 80-INTB. 60 mg of the intermediate methyl (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)-5-fluorophenyl)-4-(2,6-diazaspiro[3.4]octan-2-yl)butanoate (81-INTA) was obtained as a light yellow oil. LC-MS: ESI m/z = 401.2 [M+H] + . 30 mg of a yellow oily product was obtained. LC-MS: ESI m/z = 547.5 [M+H] + .
中间体82-INTB：甲基(S)-3-(3-溴-5-(叔-丁基)苯基)-4-(6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯的制备
Intermediate 82-INTB: Preparation of methyl (S)-3-(3-bromo-5-(tert-butyl)phenyl)-4-(6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-2-yl)butanoate
以叔-丁基(E)-2-(4-甲氧基-4-羰基丁-2-烯-1-基)-2,6-二氮杂螺[3.4]辛烷-6-羧酸酯为起始原料，制备方法和步骤同76-INTB的制备。得到淡黄色油状中间体甲基(S)-3-(3-溴-5-(叔-丁基)苯基)-4-(2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯(82-INTA)110mg。LC-MS:ESI m/z＝423.2[M+H]+。得到黄色油状产品120mg。LC-MS:ESI m/z＝569.5[M+H]+。Using tert-butyl (E)-2-(4-methoxy-4-carbonylbut-2-en-1-yl)-2,6-diazaspiro[3.4]octane-6-carboxylate as the starting material, the preparation method and steps are the same as those for the preparation of 76-INTB. 110 mg of the pale yellow oily intermediate methyl (S)-3-(3-bromo-5-(tert-butyl)phenyl)-4-(2,6-diazaspiro[3.4]octane-2-yl)butanoate (82-INTA) was obtained. LC-MS: ESI m/z=423.2[M+H] + . 120 mg of the yellow oily product was obtained. LC-MS: ESI m/z=569.5[M+H] + .
中间体83-INTB：甲基(S)-3-(3-(1H-吡咯-2-基)苯基)-4-(6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯的制备
Intermediate 83-INTB: Preparation of methyl (S)-3-(3-(1H-pyrrol-2-yl)phenyl)-4-(6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-2-yl)butanoate
步骤1：参见中间体1-INTB步骤4的制备方法。粗品经由正相硅胶柱色谱法纯化(DCM/MeOH＝100/0～20/1)后得到黄色固体产品苯甲基(S)-2-(2-(3-(1-(叔-丁氧基羰基)-1H-吡咯-2-基)苯基)-4-甲氧基-4-羰基丁基)-2,6-二氮杂螺[3.4]辛烷-6-羧酸酯370mg。LC-MS:ESI m/z＝587.8[M+H]+。Step 1: Refer to the preparation method of intermediate 1-INTB step 4. The crude product was purified by normal phase silica gel column chromatography (DCM/MeOH=100/0-20/1) to obtain 370 mg of yellow solid product benzyl (S)-2-(2-(3-(1-(tert-butyloxycarbonyl)-1H-pyrrol-2-yl)phenyl)-4-methoxy-4-carbonylbutyl)-2,6-diazaspiro[3.4]octane-6-carboxylate. LC-MS: ESI m/z=587.8[M+H] + .
步骤2：参见中间体J步骤2的制备方法。得到无色油状产品叔-丁基(S)-2-(3-(4-甲氧基-4-羰基-1-(2,6-二氮杂螺[3.4]辛烷-2-基)丁烷-2-基)苯基)-1H-吡咯-1-羧酸酯(83-INTA)137mg。LC-MS:ESI m/z＝454.5[M+H]+。Step 2: Refer to the preparation method of intermediate J step 2. 137 mg of colorless oily product tert-butyl (S)-2-(3-(4-methoxy-4-carbonyl-1-(2,6-diazaspiro[3.4]octan-2-yl)butan-2-yl)phenyl)-1H-pyrrole-1-carboxylate (83-INTA) was obtained. LC-MS: ESI m/z=454.5 [M+H] + .
步骤3：参见中间体5-INTB步骤3的制备方法。粗品经高效液相色谱制备得到无色油状产品叔-丁基(S)-2-(3-(4-甲氧基-4-羰基-1-(6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-2-基)丁烷-2-基)苯基)-1H-吡咯-1-羧酸酯80mg。LC-MS:ESI m/z＝600.5[M+H]+。Step 3: See the preparation method of intermediate 5-INTB step 3. The crude product was purified by high performance liquid chromatography to obtain 80 mg of colorless oily product tert-butyl (S)-2-(3-(4-methoxy-4-carbonyl-1-(6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-2-yl)butan-2-yl)phenyl)-1H-pyrrole-1-carboxylate. LC-MS: ESI m/z=600.5[M+H] + .
步骤4：将叔-丁基(S)-2-(3-(4-甲氧基-4-羰基-1-(6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-2-基)丁烷-2-基)苯基)-1H-吡咯-1-羧酸酯(40mg,68μmol)溶于5mlDCM中，室温下加入三氟乙酸(2mg)，室温搅拌1小时。浓缩反应液后粗品经高效液相色谱制备得到棕色固体产品50mg。LC-MS:ESI m/z＝500.3[M+H]+。Step 4: Dissolve tert-butyl (S)-2-(3-(4-methoxy-4-carbonyl-1-(6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-2-yl)butan-2-yl)phenyl)-1H-pyrrole-1-carboxylate (40 mg, 68 μmol) in 5 ml DCM, add trifluoroacetic acid (2 mg) at room temperature, and stir at room temperature for 1 hour. After concentrating the reaction solution, the crude product was purified by HPLC to obtain 50 mg of a brown solid product. LC-MS: ESI m/z=500.3[M+H] + .
中间体84-INTB：甲基(S)-3-(3-环丙基苯基)-4-(6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯的制备
Intermediate 84-INTB: Preparation of methyl (S)-3-(3-cyclopropylphenyl)-4-(6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-2-yl)butanoate
以叔-丁基(E)-2-(4-甲氧基-4-羰基丁-2-烯-1-基)-2,6-二氮杂螺[3.4]辛烷-6-羧酸酯和(3-环丙基苯基)硼酸为起始原料，制备方法和步骤同76-INTB的制备。得到淡黄色油状中间体甲基(S)-3-(3-环丙基苯基)-4-(2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯(84-INTA)160mg。LC-MS:ESI m/z＝329.5[M+H]+。得到黄色油状产品90mg。LC-MS:ESI m/z＝475.6[M+H]+。Using tert-butyl (E)-2-(4-methoxy-4-carbonylbut-2-en-1-yl)-2,6-diazaspiro[3.4]octane-6-carboxylate and (3-cyclopropylphenyl)boronic acid as starting materials, the preparation method and steps are the same as the preparation of 76-INTB. 160 mg of the light yellow oily intermediate methyl (S)-3-(3-cyclopropylphenyl)-4-(2,6-diazaspiro[3.4]octane-2-yl)butanoate (84-INTA) was obtained. LC-MS: ESI m/z=329.5[M+H] + . 90 mg of the yellow oily product was obtained. LC-MS: ESI m/z=475.6[M+H] + .
中间体85-INTB：甲基(S)-3-(5-溴-2-氟苯基)-4-(6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯的制备
Intermediate 85-INTB: Preparation of methyl (S)-3-(5-bromo-2-fluorophenyl)-4-(6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-2-yl)butanoate
以叔-丁基(E)-2-(4-甲氧基-4-羰基丁-2-烯-1-基)-2,6-二氮杂螺[3.4]辛烷-6-羧酸酯和(5-溴-2-氟苯基)硼酸为起始原料，制备方法和步骤同76-INTB的制备。得到淡黄色油状中间体甲基(S)-3-(5-溴-2-氟苯基)-4-(2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯(85-INTA)160mg。LC-MS:ESI m/z＝385.1[M+H]+。得到黄色油状产品110mg。LC-MS:ESI m/z＝531.5[M+H]+。Using tert-butyl (E)-2-(4-methoxy-4-carbonylbut-2-en-1-yl)-2,6-diazaspiro[3.4]octane-6-carboxylate and (5-bromo-2-fluorophenyl)boronic acid as starting materials, the preparation method and steps are the same as the preparation of 76-INTB. 160 mg of the light yellow oily intermediate methyl (S)-3-(5-bromo-2-fluorophenyl)-4-(2,6-diazaspiro[3.4]octane-2-yl)butanoate (85-INTA) was obtained. LC-MS: ESI m/z=385.1[M+H] + . 110 mg of the yellow oily product was obtained. LC-MS: ESI m/z=531.5[M+H] + .
中间体86-INTB：甲基(S)-3-(3-环丙基-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯的制备
Intermediate 86-INTB: Preparation of methyl (S)-3-(3-cyclopropyl-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-2-yl)butanoate
步骤1：参见中间体1-INTB步骤4的制备方法。粗品经由正相硅胶柱色谱法纯化(DCM:甲醇＝100:0～20:1)后得到棕色油状产品苯甲基(S)-2-(2-(3-溴-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-甲氧基-4-羰基丁基)-2,6-二氮杂螺[3.4]辛烷-6-羧酸酯400mg。LC-MS:ESI m/z＝595.4[M+H]+。Step 1: Refer to the preparation method of intermediate 1-INTB step 4. The crude product was purified by normal phase silica gel column chromatography (DCM: methanol = 100: 0 to 20: 1) to obtain 400 mg of brown oily product benzyl (S)-2-(2-(3-bromo-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-methoxy-4-carbonylbutyl)-2,6-diazaspiro[3.4]octane-6-carboxylate. LC-MS: ESI m/z = 595.4 [M+H] + .
步骤2：依次将苯甲基(S)-2-(2-(3-溴-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-甲氧基-4-羰基丁基)-2,6-二氮杂螺[3.4]辛烷-6-羧酸酯(400mg，0.7mmol)，环丙基硼酸(87mg，1mmol)，碳酸铯(547mg，1.7mmol)和钯催化剂(49mg，0.1mmol)溶于二氧六环(10ml)中。氮气保护下加热到100℃并搅拌3小时。反应液直接旋干，正相硅胶柱色谱法纯化(DCM/MeOH＝100/0～10/1)得到棕色油状产品苯甲基(S)-2-(2-(3-环丙基-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-甲氧基-4-羰基丁基)-2,6-二氮杂螺[3.4]辛烷-6-羧酸酯373mg。LC-MS:ESI m/z＝557.5[M+H]+。Step 2: Dissolve benzyl (S)-2-(2-(3-bromo-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-methoxy-4-carbonylbutyl)-2,6-diazaspiro[3.4]octane-6-carboxylate (400 mg, 0.7 mmol), cyclopropylboronic acid (87 mg, 1 mmol), cesium carbonate (547 mg, 1.7 mmol) and palladium catalyst (49 mg, 0.1 mmol) in dioxane (10 ml) in sequence. Heat to 100°C and stir for 3 hours under nitrogen protection. The reaction solution was directly spin-dried and purified by normal phase silica gel column chromatography (DCM/MeOH=100/0-10/1) to obtain 373 mg of brown oily product benzyl (S)-2-(2-(3-cyclopropyl-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-methoxy-4-carbonylbutyl)-2,6-diazaspiro[3.4]octane-6-carboxylate. LC-MS: ESI m/z=557.5[M+H] + .
步骤3：参见中间体J步骤2的制备方法。得到棕色油状产品甲基(S)-3-(3-环丙基-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯(86-INTA)270mg。LC-MS:ESI m/z＝423.5[M+H]+。Step 3: Refer to the preparation method of intermediate J step 2. 270 mg of methyl (S)-3-(3-cyclopropyl-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(2,6-diazaspiro[3.4]octan-2-yl)butanoate (86-INTA) was obtained as a brown oily product. LC-MS: ESI m/z=423.5 [M+H] + .
步骤4：参见中间体1-INTB步骤3的制备方法。粗品经正相硅胶柱色谱法纯化(DCM(1％氨水)/甲醇＝100/0～10/1)后得到棕色油状产品130mg。LC-MS:ESI m/z＝569.4[M+H]+。Step 4: See the preparation method of intermediate 1-INTB in step 3. The crude product was purified by normal phase silica gel column chromatography (DCM (1% aqueous ammonia)/methanol = 100/0 to 10/1) to obtain 130 mg of a brown oily product. LC-MS: ESI m/z = 569.4 [M+H] + .
中间体87-INTB：甲基(S)-3-(3-(5-甲基-1H-咪唑-2-基)苯基)-4-(6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯的制备
Intermediate 87-INTB: Preparation of methyl (S)-3-(3-(5-methyl-1H-imidazol-2-yl)phenyl)-4-(6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-2-yl)butanoate
以苯甲基(E)-2-(4-甲氧基-4-羰基丁-2-烯-1-基)-2,6-二氮杂螺[3.4]辛烷-6-羧酸酯和中间体L为起始原料，制备方法和步骤同83-INTB的制备。得到淡黄色油状中间体甲基(S)-3-(3-(5-甲基-1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-咪唑-2-基)苯基)-4-(2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯(87-INTA)150mg。LC-MS:ESI m/z＝499.5[M+H]+。得到黄色油状产品100mg。LC-MS:ESI m/z＝515.4[M+H]+。Benzyl (E)-2-(4-methoxy-4-carbonylbut-2-en-1-yl)-2,6-diazaspiro[3.4]octane-6-carboxylate and intermediate L were used as starting materials. The preparation method and steps were the same as those for 83-INTB. 150 mg of the intermediate methyl (S)-3-(3-(5-methyl-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)phenyl)-4-(2,6-diazaspiro[3.4]octane-2-yl)butanoate (87-INTA) was obtained as a pale yellow oil. LC-MS: ESI m/z=499.5[M+H] + . 100 mg of a yellow oily product was obtained. LC-MS: ESI m/z=515.4[M+H] + .
中间体88-INTB：甲基(S)-3-(3-(1H-咪唑-2-基)苯基)-4-(6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯的制备
Intermediate 88-INTB: Preparation of methyl (S)-3-(3-(1H-imidazol-2-yl)phenyl)-4-(6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-2-yl)butanoate
以苯甲基(E)-2-(4-甲氧基-4-羰基丁-2-烯-1-基)-2,6-二氮杂螺[3.4]辛烷-6-羧酸酯和中间体M为起始原料，制备方法和步骤同83-INTB的制备。得到淡黄色油状中间体甲基(S)-4-(2,6-二氮杂螺[3.4]辛烷-2-基)-3-(3-(1-((2-(三甲基甲硅烷基)乙氧基)甲基)-1H-咪唑-2-基)苯基)丁酸酯(88-INTA)350mg。LC-MS:ESI m/z＝485.4[M+H]+。得到黄色油状产品300mg。LC-MS:ESI m/z＝501.4[M+H]+。Benzyl (E)-2-(4-methoxy-4-carbonylbut-2-en-1-yl)-2,6-diazaspiro[3.4]octane-6-carboxylate and intermediate M were used as starting materials. The preparation method and steps were the same as those for 83-INTB. 350 mg of the intermediate methyl (S)-4-(2,6-diazaspiro[3.4]octane-2-yl)-3-(3-(1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)phenyl)butanoate (88-INTA) was obtained as a pale yellow oil. LC-MS: ESI m/z=485.4[M+H] + . 300 mg of a yellow oily product was obtained. LC-MS: ESI m/z=501.4[M+H] + .
中间体89-INTB：甲基(S)-3-(5-(叔-丁基)-2-氟苯基)-4-(6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯的制备
Intermediate 89-INTB: Preparation of methyl (S)-3-(5-(tert-butyl)-2-fluorophenyl)-4-(6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-2-yl)butanoate
步骤1：参见中间体1-INTB步骤4的制备方法。粗品经正相硅胶柱色谱法纯化(DCM/MeOH＝100/0～8/1)得到无色油状产品甲基(E)-4-(6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-2-基)丁-2-烯酸酯(89-INTA)400mg。LC-MS:ESI m/z＝357.3[M+H]+。Step 1: See the preparation method of intermediate 1-INTB step 4. The crude product was purified by normal phase silica gel column chromatography (DCM/MeOH=100/0-8/1) to obtain 400 mg of methyl (E)-4-(6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-2-yl)but-2-enoate (89-INTA) as a colorless oily product. LC-MS: ESI m/z=357.3[M+H] + .
步骤2：参见中间体1-INTB步骤4的制备方法。粗品经高效液相色谱制备得到无色油状产品21mg。LC-MS:ESI m/z＝509.6[M+H]+。1H NMR(400MHz,METHANOL-d4)δ＝7.36-7.22(m,3H),6.99(br t,J＝9.5Hz,1H),6.52(d,J＝7.2Hz,1H),3.94(s,2H),3.73-3.46(m,9H),3.44-3.39(m,2H),3.27(s,2H),3.16-3.07(m,3H),2.83(br dd,J＝6.8,15.8Hz,1H),2.75(br t,J＝6.1Hz,2H),2.67(br dd,J＝8.0,16.0Hz,1H),2.31-2.14(m,2H),1.96-1.83(m,2H),1.39-1.26(m,9H)。Step 2: Refer to the preparation method of intermediate 1-INTB step 4. The crude product was purified by HPLC to obtain 21 mg of a colorless oily product. LC-MS: ESI m/z = 509.6 [M+H] + . 1 H NMR (400 MHz, METHANOL-d 4 ) δ＝7.36-7.22 (m, 3H), 6.99 (br t, J＝9.5 Hz, 1H), 6.52 (d, J＝7.2 Hz, 1H), 3.94 (s, 2H), 3.73-3.46 (m, 9H), 3.44-3.39 (m, 2H), 3.27 (s, 2H), 3.16-3.07 (m, 3H), 2.83 (br dd, J＝6.8, 15.8 Hz, 1H), 2.75 (br t, J＝6.1 Hz, 2H), 2.67 (br dd, J＝8.0, 16.0 Hz, 1H), 2.31-2.14 (m, 2H), 1.96-1.83 (m, 2H), 1.39-1.26 (m, 9H).
中间体90-INTB：甲基(S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(6-(2-(5,6,7,8-四氢-1,8-二氮杂萘-2-基)乙基)-2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯的制备
Intermediate 90-INTB: Preparation of methyl (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(6-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)-2,6-diazaspiro[3.4]octan-2-yl)butanoate
步骤1：参见中间体20-INTB步骤1的制备方法。粗品经正相硅胶柱色谱法纯化(DCM/MeOH＝10/1)得到黄色油状产品叔-丁基(S)-7-(2-(2-(2-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-甲氧基-4-羰基丁基)-2,6-二氮杂螺[3.4]辛烷-6-基)乙基)-3,4-二氢-1,8-二氮杂萘-1(2H)-羧酸酯120mg。LC-MS:ESI m/z＝643.8[M+H]+.Step 1: See the preparation method of intermediate 20-INTB step 1. The crude product was purified by normal phase silica gel column chromatography (DCM/MeOH=10/1) to obtain 120 mg of yellow oily product tert-butyl (S)-7-(2-(2-(2-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-methoxy-4-carbonylbutyl)-2,6-diazaspiro[3.4]octan-6-yl)ethyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylate. LC-MS: ESI m/z=643.8[M+H] + .
步骤2：参见中间体20-INTB步骤2的制备方法。粗品经高效液相色谱纯化得到黄色固体产品50mg。LC-MS:ESI m/z＝543.5[M+H]+。Step 2: See the preparation method of intermediate 20-INTB step 2. The crude product was purified by HPLC to obtain 50 mg of a yellow solid product. LC-MS: ESI m/z = 543.5 [M+H] + .
中间体91-INTB：甲基(S)-3-(3-(叔-丁基)-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(6-(2-(5,6,7,8- 四氢-1,8-二氮杂萘-2-基)乙基)-2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯的制备
Intermediate 91-INTB: methyl (S)-3-(3-(tert-butyl)-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(6-(2-(5,6,7,8- Preparation of tetrahydro-1,8-naphthyridin-2-yl)ethyl)-2,6-diazaspiro[3.4]octan-2-yl)butyrate
以中间体43-INTA为起始原料，制备方法和步骤同90-INTB的制备。得到黄色油状产品400mg。LC-MS:ESI m/z＝599.6[M+H]+。The intermediate 43-INTA was used as the starting material, and the preparation method and steps were the same as those for 90-INTB to obtain 400 mg of a yellow oily product. LC-MS: ESI m/z = 599.6 [M+H] + .
中间体92-INTB：甲基(S)-3-(3-溴-5-(叔-丁基)苯基)-4-(6-(2-(5,6,7,8-四氢-1,8-二氮杂萘-2-基)乙基)-2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯的制备
Intermediate 92-INTB: Preparation of methyl (S)-3-(3-bromo-5-(tert-butyl)phenyl)-4-(6-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)-2,6-diazaspiro[3.4]octan-2-yl)butanoate
以中间体82-INTA为起始原料，制备方法和步骤同90-INTB的制备。得到黄色油状产品350mg。LC-MS:ESI m/z＝583.4[M+H]+。The intermediate 82-INTA was used as the starting material, and the preparation method and steps were the same as those for 90-INTB to obtain 350 mg of a yellow oily product. LC-MS: ESI m/z = 583.4 [M+H] + .
中间体93-INTB：甲基(S)-3-(5-溴-2-氟苯基)-4-(6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.3]庚烷-2-基)丁酸酯的制备
Intermediate 93-INTB: Preparation of methyl (S)-3-(5-bromo-2-fluorophenyl)-4-(6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.3]heptane-2-yl)butanoate
以甲基(E)-4-(2,6-二氮杂螺[3.3]庚烷-2-基)丁-2-烯酸酯为起始原料，制备方法和步骤同89-INTB的制备。得到黄色油状中间体甲基(E)-4-(6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.3]庚烷-2-基)丁-2-烯酸酯(93-INTA)220mgmg。LC-MS:ESI m/z＝343.4[M+H]+；1H NMR(400MHz,CDCl3)δ7.16(d,J＝7.21Hz,1H)6.81(m,1H)6.49(d,J＝7.34Hz,1H)5.92(br d,J＝15.77Hz,1H)5.75(br s,1H)3.74(s,3H)3.62(s,2H)3.58(s,4H)3.42(br s,2H)3.34(s,4H)3.16(br d,J＝4.52Hz,2H)2.72(br t,J＝6.11Hz,2H)1.91(m,2H)。得到黄色油状产品55mg。LC-MS:ESI m/z＝517.1[M+H]+。Using methyl (E)-4-(2,6-diazaspiro[3.3]heptane-2-yl)but-2-enoate as the starting material, the preparation method and steps were the same as those for 89-INTB to obtain 220 mgmg of the yellow oily intermediate methyl (E)-4-(6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.3]heptane-2-yl)but-2-enoate (93-INTA). LC-MS: ESI m/z=343.4 [M+H] + ; 1 H NMR (400 MHz, CDCl3) δ7.16 (d, J=7.21 Hz, 1H) 6.81 (m, 1H) 6.49 (d, J=7.34 Hz, 1H) 5.92 (br d, J=15.77 Hz, 1H) 5.75 (br s, 1H) 3.74 (s, 3H) 3.62 (s, 2H) 3.58 (s, 4H) 3.42 (br s, 2H) 3.34 (s, 4H) 3.16 (br d, J=4.52 Hz, 2H) 2.72 (br t, J=6.11 Hz, 2H) 1.91 (m, 2H). 55 mg of a yellow oily product was obtained. LC-MS: ESI m/z=517.1 [M+H] + .
中间体94-INTB：甲基(3S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(1-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-1,7-二氮杂螺[4.4]壬烷-7-基)丁酸酯的制备
Intermediate 94-INTB: Preparation of methyl (3S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(1-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-1,7-diazaspiro[4.4]nonan-7-yl)butanoate
步骤1：参见中间体3-INTB步骤3的制备方法。粗品经正相硅胶柱色谱法纯化(100％石油醚～乙酸乙酯＝1/2)得到黄色油状产品叔-丁基(E)-7-(4-甲氧基-4-羰基丁-2-烯-1-基)-1,7-二氮杂螺[4.4]壬烷-1-羧酸酯190mg。1H NMR(400MHz,CDCl3)δ6.90(m,1H)5.92(br d,J＝15.65Hz,1H)3.67(s,3H)3.24-3.49(m,2H)3.12-3.23(m,2H)2.85(m,1H)2.57-2.66(m,1H)2.45-2.54(m,2H)2.06(m,1H)1.57-1.84(m,5H)1.36-1.45(s,9H)。Step 1: Refer to the preparation method of intermediate 3-INTB step 3. The crude product was purified by normal phase silica gel column chromatography (100% petroleum ether to ethyl acetate = 1/2) to obtain 190 mg of yellow oily product tert-butyl (E)-7-(4-methoxy-4-carbonylbut-2-en-1-yl)-1,7-diazaspiro[4.4]nonane-1-carboxylate. 1 H NMR (400 MHz, CDCl3) δ 6.90 (m, 1H) 5.92 (br d, J = 15.65 Hz, 1H) 3.67 (s, 3H) 3.24-3.49 (m, 2H) 3.12-3.23 (m, 2H) 2.85 (m, 1H) 2.57-2.66 (m, 1H) 2.45-2.54 (m, 2H) 2.06 (m, 1H) 1.57-1.84 (m, 5H) 1.36-1.45 (s, 9H).
步骤2：参见中间体1-INTB步骤4的制备方法。粗品经正相硅胶柱色谱法纯化(EA)得到黄色油状产品叔-丁基7-((S)-2-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-甲氧基-4-羰基丁基)-1,7-二氮杂螺[4.4]壬烷-1-羧酸酯140mg。LC-MS:ESI m/z＝497.6[M+H]+。Step 2: Refer to the preparation method of intermediate 1-INTB step 4. The crude product was purified by normal phase silica gel column chromatography (EA) to obtain 140 mg of yellow oily product tert-butyl 7-((S)-2-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-methoxy-4-carbonylbutyl)-1,7-diazaspiro[4.4]nonane-1-carboxylate. LC-MS: ESI m/z=497.6[M+H] + .
步骤3：参见中间体3-INTB步骤2的制备方法。得到淡黄色油状产品甲基(3S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(1,7-二氮杂螺[4.4]壬烷-7-基)丁酸酯(94-INTA)160mg。LC-MS:ESI m/z＝397.3[M+H]+。Step 3: Refer to the preparation method of intermediate 3-INTB in step 2. 160 mg of methyl (3S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(1,7-diazaspiro[4.4]nonan-7-yl)butanoate (94-INTA) was obtained as a light yellow oily product. LC-MS: ESI m/z=397.3 [M+H] + .
步骤4：参见中间体5-INTB步骤3的制备方法。粗品经正相硅胶柱色谱法纯化(100％DCM～甲醇＝4/1)得到黄色油状产品200mg。LC-MS:ESI m/z＝543.6[M+H]+。Step 4: Refer to the preparation method of intermediate 5-INTB in step 3. The crude product was purified by normal phase silica gel column chromatography (100% DCM-methanol = 4/1) to obtain 200 mg of a yellow oily product. LC-MS: ESI m/z = 543.6 [M+H] + .
中间体95-INTB：甲基(S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(7-(((5,6,7,8-四氢-1,8-二氮杂萘-2-基)氨基)甲基)-3,4-二氢异喹啉-2(1H)-基)丁酸酯的制备
Intermediate 95-INTB: Preparation of methyl (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(7-(((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)amino)methyl)-3,4-dihydroisoquinolin-2(1H)-yl)butanoate
步骤1：将吡啶-2,6-二胺(5g，45.8mmol)和1,1,3,3-四甲氧基丙烷(7.5g，45.8mmol)溶于磷酸(50ml)中，氮气保护下，升温至70℃，搅拌2小时。0℃下用5M的氢氧化钠水溶液调节反应液的pH＝10，过滤并且用DCM冲洗滤饼(50ml*3)，将得到的溶液继续用DCM萃取(200ml*3)，有机相用饱和食盐水洗(100ml*2)，Na2SO4干燥，过滤，旋干即得到粗产品。粗产品用正相硅胶柱色谱法纯化(DCM/MeOH＝10/1)得到红色固体产品1,8-二氮杂萘-2-胺300mg。LC-MS:ESI m/z＝146.3[M+H]+。Step 1: Dissolve pyridine-2,6-diamine (5g, 45.8mmol) and 1,1,3,3-tetramethoxypropane (7.5g, 45.8mmol) in phosphoric acid (50ml), heat to 70°C under nitrogen protection, and stir for 2 hours. Adjust the pH of the reaction solution to 10 with 5M sodium hydroxide aqueous solution at 0°C, filter and rinse the filter cake with DCM (50ml*3), continue to extract the obtained solution with DCM (200ml*3), wash the organic phase with saturated brine (100ml*2), dry with Na 2 SO 4 , filter, and spin dry to obtain a crude product. The crude product is purified by normal phase silica gel column chromatography (DCM/MeOH=10/1) to obtain 300mg of a red solid product 1,8-diazine-2-amine. LC-MS:ESI m/z=146.3[M+H] + .
步骤2：参见中间体A-2的制备方法，得到粗产品5,6,7,8-四氢-1,8-二氮杂萘-2-胺290mg。LC-MS:ESI m/z＝151.4[M+H]+。Step 2: Referring to the preparation method of intermediate A-2, 290 mg of crude product 5,6,7,8-tetrahydro-1,8-naphthyridin-2-amine was obtained. LC-MS: ESI m/z=151.4 [M+H] + .
步骤3：参见中间体3-INTB步骤2的制备方法。得到粗产品7-溴-1,2,3,4-四氢异喹啉2.3g。LC-MS:ESI m/z＝215.1[M+H]+。Step 3: Refer to the preparation method of intermediate 3-INTB in step 2. Obtain 2.3 g of crude product 7-bromo-1,2,3,4-tetrahydroisoquinoline. LC-MS: ESI m/z=215.1 [M+H] + .
步骤4：参见中间体6-INTB步骤1的制备方法。粗品经正相硅胶柱色谱法纯化(PE/EA＝1/1)得到黄色油状产品甲基(E)-4-(7-溴-3,4-二氢异喹啉-2(1H)-基)丁-2-烯酸酯630mg。LC-MS:ESI m/z＝312.3[M+H]+。Step 4: Refer to the preparation method of intermediate 6-INTB in step 1. The crude product was purified by normal phase silica gel column chromatography (PE/EA=1/1) to obtain 630 mg of methyl (E)-4-(7-bromo-3,4-dihydroisoquinolin-2(1H)-yl)but-2-enoate as a yellow oily product. LC-MS: ESI m/z=312.3 [M+H] + .
步骤5：参见中间体1-INTB步骤4的制备方法。粗品经正相硅胶柱色谱法纯化(PE/EA＝1/1)后得到黄色油状产品甲基(S)-4-(7-溴-3,4-二氢异喹啉-2(1H)-基)-3-(3-(3,5-二甲基-1H-吡唑-1- 基)苯基)丁酸酯600mg。LC-MS:ESI m/z＝481.6[M+H]+。Step 5: Refer to the preparation method of intermediate 1-INTB in step 4. The crude product was purified by normal phase silica gel column chromatography (PE/EA=1/1) to obtain a yellow oily product methyl (S)-4-(7-bromo-3,4-dihydroisoquinolin-2(1H)-yl)-3-(3-(3,5-dimethyl-1H-pyrazole-1- 600 mg of 2-(4-(2-amino-4-phenyl)butyrate. LC-MS: ESI m/z=481.6 [M+H] + .
步骤6：参见中间体D-4的制备方法，粗品用正相硅胶柱色谱法纯化(PE/EA＝1/1)得到黄色油状产品甲基(S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(7-乙烯基-3,4-二氢异喹啉-2(1H)-基)丁酸酯340mg。LC-MS:ES m/z＝452.4[M+Na]+。Step 6: Referring to the preparation method of intermediate D-4, the crude product was purified by normal phase silica gel column chromatography (PE/EA=1/1) to obtain 340 mg of methyl (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(7-vinyl-3,4-dihydroisoquinolin-2(1H)-yl)butanoate as a yellow oily product. LC-MS: ES m/z=452.4[M+Na] + .
步骤7：参见中间体D-6的制备方法，粗品经高效液相色谱纯化得到黄色油状产品甲基(S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(7-甲酰基-3,4-二氢异喹啉-2(1H)-基)丁酸酯(95-INTA)70mg。LC-MS:ESI m/z＝432.0[M+H]+。Step 7: Referring to the preparation method of intermediate D-6, the crude product was purified by HPLC to obtain 70 mg of yellow oily product methyl (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(7-formyl-3,4-dihydroisoquinolin-2(1H)-yl)butanoate (95-INTA). LC-MS: ESI m/z=432.0 [M+H] + .
步骤8：参见中间体1-INTB步骤3的制备方法。粗品经高效液相色谱纯化得到黄色固体产品10mg。LC-MS:ESI m/z＝565.4[M+H]+。Step 8: Refer to the preparation method of intermediate 1-INTB in step 3. The crude product was purified by HPLC to obtain 10 mg of a yellow solid product. LC-MS: ESI m/z = 565.4 [M+H] + .
中间体96-INTB：甲基(S)-4-(5,5-二氟-7-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,7-二氮杂螺[3.5]壬烷-2-基)-3-(5-(3,5-二甲基-1H-吡唑-1-基)-2-氟苯基)丁酸酯的制备
Intermediate 96-INTB: Preparation of methyl (S)-4-(5,5-difluoro-7-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,7-diazaspiro[3.5]nonan-2-yl)-3-(5-(3,5-dimethyl-1H-pyrazol-1-yl)-2-fluorophenyl)butanoate
以叔-丁基5,5-二氟-2,7-二氮杂螺[3.5]壬烷-7-羧酸酯为起始原料根据中间体3-INTB步骤3的制备方法获得中间体叔-丁基(E)-5,5-二氟-2-(4-甲氧基-4-羰基丁-2-烯-1-基)-2,7-二氮杂螺[3.5]壬烷-7-羧酸酯，以此为原料后续制备方法和步骤同80-INTB的制备。得到黄色油状产品甲基(S)-4-(5,5-二氟-2,7-二氮杂螺[3.5]壬烷-2-基)-3-(5-(3,5-二甲基-1H-吡唑-1-基)-2-氟苯基)丁酸酯(96-INTA)60mg。LC-MS:ESI m/z＝451.2[M+H]+。得到黄色油状产品40mg。LC-MS:ESI m/z＝597.3[M+H]+。Using tert-butyl 5,5-difluoro-2,7-diazaspiro[3.5]nonane-7-carboxylate as the starting material, the intermediate tert-butyl (E)-5,5-difluoro-2-(4-methoxy-4-carbonylbut-2-en-1-yl)-2,7-diazaspiro[3.5]nonane-7-carboxylate was obtained according to the preparation method of step 3 of intermediate 3-INTB. The subsequent preparation method and steps were the same as those of 80-INTB. 60 mg of methyl (S)-4-(5,5-difluoro-2,7-diazaspiro[3.5]nonane-2-yl)-3-(5-(3,5-dimethyl-1H-pyrazol-1-yl)-2-fluorophenyl)butanoate (96-INTA) was obtained as a yellow oily product. LC-MS: ESI m/z=451.2[M+H] + . 40 mg of a yellow oily product was obtained. LC-MS: ESI m/z=597.3 [M+H] + .
中间体97-INTB：甲基(S)-3-(3-(叔-丁基)-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(5,5-二氟-7-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,7-二氮杂螺[3.5]壬烷-2-基)丁酸酯的制备
Intermediate 97-INTB: Preparation of methyl (S)-3-(3-(tert-butyl)-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(5,5-difluoro-7-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,7-diazaspiro[3.5]nonan-2-yl)butanoate
步骤1：参见中间体1-INTB步骤4的制备方法。粗品经高效液相色谱纯化得到黄色固体产品10mg。LC-MS:ESI m/z＝635.4[M+H]+。Step 1: Refer to the preparation method of intermediate 1-INTB step 4. The crude product was purified by HPLC to obtain 10 mg of a yellow solid product. LC-MS: ESI m/z = 635.4 [M+H] + .
中间体98-INTB：甲基(S)-3-(3-溴-5-(叔-丁基)苯基)-4-(5,5-二氟-7-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,7-二氮杂螺[3.5]壬烷-2-基)丁酸酯的制备
Intermediate 98-INTB: Preparation of methyl (S)-3-(3-bromo-5-(tert-butyl)phenyl)-4-(5,5-difluoro-7-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,7-diazaspiro[3.5]nonan-2-yl)butanoate
步骤1：参见中间体1-INTB步骤4的制备方法。粗品经高效液相色谱纯化得到黄色固体产品10mg。LC-MS:ESI m/z＝605.2[M+H]+。Step 1: Refer to the preparation method of intermediate 1-INTB step 4. The crude product was purified by HPLC to obtain 10 mg of a yellow solid product. LC-MS: ESI m/z = 605.2 [M+H] + .
中间体99-INTB：甲基(S)-3-(3-溴苯基)-4-(5,5-二氟-7-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,7-二氮杂螺[3.5]壬烷-2-基)丁酸酯的制备
Intermediate 99-INTB: Preparation of methyl (S)-3-(3-bromophenyl)-4-(5,5-difluoro-7-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,7-diazaspiro[3.5]nonan-2-yl)butanoate
步骤1：参见中间体1-INTB步骤4的制备方法。粗品经高效液相色谱纯化得到黄色固体产品10mg。LC-MS:ESI m/z＝563.2[M+H]+。Step 1: Refer to the preparation method of intermediate 1-INTB step 4. The crude product was purified by HPLC to obtain 10 mg of a yellow solid product. LC-MS: ESI m/z = 563.2 [M+H] + .
中间体100-INTB：甲基(S)-3-(5-溴-2-氟苯基)-4-(5,5-二氟-7-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,7-二氮杂螺[3.5]壬烷-2-基)丁酸酯的制备
Intermediate 100-INTB: Preparation of methyl (S)-3-(5-bromo-2-fluorophenyl)-4-(5,5-difluoro-7-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,7-diazaspiro[3.5]nonan-2-yl)butanoate
步骤1：参见中间体1-INTB步骤4的制备方法。粗品经高效液相色谱纯化得到黄色固体产品10mg。LC-MS:ESI m/z＝581.2[M+H]+。Step 1: Refer to the preparation method of intermediate 1-INTB step 4. The crude product was purified by HPLC to obtain 10 mg of a yellow solid product. LC-MS: ESI m/z = 581.2 [M+H] + .
中间体101-INTB:甲基(S)-4-(5,5-二氟-7-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,7-二氮杂螺[3.5]壬烷-2-基)-3-(3-(2-甲氧基乙氧基)苯基)丁酸酯的制备
Intermediate 101-INTB: Preparation of methyl (S)-4-(5,5-difluoro-7-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,7-diazaspiro[3.5]nonan-2-yl)-3-(3-(2-methoxyethoxy)phenyl)butyrate
步骤1：参见中间体1-INTB步骤4的制备方法。粗品经高效液相色谱纯化得到黄色固体产品10mg。LC-MS:ESI m/z＝559.3[M+H]+。Step 1: Refer to the preparation method of intermediate 1-INTB step 4. The crude product was purified by HPLC to obtain 10 mg of a yellow solid product. LC-MS: ESI m/z = 559.3 [M+H] + .
中间体102-INTB：甲基(S)-4-(5,5-二氟-7-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,7-二氮杂螺[3.5]壬烷-2-基)-3-(5-(3,5-二甲基-1H-吡唑-1-基)吡啶-3-基)丁酸酯的制备
Intermediate 102-INTB: Preparation of methyl (S)-4-(5,5-difluoro-7-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,7-diazaspiro[3.5]nonan-2-yl)-3-(5-(3,5-dimethyl-1H-pyrazol-1-yl)pyridin-3-yl)butanoate
步骤1：参见中间体1-INTB步骤4的制备方法。粗品经高效液相色谱纯化得到黄色固体产品甲基(S)-3-(5-溴吡啶-3-基)-4-(5,5-二氟-7-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,7-二氮杂螺[3.5]壬烷-2-基)丁酸酯50mg。LC-MS:ESI m/z＝564.2[M+H]+。 Step 1: See the preparation method of intermediate 1-INTB step 4. The crude product was purified by HPLC to obtain 50 mg of methyl (S)-3-(5-bromopyridin-3-yl)-4-(5,5-difluoro-7-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,7-diazaspiro[3.5]nonane-2-yl)butanoate as a yellow solid product. LC-MS: ESI m/z=564.2[M+H] + .
步骤2：参见中间体44-INTB步骤2的制备方法。粗品经高效液相色谱纯化得到黄色固体产品15mg。LC-MS:ESI m/z＝580.3[M+H]+。Step 2: See the preparation method of intermediate 44-INTB step 2. The crude product was purified by HPLC to obtain 15 mg of a yellow solid product. LC-MS: ESI m/z = 580.3 [M+H] + .
中间体103-INTB：甲基(S)-4-(5,5-二氟-7-(2-(5,6,7,8-四氢-1,8-二氮杂萘-2-基)乙基)-2,7-二氮杂螺[3.5]壬烷-2-基)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)丁酸酯的制备
Intermediate 103-INTB: Preparation of methyl (S)-4-(5,5-difluoro-7-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)-2,7-diazaspiro[3.5]nonan-2-yl)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)butanoate
步骤1：参见中间体20-INTB步骤1的制备方法。得到黄色油状产品叔-丁基7-(2-(2-(叔-丁氧基羰基)-5,5-二氟-2,7-二氮杂螺[3.5]壬烷-7-基)乙基)-3,4-二氢-1,8-二氮杂萘-1(2H)-羧酸酯。LC-MS:ESI m/z＝523.3[M+H]+。Step 1: Refer to the preparation method of intermediate 20-INTB step 1. Obtain yellow oily product tert-butyl 7-(2-(2-(tert-butoxycarbonyl)-5,5-difluoro-2,7-diazaspiro[3.5]nonan-7-yl)ethyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylate. LC-MS: ESI m/z=523.3[M+H] + .
步骤2：参见中间体3-INTB步骤2的制备方法。得到黄色油状产品7-(2-(5,5-二氟-2,7-二氮杂螺[3.5]壬烷-7-基)乙基)-1,2,3,4-四氢-1,8-二氮杂萘。LC-MS:ESI m/z＝323.3[M+H]+。Step 2: Refer to the preparation method of intermediate 3-INTB step 2. Obtain yellow oily product 7-(2-(5,5-difluoro-2,7-diazaspiro[3.5]nonan-7-yl)ethyl)-1,2,3,4-tetrahydro-1,8-naphthyridine. LC-MS: ESI m/z=323.3 [M+H] + .
步骤3：参见中间体3-INTB步骤3的制备方法。得到黄色油状产品甲基(E)-4-(5,5-二氟-7-(2-(5,6,7,8-四氢-1,8-二氮杂萘-2-基)乙基)-2,7-二氮杂螺[3.5]壬烷-2-基)丁-2-烯酸酯(103-INTA)。LC-MS:ESI m/z＝421.2[M+H]+。Step 3: Refer to the preparation method of intermediate 3-INTB step 3. A yellow oily product, methyl (E)-4-(5,5-difluoro-7-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)-2,7-diazaspiro[3.5]nonane-2-yl)but-2-enoate (103-INTA), was obtained. LC-MS: ESI m/z=421.2 [M+H] + .
步骤4：参见中间体1-INTB步骤4的制备方法。得到淡黄色固体产品。LC-MS:ESI m/z＝593.3[M+H]+。Step 4: Refer to the preparation method of intermediate 1-INTB step 4. A pale yellow solid product is obtained. LC-MS: ESI m/z=593.3 [M+H] + .
中间体104-INTB：甲基(3S)-3-(3-(叔-丁基)-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(5-氟-7-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,7-二氮杂螺[3.5]壬烷-2-基)丁酸酯的制备
Intermediate 104-INTB: Preparation of methyl (3S)-3-(3-(tert-butyl)-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(5-fluoro-7-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,7-diazaspiro[3.5]nonan-2-yl)butanoate
步骤1：参见中间体1-INTB步骤4的制备方法。粗品经高效液相色谱纯化得到黄色固体产品50mg。LC-MS:ESI m/z＝617.4[M+H]+。Step 1: Refer to the preparation method of intermediate 1-INTB step 4. The crude product was purified by HPLC to obtain 50 mg of a yellow solid product. LC-MS: ESI m/z = 617.4 [M+H] + .
中间体105-INTB：甲基(3S)-3-(3-溴-5-(叔-丁基)苯基)-4-(5-氟-7-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,7-二氮杂螺[3.5]壬烷-2-基)丁酸酯的制备
Intermediate 105-INTB: Preparation of methyl (3S)-3-(3-bromo-5-(tert-butyl)phenyl)-4-(5-fluoro-7-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,7-diazaspiro[3.5]nonan-2-yl)butanoate
步骤1：参见中间体1-INTB步骤4的制备方法。粗品经高效液相色谱纯化得到黄色固体 产品50mg。LC-MS:ESI m/z＝601.2[M+H]+。Step 1: See the preparation method of intermediate 1-INTB step 4. The crude product was purified by HPLC to obtain a yellow solid Product 50 mg. LC-MS: ESI m/z=601.2 [M+H] + .
中间体106-INTB：甲基(3S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(5-氟-7-(2-(5,6,7,8-四氢-1,8-二氮杂萘-2-基)乙基)-2,7-二氮杂螺[3.5]壬烷-2-基)丁酸酯的制备
Intermediate 106-INTB: Preparation of methyl (3S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(5-fluoro-7-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)-2,7-diazaspiro[3.5]nonan-2-yl)butanoate
以叔-丁基5-氟-2,7-二氮杂螺[3.5]壬烷-2-羧酸酯为起始原料，制备方法和步骤同103-INTB的制备。得到黄色油状产品甲基(E)-4-(5-氟-7-(2-(5,6,7,8-四氢-1,8-二氮杂萘-2-基)乙基)-2,7-二氮杂螺[3.5]壬烷-2-基)丁-2-烯酸酯(106-INTA)60mg。LC-MS:ESI m/z＝403.2[M+H]+。得到黄色油状产品40mg。LC-MS:ESI m/z＝575.3[M+H]+。Using tert-butyl 5-fluoro-2,7-diazaspiro[3.5]nonane-2-carboxylate as the starting material, the preparation method and steps are the same as those for 103-INTB. 60 mg of methyl (E)-4-(5-fluoro-7-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)-2,7-diazaspiro[3.5]nonane-2-yl)but-2-enoate (106-INTA) was obtained as a yellow oily product. LC-MS: ESI m/z=403.2[M+H] + . 40 mg of a yellow oily product was obtained. LC-MS: ESI m/z=575.3[M+H] + .
中间体107-INTB：甲基(S)-4-(8,8-二氟-6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-2-基)-3-(5-(3,5-二甲基-1H-吡唑-1-基)-2-氟苯基)丁酸酯的制备
Intermediate 107-INTB: Preparation of methyl (S)-4-(8,8-difluoro-6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-2-yl)-3-(5-(3,5-dimethyl-1H-pyrazol-1-yl)-2-fluorophenyl)butanoate
中间体107-INTA的制备：以叔-丁基8,8-二氟-2,6-二氮杂螺[3.4]辛烷-2-羧酸酯为起始原料，制备方法和步骤同103-INTA的制备。Preparation of intermediate 107-INTA: Using tert-butyl 8,8-difluoro-2,6-diazaspiro[3.4]octane-2-carboxylate as the starting material, the preparation method and steps are the same as those for 103-INTA.
步骤1：参见中间体1-INTB步骤4的制备方法。粗品经高效液相色谱纯化得到黄色固体产品甲基(S)-3-(5-溴-2-氟苯基)-4-(8,8-二氟-6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯50mg。LC-MS:ESI m/z＝567.2[M+H]+。Step 1: Refer to the preparation method of intermediate 1-INTB step 4. The crude product was purified by HPLC to obtain 50 mg of yellow solid product methyl (S)-3-(5-bromo-2-fluorophenyl)-4-(8,8-difluoro-6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-2-yl)butanoate. LC-MS: ESI m/z=567.2[M+H] + .
步骤2：参见中间体80-INTB步骤2的制备方法。粗品经正相硅胶柱色谱法纯化(DCM/MeOH＝10/1)得到黄色油状产品20mg。LC-MS:ESI m/z＝583.3[M+H]+。Step 2: See the preparation method of intermediate 80-INTB step 2. The crude product was purified by normal phase silica gel column chromatography (DCM/MeOH=10/1) to obtain 20 mg of a yellow oily product. LC-MS: ESI m/z=583.3 [M+H] + .
中间体108-INTB：甲基(S)-3-(3-(叔-丁基)-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(8,8-二氟-6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯的制备
Intermediate 108-INTB: Preparation of methyl (S)-3-(3-(tert-butyl)-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(8,8-difluoro-6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-2-yl)butanoate
步骤1：参见中间体1-INTB步骤4的制备方法。粗品经高效液相色谱纯化得到黄色固体产品50mg。LC-MS:ESI m/z＝621.4[M+H]+。Step 1: Refer to the preparation method of intermediate 1-INTB step 4. The crude product was purified by HPLC to obtain 50 mg of a yellow solid product. LC-MS: ESI m/z = 621.4 [M+H] + .
中间体109-INTB：甲基(S)-3-(3-溴-5-(叔-丁基)苯基)-4-(8,8-二氟-6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯的制备
Intermediate 109-INTB: Preparation of methyl (S)-3-(3-bromo-5-(tert-butyl)phenyl)-4-(8,8-difluoro-6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-2-yl)butanoate
步骤1：参见中间体1-INTB步骤4的制备方法。粗品经高效液相色谱纯化得到黄色固体产品50mg。LC-MS:ESI m/z＝605.4[M+H]+。Step 1: Refer to the preparation method of intermediate 1-INTB step 4. The crude product was purified by HPLC to obtain 50 mg of a yellow solid product. LC-MS: ESI m/z = 605.4 [M+H] + .
中间体110-INTB：甲基(S)-4-(8,8-二氟-6-(2-(5,6,7,8-四氢-1,8-二氮杂萘-2-基)乙基)-2,6-二氮杂螺[3.4]辛烷-2-基)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)丁酸酯的制备
Intermediate 110-INTB: Preparation of methyl (S)-4-(8,8-difluoro-6-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)-2,6-diazaspiro[3.4]octan-2-yl)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)butanoate
以叔-丁基8,8-二氟-2,6-二氮杂螺[3.4]辛烷-2-羧酸酯为起始原料，制备方法和步骤同103-INTB的制备。得到黄色油状产品甲基(E)-4-(8,8-二氟-6-(2-(5,6,7,8-四氢-1,8-二氮杂萘-2-基)乙基)-2,6-二氮杂螺[3.4]辛烷-2-基)丁-2-烯酸酯(110-INTA)60mg。LC-MS:ESI m/z＝407.2[M+H]+。得到黄色油状产品40mg。LC-MS:ESI m/z＝579.3[M+H]+。Using tert-butyl 8,8-difluoro-2,6-diazaspiro[3.4]octane-2-carboxylate as the starting material, the preparation method and steps are the same as those for 103-INTB. 60 mg of methyl (E)-4-(8,8-difluoro-6-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)-2,6-diazaspiro[3.4]octane-2-yl)but-2-enoate (110-INTA) was obtained as a yellow oily product. LC-MS: ESI m/z=407.2[M+H] + . 40 mg of a yellow oily product was obtained. LC-MS: ESI m/z=579.3[M+H] + .
中间体111-INTB：甲基(3S)-3-(3-(叔-丁基)-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(8-氟-6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯的制备
Intermediate 111-INTB: Preparation of methyl (3S)-3-(3-(tert-butyl)-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(8-fluoro-6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-2-yl)butanoate
以叔-丁基8-氟-2,6-二氮杂螺[3.4]辛烷-6-羧酸酯为起始原料，制备方法和步骤同67-INTB的制备。得到淡黄色油状中间体甲基(3S)-3-(3-(叔-丁基)-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(8-氟-2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯(111-INTA)100mg。LC-MS:ESI m/z＝457.3[M+H]+。得到黄色油状产品85mg。LC-MS:ESI m/z＝603.4[M+H]+。Using tert-butyl 8-fluoro-2,6-diazaspiro[3.4]octane-6-carboxylate as the starting material, the preparation method and steps are the same as those for 67-INTB. 100 mg of the pale yellow oily intermediate methyl (3S)-3-(3-(tert-butyl)-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(8-fluoro-2,6-diazaspiro[3.4]octane-2-yl)butanoate (111-INTA) was obtained. LC-MS: ESI m/z=457.3[M+H] + . 85 mg of a yellow oily product was obtained. LC-MS: ESI m/z=603.4[M+H] + .
中间体112-INTB：甲基(3S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(8-氟-6-(2-(5,6,7,8-四氢-1,8-二氮杂萘-2-基)乙基)-2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯的制备
Intermediate 112-INTB: Preparation of methyl (3S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(8-fluoro-6-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)-2,6-diazaspiro[3.4]octan-2-yl)butanoate
步骤1：参见中间体1-INTB步骤4的制备。粗品经高效液相色谱纯化得到黄色固体产品50mg。LC-MS:ESI m/z＝561.3[M+H]+。 Step 1: Refer to the preparation of intermediate 1-INTB in step 4. The crude product was purified by HPLC to obtain 50 mg of a yellow solid product. LC-MS: ESI m/z = 561.3 [M+H] + .
中间体113-INTB：甲基(S)-3-(3-(叔-丁基)-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(8,8-二氟-2-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-6-基)丁酸酯的制备
Intermediate 113-INTB: Preparation of methyl (S)-3-(3-(tert-butyl)-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(8,8-difluoro-2-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-6-yl)butanoate
以叔-丁基(E)-8,8-二氟-6-(4-甲氧基-4-羰基丁-2-烯-1-基)-2,6-二氮杂螺[3.4]辛烷-2-羧酸酯和中间体I为起始原料，制备方法和步骤同76-INTB的制备。得到淡黄色油状中间体甲基(S)-3-(3-(叔-丁基)-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(8,8-二氟-2,6-二氮杂螺[3.4]辛烷-6-基)丁酸酯(113-INTA)290mg。LC-MS:ESI m/z＝475.3[M+H]+。得到黄色油状产品150mg。LC-MS:ESI m/z＝621.4[M+H]+。Using tert-butyl (E)-8,8-difluoro-6-(4-methoxy-4-carbonylbut-2-en-1-yl)-2,6-diazaspiro[3.4]octane-2-carboxylate and intermediate I as starting materials, the preparation method and steps are the same as the preparation of 76-INTB. The intermediate methyl (S)-3-(3-(tert-butyl)-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(8,8-difluoro-2,6-diazaspiro[3.4]octane-6-yl)butanoate (113-INTA) 290 mg was obtained as a pale yellow oil. LC-MS: ESI m/z=475.3[M+H] + . 150 mg of a yellow oily product was obtained. LC-MS: ESI m/z=621.4[M+H] + .
中间体114-INTB：甲基(S)-4-(8,8-二氟-2-(2-(5,6,7,8-四氢-1,8-二氮杂萘-2-基)乙基)-2,6-二氮杂螺[3.4]辛烷-6-基)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)丁酸酯的制备
Intermediate 114-INTB: Preparation of methyl (S)-4-(8,8-difluoro-2-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)-2,6-diazaspiro[3.4]octan-6-yl)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)butanoate
以叔-丁基8,8-二氟-2,6-二氮杂螺[3.4]辛烷-6-羧酸酯为起始原料，制备方法和步骤同103-INTB的制备。得到黄色油状产品甲基(E)-4-(8,8-二氟-2-(2-(5,6,7,8-四氢-1,8-二氮杂萘-2-基)乙基)-2,6-二氮杂螺[3.4]辛烷-6-基)丁-2-烯酸酯(114-INTA)60mg。得到黄色油状产品40mg。LC-MS:ESI m/z＝579.3[M+H]+。Using tert-butyl 8,8-difluoro-2,6-diazaspiro[3.4]octane-6-carboxylate as the starting material, the preparation method and steps are the same as those for 103-INTB. 60 mg of methyl (E)-4-(8,8-difluoro-2-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)-2,6-diazaspiro[3.4]octane-6-yl)but-2-enoate (114-INTA) was obtained as a yellow oily product. 40 mg of the yellow oily product was obtained. LC-MS: ESI m/z=579.3[M+H] + .
中间体115-INTB：甲基(3S)-3-(3-(叔-丁基)-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(8-氟-2-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-6-基)丁酸酯的制备
Intermediate 115-INTB: Preparation of methyl (3S)-3-(3-(tert-butyl)-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(8-fluoro-2-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-6-yl)butanoate
以叔-丁基8-氟-2,6-二氮杂螺[3.4]辛烷-2-羧酸酯为起始原料，制备方法和步骤同67-INTB的制备。得到淡黄色油状中间体甲基甲基(3S)-3-(3-(叔-丁基)-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(8-氟-2,6-二氮杂螺[3.4]辛烷-6-基)丁酸酯(115-INTA)110mg。LC-MS:ESI m/z＝603.4[M+H]+。Using tert-butyl 8-fluoro-2,6-diazaspiro[3.4]octane-2-carboxylate as the starting material, the preparation method and steps were the same as those for 67-INTB. 110 mg of the pale yellow oily intermediate methyl (3S)-3-(3-(tert-butyl)-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(8-fluoro-2,6-diazaspiro[3.4]octane-6-yl)butanoate (115-INTA) was obtained. LC-MS: ESI m/z=603.4[M+H] + .
中间体116-INTB：甲基(3S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(8-氟-2-(2-(5,6,7,8-四氢-1,8-二氮杂萘-2-基)乙基)-2,6-二氮杂螺[3.4]辛烷-6-基)丁酸酯的制备
Intermediate 116-INTB: Preparation of methyl (3S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(8-fluoro-2-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)-2,6-diazaspiro[3.4]octan-6-yl)butanoate
以叔-丁基8-氟-2,6-二氮杂螺[3.4]辛烷-6-羧酸酯为起始原料，制备方法和步骤同103-INTB的制备。得到黄色油状产品甲基(E)-4-(8-氟-2-(2-(5,6,7,8-四氢-1,8-二氮杂萘-2-基)乙基)-2,6-二氮杂螺[3.4]辛烷-6-基)丁-2-烯酸酯(116-INTA)60mg。LC-MS:ESI m/z＝561.3[M+H]+。中间体117-INTB：甲基(S)-3-(3-环丙基-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(8,8-二氟-6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯的制备
Using tert-butyl 8-fluoro-2,6-diazaspiro[3.4]octane-6-carboxylate as the starting material, the preparation method and steps were the same as those for 103-INTB. A yellow oily product, methyl (E)-4-(8-fluoro-2-(2-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)ethyl)-2,6-diazaspiro[3.4]octane-6-yl)but-2-enoate (116-INTA) (60 mg), was obtained. LC-MS: ESI m/z = 561.3 [M+H] + . Intermediate 117-INTB: Preparation of methyl (S)-3-(3-cyclopropyl-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(8,8-difluoro-6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-2-yl)butanoate
步骤1：以叔-丁基(E)-8,8-二氟-2-(4-甲氧基-4-羰基丁-2-烯-1-基)-2,6-二氮杂螺[3.4]辛烷-6-羧酸酯为起始原料，参见中间体1-INTB步骤4的制备方法。粗品经快速柱层析纯化得到中间体叔-丁基(S)-2-(2-(3-环丙基-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-甲氧基-4-羰基丁基)-8,8-二氟-2,6-二氮杂螺[3.4]辛烷-6-羧酸酯30mg。LC-MS:ESI m/z＝559[M+H]+。Step 1: Using tert-butyl (E)-8,8-difluoro-2-(4-methoxy-4-carbonylbut-2-en-1-yl)-2,6-diazaspiro[3.4]octane-6-carboxylate as the starting material, refer to the preparation method of intermediate 1-INTB step 4. The crude product was purified by flash column chromatography to obtain 30 mg of the intermediate tert-butyl (S)-2-(2-(3-cyclopropyl-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-methoxy-4-carbonylbutyl)-8,8-difluoro-2,6-diazaspiro[3.4]octane-6-carboxylate. LC-MS: ESI m/z=559[M+H]+.
步骤2：参见中间体3-INTB步骤2的制备方法。粗品30mg直接用于下一步。LC-MS:ESI m/z＝459[M+H]+。Step 2: Refer to the preparation method of intermediate 3-INTB step 2. 30 mg of the crude product was used directly in the next step. LC-MS: ESI m/z = 459 [M+H] + .
步骤3：将甲基(S)-3-(3-环丙基-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(8,8-二氟-2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯(30mg，0.1mmol)溶于DCM(1.5ml)中，向溶液中依次加入A-3(16mg，0.1mmol)、十八烷基三甲基溴化铵(STAB，42mg，0.2mmol)以及一滴醋酸，常温搅拌2小时。LCMS监测反应完全，加入水，DCM萃取，有机相用Na2SO4干燥，得到产品29mg。LC-MS:ESI m/z＝605[M+H]+。Step 3: Methyl (S)-3-(3-cyclopropyl-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(8,8-difluoro-2,6-diazaspiro[3.4]octan-2-yl)butanoate (30 mg, 0.1 mmol) was dissolved in DCM (1.5 ml), A-3 (16 mg, 0.1 mmol), octadecyltrimethylammonium bromide (STAB, 42 mg, 0.2 mmol) and a drop of acetic acid were added to the solution in sequence, and stirred at room temperature for 2 hours. LCMS monitored the reaction to be complete, water was added, and DCM was extracted. The organic phase was dried over Na 2 SO 4 to obtain 29 mg of the product. LC-MS: ESI m/z=605[M+H] + .
中间体118-INTB：甲基(S)-3-(3-环丁基-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(8,8-二氟-6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯的制备
Intermediate 118-INTB: Preparation of methyl (S)-3-(3-cyclobutyl-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(8,8-difluoro-6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-2-yl)butanoate
参见117-INTB的制备方法。LC-MS:ESI m/z＝619.4[M+H]+。See the preparation method of 117-INTB. LC-MS: ESI m/z=619.4 [M+H] + .
中间体119-INTB：甲基(S)-3-(3-环戊基-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(8,8-二氟-6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯的制备
Intermediate 119-INTB: Preparation of methyl (S)-3-(3-cyclopentyl-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(8,8-difluoro-6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-2-yl)butanoate
参见117-INTB的制备方法。LC-MS:ESI m/z＝633[M+H]+。See the preparation method of 117-INTB. LC-MS: ESI m/z=633 [M+H] + .
中间体120-INTB：甲基(S)-3-(3-环己基-5-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(8,8-二氟-6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯的制备
Intermediate 120-INTB: Preparation of methyl (S)-3-(3-cyclohexyl-5-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(8,8-difluoro-6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-2-yl)butanoate
参见117-INTB的制备方法。LC-MS:ESI m/z＝633.7[M+H]+。See the preparation method of 117-INTB. LC-MS: ESI m/z=633.7 [M+H] + .
中间体121-INTB：甲基(S)-3-(5-(叔-丁基)-[1,1'-联苯基]-3-基)-4-(8,8-二氟-6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯的制备
Intermediate 121-INTB: Preparation of methyl (S)-3-(5-(tert-butyl)-[1,1'-biphenyl]-3-yl)-4-(8,8-difluoro-6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-2-yl)butanoate
参见117-INTB的制备方法。LC-MS:ESI m/z＝603[M+H]+。See the preparation method of 117-INTB. LC-MS: ESI m/z=603 [M+H] + .
中间体122-INTB：甲基(S)-3-(3,5-二-叔-丁基苯基)-4-(8,8-二氟-6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-2-基)丁酸酯的制备
Intermediate 122-INTB: Preparation of methyl (S)-3-(3,5-di-tert-butylphenyl)-4-(8,8-difluoro-6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-2-yl)butanoate
参见117-INTB的制备方法。LC-MS:ESI m/z＝583[M+H]+。See the preparation method of 117-INTB. LC-MS: ESI m/z=583 [M+H] + .
实施例1～14、实施例19-21、实施例23-25、实施例29-54、实施例62-66的合成Synthesis of Examples 1 to 14, Examples 19 to 21, Examples 23 to 25, Examples 29 to 54, Examples 62 to 66
合成方法概述：将实施例的酯中间体溶于甲醇和水(体积比5:1)溶剂中，室温下加入氢氧化锂(5个当量)。室温搅拌约16小时后，LCMS检测原料反应完全，浓缩反应混合液，所得粗品经高效液相色谱制备(Column:Kromasil C18 150*30mm*5um；Condition:water(0.2％ammonium hydroxide)-ACN；Begin B:5；End B:100；Gradient Time(min):20；100％B Hold Time(Time):5；Flow Rate(ml/min):20；Detection wavelength:220nm and 254nm)纯化得到产品。













Summary of the synthesis method: The ester intermediate of the example was dissolved in methanol and water (volume ratio 5:1) solvent, and lithium hydroxide (5 equivalents) was added at room temperature. After stirring at room temperature for about 16 hours, LCMS detected that the raw material reaction was complete, and the reaction mixture was concentrated. The crude product was purified by high performance liquid chromatography (Column: Kromasil C18 150*30mm*5um; Condition: water (0.2% ammonium hydroxide)-ACN; Begin B: 5; End B: 100; Gradient Time (min): 20; 100% B Hold Time (Time): 5; Flow Rate (ml/min): 20; Detection wavelength: 220nm and 254nm) to obtain the product.













实施例2：(S)-3-(3-(3,5-二甲基-1H-吡唑-1-基)苯基)-4-(6-((5,6,7,8-四氢-1,8-二氮杂萘-2-基)甲基)-2,6-二氮杂螺[3.4]辛烷-2-基)丁酸(化合物2)的制备
Example 2: Preparation of (S)-3-(3-(3,5-dimethyl-1H-pyrazol-1-yl)phenyl)-4-(6-((5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)methyl)-2,6-diazaspiro[3.4]octan-2-yl)butanoic acid (Compound 2)
在氮气保护,室温条件下，将中间体2-INTB(29mg,0.1mmol)溶于4N HCl/dioxane(1mL)中，反应2h。LCMS显示反应完毕，浓缩反应液，粗品经Prep-HPLC提纯得白色固体产品10mg。LC-MS:ESI m/z:515.3[M+H]+；1HNMR(400MHz,CD3OD)δ7.48(t,J＝8.0Hz,1H),7.36-7.30(m,4H),6.53(d,J＝7.2Hz,1H),6.07(s,1H),3.85(d,J＝10.8Hz,6H),3.44-3.40(m,3H),3.38-3.30(m,2H),3.19(s,2H),3.01(t,J＝6.8Hz,2H),2.77-2.70(m,3H),2.63-2.58(m,1H),2.27(s,3H),2.25(s,3H),2.27-2.23(m,2H),1.91-1.85(m,2H)。Under nitrogen protection and room temperature, the intermediate 2-INTB (29 mg, 0.1 mmol) was dissolved in 4N HCl/dioxane (1 mL) and reacted for 2 h. LCMS showed that the reaction was complete, and the reaction solution was concentrated. The crude product was purified by Prep-HPLC to obtain 10 mg of a white solid product. LC-MS: ESI m/z: 515.3 [M+H] + ; 1 HNMR (400 MHz, CD 3 OD) δ7.48 (t, J = 8.0 Hz, 1H), 7.36-7.30 (m, 4H), 6.53 (d, J = 7.2 Hz, 1H), 6.07 (s, 1H), 3.85 (d, J = 10.8 Hz, 6H), 3.44-3.40 (m, 3H), 3.38-3.30 (m, 2H), 3.19 (s, 2H), 3.01 (t, J = 6.8 Hz, 2H), 2.77-2.70 (m, 3H), 2.63-2.58 (m, 1H), 2.27 (s, 3H), 2.25 (s, 3H), 2.27-2.23 (m, 2H), 1.91-1.85 (m, 2H).
实施例15～18、实施例22、实施例26-28、实施例55-61的合成Synthesis of Examples 15-18, Example 22, Examples 26-28, Examples 55-61
合成方法概述：将氢氧化钠溶于50％羟胺水溶液中，氮气保护下，降温至0℃。之后，将酯中间体溶于甲醇/THF(体积比1:1)中，并缓慢滴加入上述反应液中，室温继续搅拌2小时。LCMS检测原料反应完全，浓缩反应混合物，粗品经高效液相色谱(Column:Kromasil C18 150*30mm*5um；Condition:water(0.2％ammonium hydroxide)-ACN；Begin B:5；End B:100；Gradient Time(min):20；100％B Hold Time(Time):5；Flow Rate(ml/min):20；Detection wavelength:220nm and 254nm)纯化得到产品。


Synthesis method overview: Sodium hydroxide was dissolved in 50% hydroxylamine aqueous solution, and the temperature was lowered to 0°C under nitrogen protection. After that, the ester intermediate was dissolved in methanol/THF (volume ratio 1:1) and slowly added dropwise to the above reaction solution, and the mixture was stirred at room temperature for 2 hours. LCMS detected that the raw material reaction was complete, the reaction mixture was concentrated, and the crude product was purified by high performance liquid chromatography (Column: Kromasil C18 150*30mm*5um; Condition: water(0.2％ammonium hydroxide)-ACN; Begin B:5; End B:100; Gradient Time(min):20; 100％B Hold Time(Time):5; Flow Rate(ml/min):20; Detection wavelength:220nm and 254nm) to obtain the product.


效果实施例1：化合物与αVβ1，αVβ6和αVβ8蛋白的结合测试Effect Example 1: Binding test of compound to αVβ1, αVβ6 and αVβ8 proteins
1.1荧光各异向性测试原理(Fluorescence Anisotropy)1.1 Fluorescence Anisotropy Test Principle
基于的原理是通过检测荧光素标记的小分子与其它分子相互作用前后分子量的变化，计算水平方向及垂直方向的荧光偏振值作相关分析。如果被荧光标记小分子与大分子之间的结合平衡建立后，它受激发时运动慢，测得的荧光偏振光值会增高。如果荧光标记小分子与大分子之间的结合被其它配基取代，它在游离状态下的旋转或翻转速度会变快，发射光相对于激发光平面将去偏振化，测得的偏振光值降低，从而计算出样品的荧光各异向性。The principle is to detect the change in molecular weight of the fluorescently labeled small molecule before and after the interaction with other molecules, and calculate the fluorescence polarization values in the horizontal and vertical directions for correlation analysis. If the binding equilibrium between the fluorescently labeled small molecule and the macromolecule is established, it moves slowly when excited, and the measured fluorescence polarization light value will increase. If the binding between the fluorescently labeled small molecule and the macromolecule is replaced by other ligands, its rotation or flipping speed in the free state will become faster, the emitted light will be depolarized relative to the excitation light plane, and the measured polarization light value will decrease, thereby calculating the fluorescence anisotropy of the sample.
1.2实验方法1.2 Experimental methods
实验反应体系40ul，均设置复孔，αVβ6缓冲液为：50mM HEPES PH7.4,150mM Nacl,0.5mM CHAPS，0.4mM MgCl2，αVβ1和αVβ8缓冲液为：50mM HEPES PH7.4,150mM Nacl,0.5mM CHAPS，0.1mM MnCl2。荧光底物的工作浓度为1nM，αVβ6(R&D systems公司生产的重组人整合素，货号3817-AV)和αVβ8(R&D systems公司生产的重组人整合素，货号4135-AV)的工作浓度均为8nM；αVβ1(货号6579-AVB)工作浓度为12.5nM；化合物初筛浓度为1uM和100nM，DMSO终浓度为千分之二，所有成分在384孔板(corning货号：CLS3575)中混合室温(24℃左右)反应2小时后测定Anisotropy值，测试仪器为BioTek品牌SYNERGY neo2酶标仪，Excitation为485nM，emission为530nM。以只加缓冲液的孔为系统读数空白对照。取复孔平均值计算抑制率，化合物100nM条件下抑制率大于50％的化合物测定其IC50值。The experimental reaction system was 40ul, and duplicate wells were set up. The buffer for αVβ6 was: 50mM HEPES PH7.4, 150mM Nacl, 0.5mM CHAPS, 0.4mM MgCl2, and the buffer for αVβ1 and αVβ8 was: 50mM HEPES PH7.4, 150mM Nacl, 0.5mM CHAPS, 0.1mM MnCl2. The working concentration of the fluorescent substrate is 1nM, the working concentrations of αVβ6 (recombinant human integrin produced by R&D systems, catalog number 3817-AV) and αVβ8 (recombinant human integrin produced by R&D systems, catalog number 4135-AV) are both 8nM; the working concentration of αVβ1 (catalog number 6579-AVB) is 12.5nM; the initial screening concentrations of the compound are 1uM and 100nM, and the final concentration of DMSO is 0.2%. All components are mixed in a 384-well plate (corning catalog number: CLS3575) and reacted at room temperature (about 24°C) for 2 hours before determining the Anisotropy value. The testing instrument is the BioTek brand SYNERGY neo2 microplate reader, with an Excitation of 485nM and an emission of 530nM. The wells with only buffer added are used as blank controls for system readings. The inhibition rate is calculated by taking the average value of the duplicate wells, and the IC 50 value of the compound with an inhibition rate greater than 50% under the condition of 100nM of the compound is determined.
选用PLN-74809和GSK-3008348作为本实验的对照化合物。PLN-74809(CAS：2376257-44-0)以及GSK-3008348(CAS：1629249-33-7)是两种极具潜力的整合素配体分子。实验处理方法同上。PLN-74809 and GSK-3008348 were selected as control compounds in this experiment. PLN-74809 (CAS: 2376257-44-0) and GSK-3008348 (CAS: 1629249-33-7) are two very potential integrin ligand molecules. The experimental treatment method is the same as above.
1.3计算方法1.3 Calculation method
抑制率＝(C-F)/(C-B)*100％Inhibition rate = (C-F)/(C-B)*100%
其中，C为荧光底物与蛋白完全结合的Anisotropy值，F为化合物相应浓度下的Anisotropy值，B为荧光底物Anisotropy本底值。以化合物浓度和相应抑制率值(表1)作S曲线，计算相应IC50值(表2)。各实施例按照IC50值分为三档，a：IC50<100nM；b：IC50＝100-1000nM；c：IC50>1000nM。a档实施例对目标亚型具有优异的抑制活性；b档实施例具有较强的抑制活性；c档实施例抑制活性一般。Wherein, C is the Anisotropy value of complete binding of the fluorescent substrate to the protein, F is the Anisotropy value at the corresponding concentration of the compound, and B is the background value of the Anisotropy of the fluorescent substrate. The S curve is drawn with the compound concentration and the corresponding inhibition rate value (Table 1), and the corresponding IC 50 value is calculated (Table 2). Each embodiment is divided into three levels according to the IC 50 value, a: IC 50 <100nM; b: IC 50 =100-1000nM; c: IC 50 >1000nM. The a-level embodiment has excellent inhibitory activity against the target subtype; the b-level embodiment has a strong inhibitory activity; the c-level embodiment has a general inhibitory activity.
亚型选择性的计算与评价方法如下：The calculation and evaluation method of subtype selectivity is as follows:
Fold＝IC50(亚型1)/IC50(亚型2)Fold＝IC 50 (subtype 1)/IC 50 (subtype 2)
各实施例按照Fold比值分为三档，A：Fold>4.0；B：Fold＝1.0-4.0；C：Fold<1.0。A档实施例对亚型2相比亚型1具有强选择性；B档实施例具有中等选择性；C档没有选择性。Each embodiment is divided into three levels according to the Fold ratio, A: Fold>4.0; B: Fold=1.0-4.0; C: Fold<1.0. The A-level embodiment has strong selectivity for subtype 2 compared with subtype 1; the B-level embodiment has moderate selectivity; and the C-level has no selectivity.
1.4实验结果1.4 Experimental Results
表1本发明化合物对αvβ1、αvβ6和αvβ8亚型抑制活性的效果评价(抑制率)Table 1 Evaluation of the inhibitory activity of the compounds of the present invention on αvβ1, αvβ6 and αvβ8 subtypes (inhibition rate)
注A：抑制率≥50％；B：抑制率<50％；N/A表示该浓度下未做检测


Note: A: inhibition rate ≥ 50%; B: inhibition rate <50%; N/A means no test was performed at this concentration


表2本发明化合物对αvβ1、αvβ6和αvβ8亚型抑制活性(IC50值范围)以及化合物的αvβ1、αvβ8分别对αvβ6的选择性效果评价Table 2 Inhibitory activity (IC 50 range) of the compounds of the present invention against αvβ1, αvβ6 and αvβ8 subtypes and evaluation of the selective effects of αvβ1 and αvβ8 on αvβ6
注a：IC50<100nM；b：IC50＝100-1000nM；c：IC50>1000nM；A：Fold>4.0，说明化合物对某一亚型具有高选择性；B：Fold＝1.0-4.0，说明化合物对某一亚型具有中等选择性；C：Fold<1.0，说明化合物对某一亚型无选择性；N/A表示未做检测；


Note: a: IC50 < 100 nM; b: IC50 = 100-1000 nM; c: IC50 > 1000 nM; A: Fold > 4.0, indicating that the compound has high selectivity for a certain subtype; B: Fold = 1.0-4.0, indicating that the compound has moderate selectivity for a certain subtype; C: Fold < 1.0, indicating that the compound has no selectivity for a certain subtype; N/A means no test was performed;


如表1和表2所示，实施例分别对αvβ1、αvβ6和αvβ8亚型中的一个、两个或三个亚型均显现出不同的抑制效果，尤其对αvβ6单个亚型以及αvβ1、αvβ6两个亚型显示出优异的抑制作用和选择性，如实施例43、44、71、72、96-103(aaaCA型)等对αvβ1和αvβ6均有优异的抑制作用且对αvβ8有很好的选择性，选择性远优于阳性参照PLN-74809。实施例1、5、49-50、52-54、75、77-78等是对αvβ6具有极佳的抑制活性和特异的选择性。同时也筛选到对αvβ1、αvβ6和αvβ8均有抑制作用且没有选择性的化合物，且与阳性参照GSK3008348的活性和选择性相当，如实施例82、92、113、115等。由此可知本发明中的化合物对整合素αvβ1、αvβ6和αvβ8亚型具有优异的抑制活性和选择性，相较同类型分子具有优势。As shown in Table 1 and Table 2, the examples show different inhibitory effects on one, two or three subtypes of αvβ1, αvβ6 and αvβ8, respectively, and especially show excellent inhibitory effects and selectivity on the single subtype of αvβ6 and the two subtypes of αvβ1 and αvβ6, such as Examples 43, 44, 71, 72, 96-103 (aaaCA type) and the like have excellent inhibitory effects on both αvβ1 and αvβ6 and good selectivity for αvβ8, and the selectivity is much better than the positive reference PLN-74809. Examples 1, 5, 49-50, 52-54, 75, 77-78 and the like have excellent inhibitory activity and specific selectivity for αvβ6. At the same time, compounds that have inhibitory effects on αvβ1, αvβ6 and αvβ8 and are not selective were also screened, and the activity and selectivity are comparable to the positive reference GSK3008348, such as Examples 82, 92, 113, 115 and the like. It can be seen that the compounds of the present invention have excellent inhibitory activity and selectivity against integrin αvβ1, αvβ6 and αvβ8 subtypes, and have advantages over molecules of the same type.
效果实施例2：化合物对纤维化蛋白α-SMA、Fibronectin的促降解作用Effect Example 2: Effect of Compounds on Degradation of Fibrin Protein α-SMA and Fibronectin
2.1实验目的2.1 Experimental Purpose
纤维母细胞(Fibroblast，Fb)是纤维化疾病中最主要的效应细胞，被活化时可发生功能和表型改变，转分化为肌纤维母细胞(Myofibroblast，MFb)，具有收缩潜能及较强的胶原合成能力，是诱发纤维化病变的主要细胞过程。该细胞活化的表型特点在于表达α平滑肌肌动蛋白(α-Smooth muscle actin，α-SMA)和纤连蛋白(Fibronectin，FN)，因此可以通过这两种 蛋白的表达量变化，验证整合素配体对纤维母细胞活化的抑制作用。Fibroblasts (Fb) are the main effector cells in fibrotic diseases. When activated, they can undergo functional and phenotypic changes and transdifferentiate into myofibroblasts (MFb), which have contractile potential and strong collagen synthesis ability. This is the main cellular process that induces fibrotic lesions. The phenotypic characteristics of activated cells are the expression of α-smooth muscle actin (α-Smooth muscle actin, α-SMA) and fibronectin (Fibronectin, FN), so they can be activated by these two proteins. The changes in protein expression levels were used to verify the inhibitory effect of integrin ligands on fibroblast activation.
2.2实验材料与方法2.2 Experimental Materials and Methods
细胞株：人胎儿肺成纤维母细胞(HFL-1)；TGF-β：R&D公司；培养基：Hyclone公司F12K培养基；血清：Gibco公司；Fibronectin抗体：abcam公司，货号：ab45688；α-SMA抗体：abcam公司，货号：ab124964。Cell line: human fetal lung fibroblasts (HFL-1); TGF-β: R&D; culture medium: F12K culture medium from Hyclone; serum: Gibco; Fibronectin antibody: abcam, catalog number: ab45688; α-SMA antibody: abcam, catalog number: ab124964.
HFL-1细胞接种于含10％FBS的F12K培养基中常规培养。不同化合物处理时按1×106细胞/孔接种6孔板，接种后在正常培养条件下培养24h，换成无血清培养基，以赋形剂DMSO和不同浓度的化合物(每个化合物3个浓度，每个浓度3个复孔)预处理2h，然后添加10ng/ml的TGF-β重组蛋白处理24h，处理后回收细胞作Fibronectin和α-SMA的蛋白免疫印迹(Western blotting，WB)检测。HFL-1 cells were inoculated in F12K medium containing 10% FBS and cultured routinely. When treated with different compounds, 1×106 cells/well were inoculated in 6-well plates, cultured under normal culture conditions for 24 hours after inoculation, and then replaced with serum-free medium. The excipient DMSO and different concentrations of compounds (3 concentrations for each compound, 3 replicates for each concentration) were pretreated for 2 hours, and then 10 ng/ml of TGF-β recombinant protein was added for 24 hours. After treatment, the cells were recovered for protein immunoblotting (Western blotting, WB) detection of Fibronectin and α-SMA.
选用吡非尼酮(Pirfenidone)和GSK-3008348作为本实验的对照化合物。吡非尼酮(CAS：53179-13-8)是一种用途广泛的抗纤维化药物，GSK-3008348(CAS：1629249-33-7)是由葛兰素史克公司(GSK Plc.)开发的一种极具潜力的整合素配体分子。吡非尼酮药物浓度0.5mg/mL，GSK-3008348药物浓度与受试化合物相同(3个浓度，每个浓度3个复孔)，处理方法同上。Pirfenidone and GSK-3008348 were selected as control compounds for this experiment. Pirfenidone (CAS: 53179-13-8) is a widely used anti-fibrotic drug, and GSK-3008348 (CAS: 1629249-33-7) is a highly potential integrin ligand molecule developed by GlaxoSmithKline (GSK Plc.). The drug concentration of pirfenidone was 0.5 mg/mL, and the drug concentration of GSK-3008348 was the same as that of the test compound (3 concentrations, 3 replicates for each concentration), and the treatment method was the same as above.
2.3 WB实验步骤2.3 WB experimental steps
蛋白提取：PBS清洗细胞，每孔加入100ul RIPA裂解液，用细胞刮将细胞刮下后收集到EP管中，冰浴30min，充分裂解后，13000g离心10min，取上清置新EP管中。蛋白定量依照BCA蛋白浓度测定试剂盒(增强型)说明书进行操作。根据定量结果，稀释样本，使蛋白浓度一致。加入上样缓冲液混匀，沸水浴加热3-5分钟，充分变性蛋白，冷却到室温后上样。Protein extraction: Wash the cells with PBS, add 100ul RIPA lysis buffer to each well, scrape the cells with a cell scraper and collect them in an EP tube, place on ice for 30 minutes, centrifuge at 13000g for 10 minutes after sufficient lysis, and take the supernatant and place it in a new EP tube. Protein quantification is performed according to the instructions of the BCA protein concentration assay kit (enhanced). According to the quantitative results, dilute the sample to make the protein concentration consistent. Add the loading buffer and mix well, heat in a boiling water bath for 3-5 minutes to fully denature the protein, and load the sample after cooling to room temperature.
做胶：注入分离胶，预留1/5的空间，并用去离子水补满。室温静置1个小时之后，倒掉去离子水，并用吸水滤纸吸干，然后再用浓缩胶填满；接着迅速插入梳子，并注意用剩余的浓缩胶排除气泡，再室温静置30min即可。Make gel: inject separation gel, leave 1/5 of the space, and fill it up with deionized water. After standing at room temperature for 1 hour, pour out the deionized water, dry it with absorbent filter paper, and then fill it up with concentrated gel; then quickly insert the comb, and pay attention to remove bubbles with the remaining concentrated gel, and then stand at room temperature for 30 minutes.
加样/电泳/转膜：各样品的总蛋白的上样量为25ug，用加样枪加至样品孔中。电泳：70V跑至浓缩胶下层分离胶上层，再120V跑完。转膜：先将0.45um PVDF膜、滤纸、海绵泡在转移缓冲液中，将PVDF膜做好标记；电泳完毕后，选取合适的胶；制作转膜的三明治，上架，注满电转缓冲液，恒流230mA，120min。Sample loading/electrophoresis/transfer: The total protein loading amount of each sample is 25ug, which is added to the sample well with a sample gun. Electrophoresis: Run at 70V to the lower layer of the concentrated gel and the upper layer of the separation gel, and then run at 120V to the end. Transfer: First soak the 0.45um PVDF membrane, filter paper, and sponge in the transfer buffer, and mark the PVDF membrane; after the electrophoresis is completed, select the appropriate gel; make a transfer sandwich, put it on the rack, fill it with electrotransfer buffer, and run at a constant current of 230mA for 120min.
抗原封闭与抗体孵育：先用PBS清洗膜，加入Western封闭液(5％nonfat-milk)，室温摇床孵育1h。一抗孵育：分别加入一抗α-SMA、Fibronectin和内参Actin，4℃过夜；洗涤一抗，加入TBST，室温摇床洗3次，每次5min。二抗孵育：加入相应的二抗，室温下摇动孵育1h。洗涤二抗，加入TBST，室温摇床洗4次，每次6min。最后，荧光成像仪检测，结果定量读取蛋白条带的光密度值。以actin条带光密度值为内参，比较各个目的蛋白的表达情况。Antigen blocking and antibody incubation: First wash the membrane with PBS, add Western blocking solution (5% nonfat-milk), and incubate on a shaker at room temperature for 1 hour. Primary antibody incubation: Add primary antibodies α-SMA, Fibronectin and internal reference Actin respectively, overnight at 4°C; wash the primary antibody, add TBST, and wash 3 times on a shaker at room temperature, 5 minutes each time. Secondary antibody incubation: Add the corresponding secondary antibody and incubate at room temperature with shaking for 1 hour. Wash the secondary antibody, add TBST, and wash 4 times on a shaker at room temperature, 6 minutes each time. Finally, the fluorescence imager is used for detection, and the optical density value of the protein band is quantitatively read. The optical density value of the actin band is used as the internal reference to compare the expression of each target protein.
由于α-SMA与内参蛋白Actin分子量相近，本实验采用抗体剥离法(Antibody Stripping)，首先检测α-SMA蛋白，接着使用剥离缓冲液去除α-SMA一抗，再检测Actin蛋白。两种抗体的孵育方法同上。Since α-SMA and the internal reference protein Actin have similar molecular weights, this experiment uses the Antibody Stripping method, first detecting the α-SMA protein, then using the stripping buffer to remove the α-SMA primary antibody, and then detecting the Actin protein. The incubation methods for the two antibodies are the same as above.
2.4实验结果：2.4 Experimental results:
实施例1、2、7、15、25对纤维化蛋白α-SMA、Fibronectin具有显著的促降解作用(如图1所示)。其中实施例2、7、15、25显著降低了α-SMA蛋白的表达量，对纤维化蛋白α-SMA具有极佳的促降解作用，显著优于阳性参照吡非尼酮以及GSK-3008348，表现出优异的抗纤维化效果；实施例1、2、7、25显著降低了Fibronectin蛋白的表达量，对纤维化蛋白Fibronectin具有极佳的促降解作用，同样优于阳性参照吡非尼酮以及GSK-3008348。实施例2、7、25均对α-SMA和Fibronectin两种蛋白具有极佳的促降解作用，且效果均显著优于阳性参照GSK-3008348和吡非尼酮。说明本发明中的整合素αvβ1、6、8的配体对纤维母细胞分泌α-SMA蛋白、Fibronectin蛋白以及活化有显著的抑制作用，具有成为抗纤维化药物的潜力。 Embodiments 1, 2, 7, 15, and 25 have significant promoting degradation effects on fibrosis proteins α-SMA and Fibronectin (as shown in Figure 1). Among them, Embodiments 2, 7, 15, and 25 significantly reduce the expression of α-SMA protein, have an excellent promoting degradation effect on fibrosis protein α-SMA, and are significantly better than the positive references pirfenidone and GSK-3008348, showing excellent anti-fibrosis effects; Embodiments 1, 2, 7, and 25 significantly reduce the expression of Fibronectin protein, have an excellent promoting degradation effect on fibrosis protein Fibronectin, and are also better than the positive references pirfenidone and GSK-3008348. Embodiments 2, 7, and 25 all have excellent promoting degradation effects on both α-SMA and Fibronectin proteins, and the effects are significantly better than the positive references GSK-3008348 and pirfenidone. This indicates that the ligands of integrins αvβ1, 6, and 8 in the present invention have a significant inhibitory effect on the secretion of α-SMA protein and Fibronectin protein as well as the activation of fibroblasts, and have the potential to become anti-fibrosis drugs.
效果实施例3：化合物在人、大鼠、小鼠、犬、猴中的肝微粒体代谢稳定性Effect Example 3: Metabolic Stability of Compounds in Liver Microsomes of Humans, Rats, Mice, Dogs, and Monkeys
3.1溶液配制3.1 Solution preparation
称取1mg测试化合物(底物)，溶解在DMSO中至10mM，在0.6％乙腈-水溶液中稀释至10μM。配制肝微粒体实验体系，其中NADPH生成系统，仅包括葡萄糖-6-磷酸酯，NADP+，葡萄糖-6-磷酸脱氢酶，按如下表3所示准备储液。Weigh 1 mg of the test compound (substrate), dissolve it in DMSO to 10 mM, and dilute it to 10 μM in 0.6% acetonitrile-water solution. Prepare the liver microsome experimental system, in which the NADPH generation system only includes glucose-6-phosphate, NADP+, and glucose-6-phosphate dehydrogenase, and prepare the stock solution as shown in Table 3 below.
表3肝微粒体实验体系(总体积200μL)
Table 3 Liver microsome experimental system (total volume 200 μL)
反应时间0、10、20、30minReaction time 0, 10, 20, 30 min
3.2样品处理3.2 Sample processing
在96孔板中加入底物、肝微粒体、MgCl2、磷酸钾缓冲液、水，轻轻涡旋混合均匀。将微粒体混合物在37℃水浴中预温育5min，再通过加入NADPH生成系统引发反应。孵育0、10、20、30min后，将40μL反应混合物与100μL乙腈(带内标100ng/mL)混合，终止反应。孵育结束后混合物在4℃下以4600rpm离心10min。取60μL上清液，用水1:1稀释，进样5μL进行LC-MS/MS分析。Substrate, liver microsomes, MgCl 2 , potassium phosphate buffer, and water were added to a 96-well plate and gently vortexed to mix. The microsome mixture was pre-incubated in a 37°C water bath for 5 min, and the reaction was initiated by adding the NADPH generating system. After incubation for 0, 10, 20, and 30 min, 40 μL of the reaction mixture was mixed with 100 μL of acetonitrile (with internal standard 100 ng/mL) to terminate the reaction. After the incubation, the mixture was centrifuged at 4600 rpm for 10 min at 4°C. 60 μL of the supernatant was taken, diluted 1:1 with water, and 5 μL was injected for LC-MS/MS analysis.
3.3色谱条件3.3 Chromatographic conditions
采用Waters XBridge C18色谱柱(50mm×2.1mm，5μm)，进样量5μL。流速1mL/min，运行时间1.5min。流动相A为含0.1％甲酸的水，B为含0.1％甲酸的乙腈，梯度洗脱程序如下表4所示：A Waters XBridge C18 column (50 mm × 2.1 mm, 5 μm) was used with an injection volume of 5 μL. The flow rate was 1 mL/min and the running time was 1.5 min. The mobile phase A was water containing 0.1% formic acid, and B was acetonitrile containing 0.1% formic acid. The gradient elution program is shown in Table 4 below:
表4梯度洗脱程序
Table 4 Gradient elution program
3.4质谱条件3.4 Mass spectrometry conditions
采用高效液相色谱串联质谱(LC-MS/MS)，型号API 4500(AB Sciex公司)。采用电喷雾电离(ESI)条件，多反应监测(MRM)模式检测洗脱化合物。干气温度450℃，压力50psi，雾化气体压力20psi。High performance liquid chromatography tandem mass spectrometry (LC-MS/MS) was used, model API 4500 (AB Sciex). Electrospray ionization (ESI) conditions and multiple reaction monitoring (MRM) mode were used to detect the eluted compounds. The dry gas temperature was 450°C, the pressure was 50 psi, and the nebulizing gas pressure was 20 psi.
3.5实验结论3.5 Experimental Conclusion
代谢稳定性结果如下表5所示。The metabolic stability results are shown in Table 5 below.
表5化合物在5个种属肝微粒体中测试的半衰期

Table 5 Half-lives of compounds tested in liver microsomes of five species

如表5所示，本发明中化合物在人、小鼠、大鼠、犬、猴的肝微粒体中代谢稳定，其中实施例1、2、6、72、73在人肝微粒体中的半衰期优于阳性参照PLN-74809，实施例2、6、73、117在犬肝微粒体中的半衰期要优于PLN-74809，本发明中其他化合物也具有类似的理化性质。As shown in Table 5, the compounds of the present invention are metabolically stable in the liver microsomes of humans, mice, rats, dogs and monkeys, among which Examples 1, 2, 6, 72 and 73 have better half-lives in human liver microsomes than the positive reference PLN-74809, and Examples 2, 6, 73 and 117 have better half-lives in dog liver microsomes than PLN-74809. Other compounds of the present invention also have similar physicochemical properties.
效果实施例4：化合物在大鼠体内的药代动力学Effect Example 4: Pharmacokinetics of Compounds in Rats
4.1实验方法：4.1 Experimental methods:
4.1.1溶液配制及动物实验4.1.1 Solution preparation and animal experiments
雄性SD大鼠，体重200g左右，购自上海西普尔-必凯实验动物有限公司，每组3只。每个受试化合物分别进行口服给药(PO)与静脉注射(IV)两组试验。Male SD rats, weighing about 200 g, were purchased from Shanghai Xipuer-Bikai Experimental Animal Co., Ltd., with 3 rats in each group. Each test compound was administered orally (PO) and intravenously (IV).
注射剂量1mg/kg，受试化合物使用相同溶剂配成0.1mg/mL溶液，按10mL/kg体积进行尾静脉注射。给药后5、15、30、60、120、240、480、720、1440min，采血测定血药浓度。The injection dose was 1 mg/kg, and the test compound was prepared into a 0.1 mg/mL solution using the same solvent and injected into the tail vein at a volume of 10 mL/kg. Blood was collected at 5, 15, 30, 60, 120, 240, 480, 720, and 1440 minutes after administration to determine the blood drug concentration.
口服剂量10mg/kg，受试化合物先用溶剂(DMA:Solutol-HS-15:Saline＝10:10:80)配成1mg/mL溶液，按10mL/kg体积进行灌胃。给药后15、30、60、120、240、480、720、1440min(分钟)，采血测定血药浓度。The oral dose was 10 mg/kg. The test compound was first prepared into a 1 mg/mL solution with a solvent (DMA: Solutol-HS-15: Saline = 10:10:80) and then gavaged at a volume of 10 mL/kg. Blood was collected at 15, 30, 60, 120, 240, 480, 720, and 1440 minutes after administration to determine the blood drug concentration.
精密称定1mg测试化合物，以甲醇/水溶液(MeOH:H2O＝4:1，v/v)溶解为100μg/mL的储备液。取适量储备液，用甲醇/水溶液稀释成质量浓度分别为20、40、100、200、400、1000ng/mL，2、4、10、20、40、100μg/mL的系列标准溶液。另制备质量浓度分别为2、5μg/mL的QC溶液，备用。Accurately weigh 1 mg of the test compound and dissolve it in methanol/water solution (MeOH:H2O=4:1, v/v) to make a 100 μg/mL stock solution. Take an appropriate amount of the stock solution and dilute it with methanol/water solution to a series of standard solutions with mass concentrations of 20, 40, 100, 200, 400, 1000 ng/mL, 2, 4, 10, 20, 40, 100 μg/mL. Also prepare QC solutions with mass concentrations of 2 and 5 μg/mL for use.
取各浓度标准溶液和QC溶液2.5μL，溶于47.5μL小鼠空白血浆，移入96孔板，配制成质量浓度分别为1、2、5、10、20、50、100、200、500、1000、2000、5000ng/mL的标准血浆样品，以及质量浓度为100、250ng/mL的QC血浆样品，备用。Take 2.5 μL of each concentration standard solution and QC solution, dissolve in 47.5 μL of mouse blank plasma, transfer into a 96-well plate, and prepare standard plasma samples with mass concentrations of 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, and 5000 ng/mL, as well as QC plasma samples with mass concentrations of 100 and 250 ng/mL for later use.
4.1.2血浆样品处理4.1.2 Plasma sample processing
每次采血体积200μL左右，保存于EDTA-2K抗凝试管，5500rpm离心10min分离血浆。取20μL的各时间点测试血浆样品、标准血浆样品、QC血浆样品，分别与100μL乙腈(带内标100ng/mL)混合，室温振荡10min，然后在4℃下以3700rpm离心18min。取60μL上清液，用水1:1稀释，室温振荡10min，然后进样5μL进行LC-MS/MS分析。Each blood collection volume was about 200 μL, stored in an EDTA-2K anticoagulation tube, and centrifuged at 5500 rpm for 10 min to separate plasma. Take 20 μL of the test plasma sample, standard plasma sample, and QC plasma sample at each time point, mix them with 100 μL acetonitrile (with internal standard 100 ng/mL), shake at room temperature for 10 min, and then centrifuge at 3700 rpm for 18 min at 4 ° C. Take 60 μL of the supernatant, dilute it 1:1 with water, shake it at room temperature for 10 min, and then inject 5 μL for LC-MS/MS analysis.
4.2.样品测定方法4.2. Sample determination method
4.2.1色谱条件4.2.1 Chromatographic conditions
与效果实施例3的色谱条件相同。The chromatographic conditions are the same as those in Effect Example 3.
4.2.2质谱条件4.2.2 Mass spectrometry conditions
采用高效液相色谱串联质谱(LC-MS/MS)，型号API 4500(AB Sciex公司)。采用电喷雾电离(ESI)条件，多反应监测(MRM)模式检测洗脱化合物。离子源温度为450℃，喷雾气、辅助加热气压力均为50psi，气帘气压力为20psi，碰撞室出口电压13.0V。内标及受试化合物的去簇电压分别为79、130、135eV，碰撞能量分别为19、24、41eV。High performance liquid chromatography tandem mass spectrometry (LC-MS/MS), model API 4500 (AB Sciex), was used. Electrospray ionization (ESI) conditions and multiple reaction monitoring (MRM) mode were used to detect the eluted compounds. The ion source temperature was 450°C, the spray gas and auxiliary heating gas pressures were both 50 psi, the curtain gas pressure was 20 psi, and the collision chamber outlet voltage was 13.0 V. The declustering voltages of the internal standard and the test compound were 79, 130, and 135 eV, respectively, and the collision energies were 19, 24, and 41 eV, respectively.
4.3实验结论4.3 Experimental Conclusion
药代动力学结果如下表6所示。The pharmacokinetic results are shown in Table 6 below.
表6实施例71和72在大鼠体内的药代动力学参数

Table 6 Pharmacokinetic parameters of Examples 71 and 72 in rats

如表6所示，实施例71和72大鼠静脉注射给药后，末端消除半衰期分别为1.5和0.4小时，注射AUCINF分别为4644h*nM和6511h*nM。灌胃给药后，两者末端消除半衰期分别为3.0和0.7小时，达峰时间分别为0.5和0.4小时，达峰浓度分别为4622和15946nM，口服AUCINF分别为12994h*nM和31406h*nM，大鼠吸收很好，口服生物利用度分别为28％和48％，结果显著优于阳性参照PLN-74809的口服生物利用度(大鼠实测数据仅为2％)。本发明中的其他化合物也具有类似的理化性质，具有极佳的成药性潜力和优秀的体内代谢特性。As shown in Table 6, after intravenous administration to rats of Examples 71 and 72, the terminal elimination half-life was 1.5 and 0.4 hours, respectively, and the injection AUC INF was 4644h*nM and 6511h*nM, respectively. After oral administration, the terminal elimination half-life of the two was 3.0 and 0.7 hours, the peak time was 0.5 and 0.4 hours, the peak concentration was 4622 and 15946nM, respectively, and the oral AUC INF was 12994h*nM and 31406h*nM, respectively. The rats absorbed well, and the oral bioavailability was 28% and 48%, respectively, which was significantly better than the oral bioavailability of the positive reference PLN-74809 (the actual measured data of rats was only 2%). Other compounds in the present invention also have similar physical and chemical properties, and have excellent drug-forming potential and excellent in vivo metabolic characteristics.
效果实施例5：化合物对博来霉素诱导的大鼠特发性肺纤维化(IPF)的作用Effect Example 5: Effect of the compound on bleomycin-induced idiopathic pulmonary fibrosis (IPF) in rats
5.1实验方法：5.1 Experimental methods:
第1天(Day 1)，在麻醉状态下通过气管插管向大鼠(SD)施用3mg/kg博来霉素(购自日本化药株式会社)，在左侧肺脏诱发IPF病变，建立大鼠单侧肺纤维化模型。在第2天至第15天进行连续给药，每日一次。末次给药后次日(Day 16)将所有大鼠安乐死，心脏灌流后取全肺并固定于10％福尔马林中。然后石蜡包埋，使用切片机(RM2235，莱卡LEICA)进行精密切片。组织切片后续进行H&E染色以及Masson Trichrome染色，分别评价炎细胞浸润的病理变化以及胶原沉积引起的纤维化变化。On day 1 (Day 1), 3 mg/kg bleomycin (purchased from Nippon Kayaku Co., Ltd.) was administered to rats (SD) through endotracheal intubation under anesthesia to induce IPF lesions in the left lung and establish a rat unilateral pulmonary fibrosis model. Continuous administration was performed from day 2 to day 15, once a day. The day after the last administration (Day 16), all rats were euthanized, and the whole lungs were removed after cardiac perfusion and fixed in 10% formalin. Then they were paraffin embedded and precisely sectioned using a microtome (RM2235, Leica). The tissue sections were subsequently stained with H&E and Masson Trichrome to evaluate the pathological changes of inflammatory cell infiltration and fibrosis changes caused by collagen deposition, respectively.
本实验共分五组，即假手术组(G-1)，模型组(G-2)，尼达尼布对照组(G-3)，实施例71低剂量组(G-4)、高剂量组(G-5)，如下表7所示。This experiment was divided into five groups, namely, sham operation group (G-1), model group (G-2), nintedanib control group (G-3), Example 71 low-dose group (G-4), and high-dose group (G-5), as shown in Table 7 below.
表7实验分组和给药计划
Table 7 Experimental groups and dosing schedule
实验检测指标包括动物体重和组织病理学评分。动物体重每天观察并记录1次，计算体重增长率。组织病理学评分标准如下：The experimental test indicators include animal weight and histopathological score. The animal weight is observed and recorded once a day, and the weight growth rate is calculated. The histopathological scoring criteria are as follows:
H&E染色切片中，在病变区域内和病变区域边缘随机分散各选择5个视野，对终末细支气管和伴行微小肺动脉损伤和炎症变化分别进行半定量评分。共评价支气管损伤、支气管炎症、小动脉损伤、小动脉炎症4项指标，每项0～3分，累加为0～12分，其中0分为无病理改变，12分为病变最严重。In the H&E-stained sections, 5 fields of view were randomly selected in the lesion area and at the edge of the lesion area, and semi-quantitative scores were performed on the damage and inflammatory changes of the terminal bronchioles and the accompanying small pulmonary arteries. A total of 4 indicators were evaluated, including bronchial damage, bronchial inflammation, arteriolar damage, and arteriolar inflammation, with each item ranging from 0 to 3 points, and the cumulative score was 0 to 12 points, where 0 was no pathological change and 12 was the most severe lesion.
Masson Trichrome染色切片中，在病变区域内随机选择10个面积大小为1mm2的视野，病理学家根据Ashcroft评分系统在双盲条件下进行半定量评分。Ashcroft评分共0～8分，其中0分为无纤维化病变，8分为完全纤维化。In the Masson Trichrome-stained sections, 10 fields of view with an area of 1 mm2 were randomly selected within the lesion area, and pathologists performed semi-quantitative scoring under double-blind conditions according to the Ashcroft scoring system. The Ashcroft score ranges from 0 to 8, with 0 for no fibrosis and 8 for complete fibrosis.
5.2数据分析方法：5.2 Data Analysis Methods:
数据在Office Excel和GraphPad Prism软件中进行整理，数据以Mean±SEM(标准误)表示。利用One-way ANOVA和T-test进行分析，分析方法选用Tukey进行组间比较差异显著性检验；两组间进行比较时，用T-test进行两组间双尾检测，当p<0.05时，两组之间具有显著性差异。 Data were collated in Office Excel and GraphPad Prism software, and data were expressed as Mean ± SEM (standard error). One-way ANOVA and T-test were used for analysis, and Tukey's test for significant difference between groups was used for analysis; when two groups were compared, T-test was used for two-tailed test between the two groups, and when p < 0.05, there was a significant difference between the two groups.
5.3实验结果讨论5.3 Discussion of Experimental Results
5.3.1体重及体重变化5.3.1 Body weight and weight changes
博来霉素诱导的肺损伤模型，大鼠体重会在术后3～4天逐渐降低直至最低点，随后会逐渐恢复，该现象为此模型的正常表现。在连续给药14天中，实施例71低剂量组、高剂量组的大鼠体重恢复明显更快(图2)，体重增长率高于模型组并显著优于尼达尼布组(图3)，表明在肺纤维化损伤中，实施例71有助于改善动物整体状态，帮助大鼠更快恢复体重，且未见明显副作用。In the bleomycin-induced lung injury model, the weight of rats gradually decreased to the lowest point 3 to 4 days after surgery, and then gradually recovered. This phenomenon is a normal manifestation of this model. During the 14-day continuous administration, the weight of rats in the low-dose group and the high-dose group of Example 71 recovered significantly faster (Figure 2), and the weight growth rate was higher than that of the model group and significantly better than that of the nintedanib group (Figure 3), indicating that in pulmonary fibrosis injury, Example 71 helps to improve the overall state of the animals, helps rats recover their weight faster, and no obvious side effects are observed.
5.3.2实施例71可减轻博来霉素诱导的左肺组织支气管、小动脉损伤及炎症浸润。5.3.2 Example 71 can reduce bleomycin-induced bronchial and arteriolar damage and inflammatory infiltration in the left lung tissue.
在显微镜下观察H&E染色切片，比较左肺支气管、肺小血管、肺泡组织周围病变及炎性细胞浸润程度。由图4可知，实施例71的低剂量组(D)和高剂量组(E)与模型组H&E stained sections were observed under a microscope to compare the lesions around the left lung bronchi, pulmonary small blood vessels, and alveolar tissue and the degree of inflammatory cell infiltration. As shown in Figure 4, the low-dose group (D) and high-dose group (E) of Example 71 were significantly different from the model group.
(B)相比虽仍有部分炎性细胞侵入组织内部的现象，但程度已经减轻，这与尼达尼布组(B) Although some inflammatory cells still invaded the tissue, the extent was reduced, which was different from the nintedanib group.
(C)的程度相当。由图5的分析也发现，实施例71的两个剂量组的损伤评分与模型组相比均有显著降低(p<0.001)，与阳性参照尼达尼布组降低(p<0.001)程度相当。The analysis of Figure 5 also found that the injury scores of the two dosage groups of Example 71 were significantly reduced compared with the model group (p<0.001), which was comparable to the reduction in the positive reference nintedanib group (p<0.001).
由图6可知，实施例71低剂量组(D)和高剂量组(E)能够减轻病灶边缘的肺组织炎症程度，显著优于模型组(B)，其中实施例71高剂量组(E)对炎症的减轻程度要优于低剂量组和阳性参照尼达尼布组(C)。由图7的分析也发现，实施例71的两个剂量组损伤评分与模型组比均有显著降低，其中高剂量组的损伤评分显著降低(p<0.001)，高剂量组的损伤评分远优于低剂量组(p<0.01)和阳性参照尼达尼布组(p<0.01)，低剂量组的评分与尼达尼布组相当。As shown in Figure 6, the low-dose group (D) and high-dose group (E) of Example 71 can reduce the degree of inflammation of the lung tissue at the edge of the lesion, which is significantly better than the model group (B), and the high-dose group (E) of Example 71 is better than the low-dose group and the positive reference nintedanib group (C) in reducing inflammation. The analysis of Figure 7 also found that the injury scores of the two dose groups of Example 71 were significantly lower than those of the model group, and the injury score of the high-dose group was significantly reduced (p<0.001), and the injury score of the high-dose group was much better than the low-dose group (p<0.01) and the positive reference nintedanib group (p<0.01), and the score of the low-dose group was equivalent to that of the nintedanib group.
如图8所示，实施例71高剂量组(E)肺泡壁增厚程度最轻，在箭头所指处出现轻微的肺泡壁增厚，病理变化程度明显低于模型组(B)，同样低于实施例71低剂量组(D)和尼达尼布组(C)，实施例71低剂量组与尼达尼布组的减轻程度相当。这表明实施例71高剂量组比阳性参照尼达尼布组更具优势，药效更显著。As shown in Figure 8, the alveolar wall thickening in the high-dose group (E) of Example 71 was the mildest, with slight alveolar wall thickening at the arrow point, and the degree of pathological changes was significantly lower than that in the model group (B), and also lower than that in the low-dose group (D) and the nintedanib group (C) of Example 71. The degree of reduction in the low-dose group of Example 71 was comparable to that in the nintedanib group. This indicates that the high-dose group of Example 71 is more advantageous than the positive reference nintedanib group, and the drug effect is more significant.
5.3.3实施例71可减轻博来霉素诱导的肺脏纤维化改变5.3.3 Example 71 can alleviate bleomycin-induced pulmonary fibrosis changes
在显微镜下通过Masson染色切片，观察肺组织中纤维化胶原的沉积程度，并进行纤维化评分。如图9所示，实施例71低剂量组(D)和实施例71高剂量组(E)中的纤维化程度与模型组(B)相比明显减轻，且实施例71的两个剂量组的减轻程度均优于尼达尼布组(C)。由图10的肺纤维化评分也发现，与模型组相比，实施例71低剂量组纤维化评分显著降低(p<0.001)，实施例71高剂量组纤维化评分显著降低(p<0.001)，且均优于阳性参照尼达尼布组纤维化评分(p<0.05)。The deposition degree of fibrotic collagen in lung tissue was observed by Masson staining under a microscope, and fibrosis scores were performed. As shown in Figure 9, the degree of fibrosis in the low-dose group (D) of Example 71 and the high-dose group (E) of Example 71 was significantly reduced compared with the model group (B), and the degree of reduction in the two dosage groups of Example 71 was better than that in the nintedanib group (C). It was also found from the pulmonary fibrosis score of Figure 10 that the fibrosis score of the low-dose group of Example 71 was significantly reduced (p<0.001) compared with the model group, and the fibrosis score of the high-dose group of Example 71 was significantly reduced (p<0.001), and was better than the positive reference nintedanib group fibrosis score (p<0.05).
图11为假手术组、模型组、尼达尼布组、实施例71低剂量组、实施例71高剂量组分别统计纤维化轻症、重症占比的结果。其中，纤维化评分≤3分可视为轻症；评分≥4分可视为重症。如图所示，与模型组相比实施例71低剂量组重症占比显著降低(p<0.001)，实施例71高剂量组重症占比显著降低(p<0.001)。与阳性参照尼达尼布组相比，实施例71高、低两个剂量组重症占比均明显低于尼达尼布组，实施例71的高剂量组重症占比要低于低剂量组，且显著低于尼达尼布组。这表明实施例71高剂量组中超过80％大鼠为轻症，药效极其显著。由图9、10和11说明实施例71具有显著的抗肺纤维化效果。Figure 11 is the result of statistical analysis of the proportion of mild and severe fibrosis in the sham operation group, model group, nintedanib group, low-dose group of Example 71, and high-dose group of Example 71. Among them, fibrosis scores ≤ 3 points can be regarded as mild; scores ≥ 4 points can be regarded as severe. As shown in the figure, compared with the model group, the proportion of severe cases in the low-dose group of Example 71 was significantly reduced (p<0.001), and the proportion of severe cases in the high-dose group of Example 71 was significantly reduced (p<0.001). Compared with the positive reference nintedanib group, the proportion of severe cases in the high and low dose groups of Example 71 was significantly lower than that in the nintedanib group, and the proportion of severe cases in the high-dose group of Example 71 was lower than that in the low-dose group, and significantly lower than that in the nintedanib group. This shows that more than 80% of the rats in the high-dose group of Example 71 were mild cases, and the efficacy was extremely significant. Figures 9, 10 and 11 illustrate that Example 71 has a significant anti-pulmonary fibrosis effect.
图12为Masson染色中，各组别大鼠肺泡组织中胶原沉积程度。如图所示，实施例71低剂量组(D)和实施例71高剂量组(E)胶原沉积最轻，程度明显低于模型组(B)和尼 达尼布组(C)。由图13也可知，与模型组相比，实施例71高、低两个剂量组胶原沉积面积显著降低(均为p<0.001)，降低程度均显著优于尼达尼布组(p<0.05)。实施例71高剂量组胶原沉积面积降低程度更为显著，这表明实施例71同样能够有效降低肺胶原沉积程度。FIG12 shows the degree of collagen deposition in the alveolar tissue of rats in each group in Masson staining. As shown in the figure, the collagen deposition in the low-dose group (D) of Example 71 and the high-dose group (E) of Example 71 was the lightest, and the degree was significantly lower than that in the model group (B) and the nivolumab group (E). Nintedanib group (C). As shown in Figure 13, compared with the model group, the collagen deposition area of the high and low dose groups of Example 71 was significantly reduced (both p < 0.001), and the degree of reduction was significantly better than that of the nintedanib group (p < 0.05). The degree of reduction in collagen deposition area in the high dose group of Example 71 was more significant, indicating that Example 71 can also effectively reduce the degree of lung collagen deposition.
效果实施例6：化合物对DDC诱导的小鼠原发性硬化性胆管炎(PSC)的作用Effect Example 6: Effect of the compound on DDC-induced primary sclerosing cholangitis (PSC) in mice
6.1实验方法6.1 Experimental methods
第1天，小鼠(C57BL/6J)按照体重进行随机分组。第2天起将除正常对照组外的其它所有笼盒内的正常饲料换成含有0.1％DDC(3,5-二乙氧基碳基-1,4-二氢-2,4,6-三甲基吡啶)的添加料诱导小鼠硬化性胆管炎(PSC)，正常对照组提供正常的饮食和饮水，连续3周(Day 2-Day 22)。第2天起每天按不同组别给予相应药物，连续3周(Day 2-Day 22)。末次给药后次日(Day 23)，小鼠取血，将血液样本在4℃、1500×g离心后分离血清，冻于-80℃保存。小鼠取血后安乐死，分离完整肝脏并称重，随后切取1/2肝大叶，置于10％福尔马林(甲醛)溶液中固定48h，后续进行病理学检测。On day 1, mice (C57BL/6J) were randomly divided into groups according to body weight. From day 2, the normal feed in all cages except the normal control group was replaced with feed containing 0.1% DDC (3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine) to induce mouse sclerosing cholangitis (PSC). The normal control group was provided with normal diet and drinking water for 3 consecutive weeks (Day 2-Day 22). From day 2, the corresponding drugs were given to different groups every day for 3 consecutive weeks (Day 2-Day 22). The day after the last administration (Day 23), the mice were bled, and the blood samples were centrifuged at 4°C and 1500×g to separate the serum and frozen at -80°C. After the blood was drawn, the mice were euthanized, the whole liver was separated and weighed, and then 1/2 of the liver lobe was cut and placed in 10% formalin (formaldehyde) solution for fixation for 48h, and then pathological examination was performed.
实验检测指标包括动物体重、肝重、血清肝功能指标、组织病理学评分。动物体重每天观察并记录1次，比较体重变化百分比。使用自动生化检测仪测定小鼠血样中谷丙转氨酶(ALT)、碱性磷酸酶(ALP)、谷草转氨酶(AST)、总胆汁酸(TBA)、总胆红素(TBIL)等水平。The experimental test indicators included animal body weight, liver weight, serum liver function indexes, and histopathological scores. The animal body weight was observed and recorded once a day, and the percentage of body weight change was compared. An automatic biochemical detector was used to measure the levels of alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), total bile acid (TBA), and total bilirubin (TBIL) in mouse blood samples.
6.2实验结果讨论6.2 Discussion of Experimental Results
小鼠DDC摄入后，肝损伤逐渐加重，出现明显的胆管硬化症状。所有喂食含DDC饲料的小鼠体重都出现了不同程度的下降，且随时间愈发严重。肝重/体重比值在模型组中出现显著升高，实施例71相比模型组均有显著的治疗效果，表明实施例71具有优秀的抗肝纤维化、抗胆管硬化和抗炎症作用。After mice ingested DDC, liver damage gradually worsened, and obvious symptoms of bile duct sclerosis appeared. The body weight of all mice fed with DDC-containing feed decreased to varying degrees, and became more serious over time. The liver weight/body weight ratio increased significantly in the model group, and Example 71 had a significant therapeutic effect compared with the model group, indicating that Example 71 has excellent anti-liver fibrosis, anti-bile duct sclerosis and anti-inflammatory effects.
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。 All documents mentioned in the present invention are cited as references in this application, just as each document is cited as references separately. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims (16)

  1. 一种如式I所示的一类双环衍生物，及其消旋体、立体异构体、互变异构体、同位素标记物、氮氧化物、溶剂化物、多晶型物、代谢产物、酯、前药或其药学上可接受的盐：
    A class of bicyclic derivatives as shown in formula I, and racemates, stereoisomers, tautomers, isotope-labeled products, nitrogen oxides, solvates, polymorphs, metabolites, esters, prodrugs or pharmaceutically acceptable salts thereof:
    其中，in,
    Y1为C1～C6亚烷基，-O-，-(C1～C6亚烷基)-O-，-NH-，-(C1～C6亚烷基)-NH-； Y1 is C1~C6 alkylene, -O-, -(C1~C6 alkylene)-O-, -NH-, -(C1~C6 alkylene)-NH-;
    Y2为C1～C6亚烷基，-O-，-(C1～C6亚烷基)-O-，-C(O)-(C1～C6亚烷基)-NH-，-NH-，-NH-(C1～C6亚烷基)-，-(C1～C6亚烷基)-NH-； Y2 is C1~C6 alkylene, -O-, -(C1~C6 alkylene)-O-, -C(O)-(C1~C6 alkylene)-NH-, -NH-, -NH-(C1~C6 alkylene)-, -(C1~C6 alkylene)-NH-;
    R1为取代或未取代的6-10元芳环，取代或未取代的5-8元杂芳环，取代或未取代的其中C环、D环各自独立地为取代或未取代的C6-C10芳环、取代或未取代的5-8元杂芳环，取代或未取代的5-8元环烷环，或取代或未取代的5-8元杂烷环； R1 is a substituted or unsubstituted 6-10 membered aromatic ring, a substituted or unsubstituted 5-8 membered heteroaromatic ring, a substituted or unsubstituted wherein the C ring and the D ring are each independently a substituted or unsubstituted C6-C10 aromatic ring, a substituted or unsubstituted 5-8 membered heteroaromatic ring, a substituted or unsubstituted 5-8 membered cycloalkane ring, or a substituted or unsubstituted 5-8 membered heteroalkane ring;
    R2为氢原子，取代或未取代的C6-C10芳环，取代或未取代的5-8元杂芳环，取代或未取代的C8-C16稠环，或-L1-L2； R2 is a hydrogen atom, a substituted or unsubstituted C6-C10 aromatic ring, a substituted or unsubstituted 5-8 membered heteroaromatic ring, a substituted or unsubstituted C8-C16 condensed ring, or -L1-L2;
    其中，-L1-选自无、-(取代或未取代的C1-C6亚烷基)-、-(取代或未取代的C1-C6亚烷氧基)-、-(取代或未取代的C1-C6亚烷硫基)-、-(取代或未取代的C3-C8环烷基)-、-(取代或未取代的C3-C8杂环烷基)-、-(取代或未取代的C6-C10芳基)-、-(取代或未取代的C5-C8杂芳基)-，Wherein, -L1- is selected from none, -(substituted or unsubstituted C1-C6 alkylene)-, -(substituted or unsubstituted C1-C6 alkyleneoxy)-, -(substituted or unsubstituted C1-C6 alkylenethio)-, -(substituted or unsubstituted C3-C8 cycloalkyl)-, -(substituted or unsubstituted C3-C8 heterocycloalkyl)-, -(substituted or unsubstituted C6-C10 aryl)-, -(substituted or unsubstituted C5-C8 heteroaryl)-,
    L2选自无、取代或未取代的C1-C6烷基、取代或未取代的C1-C6烷氧基、取代或未取代的C1-C6烷硫基、取代或未取代的C3-C8环烷基、-(C1-C3烷基)-取代或未取代的C3-C8环烷基、-(C1-C3烷氧基)-取代或未取代的C3-C8环烷基、-O-取代或未取代的C3-C8环烷基、取代或未取代的C3-C8杂烷环基、-(C1-C3烷基)-取代或未取代的C3-C8杂烷环基、-(C1-C3烷氧基)-取代或未取代的C3-C8杂烷环基、-O-取代或未取代的C3-C8杂烷环基、取代或未取代的5-8元杂芳基；L2 is selected from none, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C1-C6 alkylthio, substituted or unsubstituted C3-C8 cycloalkyl, -(C1-C3 alkyl)-substituted or unsubstituted C3-C8 cycloalkyl, -(C1-C3 alkoxy)-substituted or unsubstituted C3-C8 cycloalkyl, -O-substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C3-C8 heteroalkylcyclyl, -(C1-C3 alkyl)-substituted or unsubstituted C3-C8 heteroalkylcyclyl, -(C1-C3 alkoxy)-substituted or unsubstituted C3-C8 heteroalkylcyclyl, -O-substituted or unsubstituted C3-C8 heteroalkylcyclyl, substituted or unsubstituted 5-8 membered heteroaryl;
    X为氧原子或氮原子；X is an oxygen atom or a nitrogen atom;
    其中，当X为氧原子时，R3a为氢原子，C1～C6烷基，取代或未取代的C6-C10芳环，R3b不存在；当X为氮原子时，R3a为氢原子，羟基，C1～C6烷基，取代或未取代的C6-C10芳环，R3b为氢原子；Wherein, when X is an oxygen atom, R 3a is a hydrogen atom, a C1-C6 alkyl group, a substituted or unsubstituted C6-C10 aromatic ring, and R 3b is absent; when X is a nitrogen atom, R 3a is a hydrogen atom, a hydroxyl group, a C1-C6 alkyl group, a substituted or unsubstituted C6-C10 aromatic ring, and R 3b is a hydrogen atom;
    为取代或未取代的螺环或取代或未取代的并环； is a substituted or unsubstituted spiro ring or a substituted or unsubstituted cyclocyclic ring;
    其中，当为取代或未取代的螺环，如式Ia所示：

    Among them, when is a substituted or unsubstituted spiro ring, as shown in Formula Ia:

    A为选自下组的环：取代或未取代的四元环烷环，取代或未取代的五元环烷环，取代或未取代的六元环烷环，取代或未取代的七元环烷环，取代或未取代的八元环烷环、取代或未取代的四元杂烷环，取代或未取代的五元杂烷环，取代或未取代的六元杂烷环，取代或未取代的七元杂烷环，取代或未取代的八元杂烷环，取代或未取代的五元杂烷环并C6-C10芳香环，取代或未取代的六元杂烷环并C6-C10芳香环；A is a ring selected from the group consisting of a substituted or unsubstituted four-membered cycloalkane ring, a substituted or unsubstituted five-membered cycloalkane ring, a substituted or unsubstituted six-membered cycloalkane ring, a substituted or unsubstituted seven-membered cycloalkane ring, a substituted or unsubstituted eight-membered cycloalkane ring, a substituted or unsubstituted four-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring, a substituted or unsubstituted six-membered heteroalkane ring, a substituted or unsubstituted seven-membered heteroalkane ring, a substituted or unsubstituted eight-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring and a C6-C10 aromatic ring, and a substituted or unsubstituted six-membered heteroalkane ring and a C6-C10 aromatic ring;
    B为选自下组的环：取代或未取代的四元环烷环，取代或未取代的五元环烷环，取代或未取代的六元环烷环，取代或未取代的七元环烷环，取代或未取代的八元环烷环、取代或未取代的四元杂烷环，取代或未取代的五元杂烷环，取代或未取代的六元杂烷环，取代或未取代的七元杂烷环，取代或未取代的八元杂烷环，取代或未取代的五元杂烷环并C6-C10芳香环，取代或未取代的六元杂烷环并C6-C10芳香环；B is a ring selected from the group consisting of a substituted or unsubstituted four-membered cycloalkane ring, a substituted or unsubstituted five-membered cycloalkane ring, a substituted or unsubstituted six-membered cycloalkane ring, a substituted or unsubstituted seven-membered cycloalkane ring, a substituted or unsubstituted eight-membered cycloalkane ring, a substituted or unsubstituted four-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring, a substituted or unsubstituted six-membered heteroalkane ring, a substituted or unsubstituted seven-membered heteroalkane ring, a substituted or unsubstituted eight-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring and a C6-C10 aromatic ring, and a substituted or unsubstituted six-membered heteroalkane ring and a C6-C10 aromatic ring;
    当为取代或未取代的并环，如式Ib所示：
    when is a substituted or unsubstituted cyclic ring, as shown in Formula Ib:
    为单键或者双键； is a single bond or a double bond;
    A为选自下组的环：取代或未取代的四元环烷环，取代或未取代的五元环烷环，取代或未取代的六元环烷环，取代或未取代的七元环烷环，取代或未取代的八元环烷环、取代或未取代的四元杂烷环，取代或未取代的五元杂烷环，取代或未取代的六元杂烷环，取代或未取代的七元杂烷环，取代或未取代的八元杂烷环，取代或未取代的五元杂烷环并C6-C10芳香环，取代或未取代的六元杂烷环并C6-C10芳香环；A is a ring selected from the group consisting of a substituted or unsubstituted four-membered cycloalkane ring, a substituted or unsubstituted five-membered cycloalkane ring, a substituted or unsubstituted six-membered cycloalkane ring, a substituted or unsubstituted seven-membered cycloalkane ring, a substituted or unsubstituted eight-membered cycloalkane ring, a substituted or unsubstituted four-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring, a substituted or unsubstituted six-membered heteroalkane ring, a substituted or unsubstituted seven-membered heteroalkane ring, a substituted or unsubstituted eight-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring and a C6-C10 aromatic ring, and a substituted or unsubstituted six-membered heteroalkane ring and a C6-C10 aromatic ring;
    B为选自下组的环：取代或未取代的四元环烷环，取代或未取代的五元环烷环，取代或未取代的六元环烷环，取代或未取代的七元环烷环，取代或未取代的八元环烷环、取代或未取代的C6-C10芳香环，取代或未取代的四元杂烷环，取代或未取代的五元杂烷环，取代或未取代的六元杂烷环，取代或未取代的七元杂烷环，取代或未取代的八元杂烷环，取代或未取代的五元杂烷环并C6-C10芳香环，取代或未取代的六元杂烷环并C6-C10芳香环；B is a ring selected from the group consisting of a substituted or unsubstituted four-membered cycloalkane ring, a substituted or unsubstituted five-membered cycloalkane ring, a substituted or unsubstituted six-membered cycloalkane ring, a substituted or unsubstituted seven-membered cycloalkane ring, a substituted or unsubstituted eight-membered cycloalkane ring, a substituted or unsubstituted C6-C10 aromatic ring, a substituted or unsubstituted four-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring, a substituted or unsubstituted six-membered heteroalkane ring, a substituted or unsubstituted seven-membered heteroalkane ring, a substituted or unsubstituted eight-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring and a C6-C10 aromatic ring, and a substituted or unsubstituted six-membered heteroalkane ring and a C6-C10 aromatic ring;
    n为0、1、2或3；n is 0, 1, 2 or 3;
    其中，所述“取代”是指基团上的1-4个(优选为1、2、3或4个)氢原子各自独立地被选自下组的取代基所取代：C1-C6烷基、C3-C8环烷基、C3-C8杂环烷基、C1-C3卤代烷基、卤素、硝基、氰基、氨基、羟基、硫醇基、＝O、C1-C4羧基、C2-C4酯基、C2-C4酰胺基、C1-C6烷氧基、羧酸、C1-C4醇基、C1-C4烷胺基、-O-(CH2)m-C3-C8环烷、-O-(CH2)m-C3-C8杂烷环、-NH-(CH2)m-C3-C8环烷环、-NH-(CH2)m-C3-C8杂烷环；其中各个m各自独立地为0～3的整数；Wherein, the "substituted" means that 1 to 4 (preferably 1, 2, 3 or 4) hydrogen atoms on the group are independently replaced by substituents selected from the following group: C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, C1-C3 haloalkyl, halogen, nitro, cyano, amino, hydroxyl, thiol, =O, C1-C4 carboxyl, C2-C4 ester, C2-C4 amide, C1-C6 alkoxy, carboxylic acid, C1-C4 alcohol, C1-C4 alkylamino, -O-(CH 2 ) m -C3-C8 cycloalkane, -O-(CH 2 ) m -C3-C8 heteroalkyl ring, -NH-(CH 2 ) m -C3-C8 cycloalkane ring, -NH-(CH 2 ) m -C3-C8 heteroalkyl ring; wherein each m is independently an integer of 0 to 3;
    其中，所述的杂芳环、杂烷环或杂芳基各自独立地具有1-3个(优选为1、2或3个)选自N、O和S杂原子。Wherein, the heteroaromatic ring, heteroalkyl ring or heteroaryl group each independently has 1-3 (preferably 1, 2 or 3) heteroatoms selected from N, O and S.
  2. 如权利要求1所述的如式I所示的双环衍生物，其特征在于，为取代或未 取代的螺环，如式Ia所示：
    The bicyclic derivative as shown in formula I according to claim 1, characterized in that To replace or not Substituted spiro ring, as shown in Formula Ia:
    A为选自下组的环：取代或未取代的四元杂烷环，取代或未取代的五元杂烷环，取代或未取代的六元杂烷环，取代或未取代的七元杂烷环，取代或未取代的八元杂烷环；A is a ring selected from the group consisting of a substituted or unsubstituted four-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring, a substituted or unsubstituted six-membered heteroalkane ring, a substituted or unsubstituted seven-membered heteroalkane ring, and a substituted or unsubstituted eight-membered heteroalkane ring;
    B为选自下组的环：取代或未取代的四元杂烷环，取代或未取代的五元杂烷环，取代或未取代的六元杂烷环，取代或未取代的七元杂烷环，取代或未取代的八元杂烷环。B is a ring selected from the group consisting of a substituted or unsubstituted four-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring, a substituted or unsubstituted six-membered heteroalkane ring, a substituted or unsubstituted seven-membered heteroalkane ring, and a substituted or unsubstituted eight-membered heteroalkane ring.
  3. 如权利要求1所述的如式I所示的双环衍生物，其特征在于，所述A环和B环中的取代是指一个或多个(例如1、2、3个)氢原子被卤素或＝O取代。The bicyclic derivative as shown in formula I as claimed in claim 1, characterized in that the substitution in the A ring and the B ring refers to one or more (e.g., 1, 2, 3) hydrogen atoms being replaced by halogen or =O.
  4. 如权利要求1所述的如式I所示的双环衍生物，其特征在于，所述A环和B环中，所述取代在螺原子的邻位取代。The bicyclic derivative as shown in formula I as claimed in claim 1, characterized in that in the A ring and the B ring, the substitution is at the ortho position of the spiro atom.
  5. 如权利要求1或2中任一项所述双环衍生物，其特征在于，所述A环和B环中的杂烷环各自独立地包含1-3(优选1、2、3)个氮原子。The bicyclic derivative according to any one of claims 1 or 2, characterized in that the heteroalkyl rings in the A ring and the B ring each independently contain 1-3 (preferably 1, 2, 3) nitrogen atoms.
  6. 如权利要求3中所述双环衍生物，其特征在于，Y1通过杂烷环中的氮原子连接到A环；B环通过杂烷环中的氮原子连接到Y2。The bicyclic derivative as claimed in claim 3, characterized in that Y 1 is connected to ring A through a nitrogen atom in the heteroalkyl ring; and ring B is connected to Y 2 through a nitrogen atom in the heteroalkyl ring.
  7. 如权利要求1所述的双环衍生物，其特征在于，R1为取代或未取代的其中C环为C6-C10芳环或5-8元杂芳环，D环为取代或未取代的5-8元杂芳环或取代或未取代的5-8元杂烷环。The bicyclic derivative according to claim 1, characterized in that R 1 is substituted or unsubstituted The C ring is a C6-C10 aromatic ring or a 5-8 membered heteroaromatic ring, and the D ring is a substituted or unsubstituted 5-8 membered heteroaromatic ring or a substituted or unsubstituted 5-8 membered heteroalkyl ring.
  8. 如权利要求1所述的双环衍生物，其特征在于，R2为氢原子或-L1-L2；其中，-L1-为-(取代或未取代的苯基)-，L2为取代或未取代的5-6元含氮杂芳基。The bicyclic derivative according to claim 1, characterized in that R2 is a hydrogen atom or -L1-L2; wherein -L1- is -(substituted or unsubstituted phenyl)-, and L2 is a substituted or unsubstituted 5-6-membered nitrogen-containing heteroaryl group.
  9. 如权利要求1所述的双环衍生物，其特征在于，R2为如下式a结构：
    The bicyclic derivative according to claim 1, characterized in that R 2 is a structure of the following formula a:
    其中，Z1、Z2、Z3、Z4各自独立地为CRc或N；wherein Z 1 , Z 2 , Z 3 , and Z 4 are each independently CR c or N;
    L2为无、C1-C6烷基、取代或未取代的C1-C6烷氧基、取代或未取代的C1-C6烷硫基、取代或未取代的3-8元环烷基、取代或未取代的3-8元杂烷环基、取代或未取代的5-8元杂芳基； L2 is none, C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C1-C6 alkylthio, substituted or unsubstituted 3-8 membered cycloalkyl, substituted or unsubstituted 3-8 membered heteroalkyl ring group, substituted or unsubstituted 5-8 membered heteroaryl;
    各个Rc各自独立地为氢、卤素、C1-C6烷基、3-8元环烷基、C1-C6烷氧基；Each R c is independently hydrogen, halogen, C1-C6 alkyl, 3-8 membered cycloalkyl, C1-C6 alkoxy;
    或者，or,
    R2为如下式b结构：
    R 2 is the following structure:
    其中，Z1、Z2、Z3、Z4、Z5、Z6、Z7、Z8、Z9各自独立地为CRc或N； wherein Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , Z 6 , Z 7 , Z 8 , and Z 9 are each independently CR c or N;
    各个Rc各自独立地为氢、卤素、C1-C6烷基、C3-C8环烷基、C1-C6烷氧基。Each R c is independently hydrogen, halogen, C1-C6 alkyl, C3-C8 cycloalkyl, or C1-C6 alkoxy.
  10. 如权利要求1所述的双环衍生物，其特征在于，X为氧原子时，R3a为氢原子或C1～C3烷基，R3b不存在；X为氮原子时，R3a为氢原子、羟基或C1～C3烷基，R3b为氢原子。The bicyclic derivative according to claim 1, characterized in that when X is an oxygen atom, R 3a is a hydrogen atom or a C1-C3 alkyl group, and R 3b does not exist; when X is a nitrogen atom, R 3a is a hydrogen atom, a hydroxyl group or a C1-C3 alkyl group, and R 3b is a hydrogen atom.
  11. 如权利要求1所述的双环衍生物，其特征在于，所述化合物为如下式III-1结构：
    The bicyclic derivative according to claim 1, characterized in that the compound has the following structure:
    或者，所述化合物为如下式III-4结构：
    Alternatively, the compound is of the following formula III-4:
  12. 如权利要求1所述的双环衍生物，其特征在于，所述的双环衍生物选自下组：



    The bicyclic derivative according to claim 1, characterized in that the bicyclic derivative is selected from the group consisting of:



  13. 一种药物组合物，所述的药物组合物包括：A pharmaceutical composition, comprising:
    (a)治疗有效量的如权利要求1-12中任一项所述的双环衍生物，及其消旋体、立体异构体、互变异构体、同位素标记物、氮氧化物、溶剂化物、多晶型物、代谢产物、酯、前药或其药学上可接受的盐；和(a) a therapeutically effective amount of a bicyclic derivative according to any one of claims 1 to 12, and its racemate, stereoisomer, tautomer, isotope-labeled product, nitrogen oxide, solvate, polymorph, metabolite, ester, prodrug or a pharmaceutically acceptable salt thereof; and
    (b)药学上可接受的载体。(b) a pharmaceutically acceptable carrier.
  14. 如权利要求1-12中任一项所述的式(I)所示的双环衍生物及其消旋体、立体异构体、互变异构体、同位素标记物、氮氧化物、溶剂化物、多晶型物、代谢产物、酯、前药或其药学上可接受的盐的用途，其特征在于，用于制备治疗或预防与αvβ1、αvβ6和αvβ8整联蛋白活性或表达量相关的疾病、病症或病状的药物组合物。Use of the bicyclic derivative represented by formula (I) as described in any one of claims 1 to 12 and its racemate, stereoisomer, tautomer, isotope label, nitrogen oxide, solvate, polymorph, metabolite, ester, prodrug or pharmaceutically acceptable salt thereof, characterized in that it is used to prepare a pharmaceutical composition for treating or preventing diseases, disorders or conditions related to the activity or expression of αvβ1, αvβ6 and αvβ8 integrins.
  15. 如权利要求14所述的用途，其特征在于，所述与αvβ1、αvβ6和αvβ8整联蛋白活性或表达量相关的疾病、病症或病状选自下组：自身免疫疾病、纤维化疾病、炎症性肠病(IBD)、复发型多发性硬化症(RMS)、进展性多灶性白质脑病(PML)、溃疡性结肠炎(UC)、克罗恩病(CD)、慢性病毒性乙肝和丙肝，非酒精性脂肪肝(NAFLD)、老年性黄斑变性、糖尿病型视网膜症、视网膜血管性疾病、骨质疏松和细胞增殖性疾病，The use according to claim 14, characterized in that the disease, disorder or condition associated with the activity or expression of αvβ1, αvβ6 and αvβ8 integrins is selected from the group consisting of autoimmune diseases, fibrotic diseases, inflammatory bowel disease (IBD), relapsing multiple sclerosis (RMS), progressive multifocal leukoencephalopathy (PML), ulcerative colitis (UC), Crohn's disease (CD), chronic viral hepatitis B and C, non-alcoholic fatty liver disease (NAFLD), age-related macular degeneration, diabetic retinopathy, retinal vascular disease, osteoporosis and cell proliferative diseases,
    优选所述的纤维化疾病选自下组：肺纤维化、特发性肺纤维化、非特异性间质性肺炎(NSIP)、常规间质性肺疾病(UIP)、辐射诱发性肺纤维化、家族性肺纤维化、气道肺纤维化、慢性阻塞性肺疾病(COPD)、间质性肺病、肝纤维化、慢性肾病、肾纤维化、皮肤纤维化、系统性硬化症，或其组合，Preferably, the fibrotic disease is selected from the group consisting of pulmonary fibrosis, idiopathic pulmonary fibrosis, nonspecific interstitial pneumonia (NSIP), conventional interstitial lung disease (UIP), radiation-induced pulmonary fibrosis, familial pulmonary fibrosis, airway pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), interstitial lung disease, liver fibrosis, chronic kidney disease, renal fibrosis, skin fibrosis, systemic sclerosis, or a combination thereof,
    所述细胞增值性疾病为癌症，选自下组：乳腺癌、宫颈癌、子宫内膜癌、卵巢癌、结肠癌、直肠癌、胰腺癌、肝癌、肺癌、非小细胞肺癌、肺癌脑转移、口腔鳞状细胞癌、头癌、颈癌、头颈部鳞状细胞癌、口或鼻黏膜癌、喉癌、肾癌、肾细胞癌、卵巢癌、脾癌、小肠癌、大肠癌、胃癌、食道癌、食管癌、肺鳞状细胞癌、胆管癌、胆囊癌、黑色素瘤、尿路上皮癌、泌尿生殖道癌、生殖器癌、前列腺癌、睾丸癌、膀胱癌、血液癌、皮肤癌、骨髓癌、脑癌、中枢神经系统癌、肌肉组织癌、甲状腺癌，或其组合。The cell proliferative disease is a cancer selected from the group consisting of breast cancer, cervical cancer, endometrial cancer, ovarian cancer, colon cancer, rectal cancer, pancreatic cancer, liver cancer, lung cancer, non-small cell lung cancer, brain metastasis of lung cancer, oral squamous cell carcinoma, head cancer, neck cancer, head and neck squamous cell carcinoma, oral or nasal mucosal cancer, laryngeal cancer, kidney cancer, renal cell carcinoma, ovarian cancer, spleen cancer, small intestine cancer, large intestine cancer, stomach cancer, esophageal cancer, esophageal cancer, lung squamous cell carcinoma, bile duct cancer, gallbladder cancer, melanoma, urothelial carcinoma, urogenital tract cancer, genital cancer, prostate cancer, testicular cancer, bladder cancer, blood cancer, skin cancer, bone marrow cancer, brain cancer, central nervous system cancer, muscle tissue cancer, thyroid cancer, or a combination thereof.
  16. 一种双环衍生物，及其消旋体、立体异构体、互变异构体、同位素标记物、氮氧化物、溶剂化物、多晶型物、代谢产物、酯、前药或其药学上可接受的盐，其特征在于，所述双环衍生物具有如下式II-a、II-b、II-c所示的结构：
    A bicyclic derivative, and its racemate, stereoisomer, tautomer, isotope-labeled substance, nitrogen oxide, solvate, polymorph, metabolite, ester, prodrug or pharmaceutically acceptable salt, characterized in that the bicyclic derivative has a structure shown in the following formula II-a, II-b, II-c:
    其中，in,
    各个Y1各自独立地为C1～C6亚烷基，-O-，-(C1～C6亚烷基)-O-，-NH-，-(C1～C6亚烷基)-NH-；Each Y 1 is independently C1-C6 alkylene, -O-, -(C1-C6 alkylene)-O-, -NH-, -(C1-C6 alkylene)-NH-;
    各个Y2各自独立地为C1～C6亚烷基，-O-，-(C1～C6亚烷基)-O-，-C(O)-(C1～C6亚烷基)-NH-，-NH-，-NH-(C1～C6亚烷基)-，-(C1～C6亚烷基)-NH-；Each Y2 is independently C1~C6 alkylene, -O-, -(C1~C6 alkylene)-O-, -C(O)-(C1~C6 alkylene)-NH-, -NH-, -NH-(C1~C6 alkylene)-, -(C1~C6 alkylene)-NH-;
    各个R1各自独立地为取代或未取代的6-10元芳环，取代或未取代的5-8元杂芳环，取代或未取代的其中C环、D环各自独立地为取代或未取代的C6-C10芳环、取代或未取代的5-8元杂芳环，取代或未取代的5-8元环烷环，或取代或未取代的5-8元杂烷环；Each R 1 is independently a substituted or unsubstituted 6-10 membered aromatic ring, a substituted or unsubstituted 5-8 membered heteroaromatic ring, a substituted or unsubstituted wherein the C ring and the D ring are each independently a substituted or unsubstituted C6-C10 aromatic ring, a substituted or unsubstituted 5-8 membered heteroaromatic ring, a substituted or unsubstituted 5-8 membered cycloalkane ring, or a substituted or unsubstituted 5-8 membered heteroalkane ring;
    各个R2各自独立地为氢原子，取代或未取代的C6-C10芳环，取代或未取代的5-8元杂芳环，取代或未取代的C8-C16稠环，或-L1-L2；Each R 2 is independently a hydrogen atom, a substituted or unsubstituted C6-C10 aromatic ring, a substituted or unsubstituted 5-8 membered heteroaromatic ring, a substituted or unsubstituted C8-C16 condensed ring, or -L1-L2;
    其中，-L1-选自无、-(取代或未取代的C1-C6亚烷基)-、-(取代或未取代的C1-C6亚烷氧基)-、-(取代或未取代的C1-C6亚烷硫基)-、-(取代或未取代的C3-C8环烷基)-、-(取代或未取代的C3-C8杂环烷基)-、-(取代或未取代的C6-C10芳基)-、-(取代或未取代的C5-C8杂芳基)-，Wherein, -L1- is selected from none, -(substituted or unsubstituted C1-C6 alkylene)-, -(substituted or unsubstituted C1-C6 alkyleneoxy)-, -(substituted or unsubstituted C1-C6 alkylenethio)-, -(substituted or unsubstituted C3-C8 cycloalkyl)-, -(substituted or unsubstituted C3-C8 heterocycloalkyl)-, -(substituted or unsubstituted C6-C10 aryl)-, -(substituted or unsubstituted C5-C8 heteroaryl)-,
    L2为选自无、取代或未取代的C1-C6烷基、取代或未取代的C1-C6烷氧基、取代或未取代的C1-C6烷硫基、取代或未取代的C3-C8环烷基、-(C1-C3烷基)-取代或未取代的C3-C8环烷基、-(C1-C3烷氧基)-取代或未取代的C3-C8环烷基、-O-取代或未取代的C3-C8环烷基、取代或未取代的C3-C8杂烷环基、-(C1-C3烷基)-取代或未取代的C3-C8杂烷环基、-(C1-C3烷氧基)-取代或未取代的C3-C8杂烷环基、-O-取代或未取代的C3-C8杂烷环基、取代或未取代的5-8元杂芳基；L2 is selected from none, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C1-C6 alkylthio, substituted or unsubstituted C3-C8 cycloalkyl, -(C1-C3 alkyl)-substituted or unsubstituted C3-C8 cycloalkyl, -(C1-C3 alkoxy)-substituted or unsubstituted C3-C8 cycloalkyl, -O-substituted or unsubstituted C3-C8 cycloalkyl, substituted or unsubstituted C3-C8 heteroalkylcyclyl, -(C1-C3 alkyl)-substituted or unsubstituted C3-C8 heteroalkylcyclyl, -(C1-C3 alkoxy)-substituted or unsubstituted C3-C8 heteroalkylcyclyl, -O-substituted or unsubstituted C3-C8 heteroalkylcyclyl, substituted or unsubstituted 5-8 membered heteroaryl;
    各个R3a’各自独立地为C1-C6烷基；Each R 3a ' is independently a C1-C6 alkyl group;
    各个各自独立地为取代或未取代的螺环或取代或未取代的并环；each Each is independently a substituted or unsubstituted spiro ring or a substituted or unsubstituted cyclocyclic ring;
    其中，当为取代或未取代的螺环，如式Ia所示：
    Among them, when is a substituted or unsubstituted spiro ring, as shown in Formula Ia:
    A为选自下组的环：取代或未取代的四元环烷环，取代或未取代的五元环烷环，取代或未取代的六元环烷环，取代或未取代的七元环烷环，取代或未取代的八元环烷环、取代或未取代的四元杂烷环，取代或未取代的五元杂烷环，取代或未取代的六元杂烷环，取代或未取代的七元杂烷环，取代或未取代的八元杂烷环，取代或未取代的五元杂烷环并C6-C10芳香环，取代或未取代的六元杂烷环并C6-C10芳香环；A is a ring selected from the group consisting of a substituted or unsubstituted four-membered cycloalkane ring, a substituted or unsubstituted five-membered cycloalkane ring, a substituted or unsubstituted six-membered cycloalkane ring, a substituted or unsubstituted seven-membered cycloalkane ring, a substituted or unsubstituted eight-membered cycloalkane ring, a substituted or unsubstituted four-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring, a substituted or unsubstituted six-membered heteroalkane ring, a substituted or unsubstituted seven-membered heteroalkane ring, a substituted or unsubstituted eight-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring and a C6-C10 aromatic ring, and a substituted or unsubstituted six-membered heteroalkane ring and a C6-C10 aromatic ring;
    B为选自下组的环：取代或未取代的四元环烷环，取代或未取代的五元环烷环，取代或未取代的六元环烷环，取代或未取代的七元环烷环，取代或未取代的八元环烷环、取代或未取代的四元杂烷环，取代或未取代的五元杂烷环，取代或未取代的六元杂烷环，取代或未取代的七元杂烷环，取代或未取代的八元杂烷环，取代或未取代的五元杂烷环并C6-C10芳香环，取代或未取代的六元杂烷环并C6-C10芳香环；B is a ring selected from the group consisting of a substituted or unsubstituted four-membered cycloalkane ring, a substituted or unsubstituted five-membered cycloalkane ring, a substituted or unsubstituted six-membered cycloalkane ring, a substituted or unsubstituted seven-membered cycloalkane ring, a substituted or unsubstituted eight-membered cycloalkane ring, a substituted or unsubstituted four-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring, a substituted or unsubstituted six-membered heteroalkane ring, a substituted or unsubstituted seven-membered heteroalkane ring, a substituted or unsubstituted eight-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring and a C6-C10 aromatic ring, and a substituted or unsubstituted six-membered heteroalkane ring and a C6-C10 aromatic ring;
    当为取代或未取代的并环，如式Ib所示：
    when is a substituted or unsubstituted cyclic ring, as shown in Formula Ib:
    为单键或者双键； is a single bond or a double bond;
    A为选自下组的环：取代或未取代的四元环烷环，取代或未取代的五元环烷环，取代或未取代的六元环烷环，取代或未取代的七元环烷环，取代或未取代的八元环烷环、取代或未取代的四元杂烷环，取代或未取代的五元杂烷环，取代或未取代的六元杂烷环，取代或未取代的七元杂烷环，取代或未取代的八元杂烷环，取代或未取代的五元杂烷环并C6-C10芳香环，取代或未取代的六元杂烷环并C6-C10芳香环；A is a ring selected from the group consisting of a substituted or unsubstituted four-membered cycloalkane ring, a substituted or unsubstituted five-membered cycloalkane ring, a substituted or unsubstituted six-membered cycloalkane ring, a substituted or unsubstituted seven-membered cycloalkane ring, a substituted or unsubstituted eight-membered cycloalkane ring, a substituted or unsubstituted four-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring, a substituted or unsubstituted six-membered heteroalkane ring, a substituted or unsubstituted seven-membered heteroalkane ring, a substituted or unsubstituted eight-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring and a C6-C10 aromatic ring, and a substituted or unsubstituted six-membered heteroalkane ring and a C6-C10 aromatic ring;
    B为选自下组的环：取代或未取代的四元环烷环，取代或未取代的五元环烷环，取代或未取代的六元环烷环，取代或未取代的七元环烷环，取代或未取代的八元环烷环、取代或未取代的C6-C10芳香环，取代或未取代的四元杂烷环，取代或未取代的五元杂烷环，取代或未取代的六元杂烷环，取代或未取代的七元杂烷环，取代或未取代的八元杂烷环，取代或未取代的五元杂烷环并C6-C10芳香环，取代或未取代的六元杂烷环并C6-C10芳香环；B is a ring selected from the group consisting of a substituted or unsubstituted four-membered cycloalkane ring, a substituted or unsubstituted five-membered cycloalkane ring, a substituted or unsubstituted six-membered cycloalkane ring, a substituted or unsubstituted seven-membered cycloalkane ring, a substituted or unsubstituted eight-membered cycloalkane ring, a substituted or unsubstituted C6-C10 aromatic ring, a substituted or unsubstituted four-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring, a substituted or unsubstituted six-membered heteroalkane ring, a substituted or unsubstituted seven-membered heteroalkane ring, a substituted or unsubstituted eight-membered heteroalkane ring, a substituted or unsubstituted five-membered heteroalkane ring and a C6-C10 aromatic ring, and a substituted or unsubstituted six-membered heteroalkane ring and a C6-C10 aromatic ring;
    各个n各自独立地为0、1、2或3；Each n is independently 0, 1, 2 or 3;
    其中，所述“取代”是指基团上的1-4个(优选为1、2、3或4个)氢原子各自独立地被选自下组的取代基所取代：C1-C6烷基、C3-C8环烷基、C3-C8杂环烷基、C1-C3卤代烷基、卤素、硝基、氰基、氨基、羟基、硫醇基、＝O、C1-C4羧基、C2-C4酯基、C2-C4酰胺基、C1-C6烷氧基、羧酸、C1-C4醇基、C1-C4烷胺基、-O-(CH2)m-C3-C8环烷、-O-(CH2)m-C3-C8杂烷环、-NH-(CH2)m-C3-C8环烷、-NH-(CH2)m-C3-C8杂烷环或-N＝Ph2；其中各个m各自独立地为0～3的整数；Wherein, the "substituted" means that 1 to 4 (preferably 1, 2, 3 or 4) hydrogen atoms on the group are independently replaced by a substituent selected from the following group: C1-C6 alkyl, C3-C8 cycloalkyl, C3-C8 heterocycloalkyl, C1-C3 haloalkyl, halogen, nitro, cyano, amino, hydroxyl, thiol, =O, C1-C4 carboxyl, C2-C4 ester, C2-C4 amide, C1-C6 alkoxy, carboxylic acid, C1-C4 alcohol, C1-C4 alkylamino, -O-(CH 2 ) m -C3-C8 cycloalkane, -O-(CH 2 ) m -C3-C8 heteroalkyl ring, -NH-(CH 2 ) m -C3-C8 cycloalkane, -NH-(CH 2 ) m -C3-C8 heteroalkyl ring or -N=Ph 2 ; wherein each m is independently an integer of 0 to 3;
    其中，所述的杂芳环、杂烷环或杂芳基各自独立地具有1-3个(优选为1、2或3个)选自N、O和S杂原子；wherein the heteroaromatic ring, heteroalkyl ring or heteroaryl group each independently has 1 to 3 (preferably 1, 2 or 3) heteroatoms selected from N, O and S;
    其中，所述式II-a、式II-b、式II-c的结构均为化学稳定的结构。 Among them, the structures of Formula II-a, Formula II-b and Formula II-c are all chemically stable structures.
PCT/CN2023/124799 2022-10-17 2023-10-16 Bicyclic derivative integrin inhibitor WO2024083086A1 (en)

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