CN115433210A

CN115433210A - Preparation and application of compound with immunoregulation function

Info

Publication number: CN115433210A
Application number: CN202111602298.XA
Authority: CN
Inventors: 张毅; 程战领; 冯志勇; 江磊; 尚珂; 寿建勇; 汪兵; 徐雪丽; 徐圆; 张兴; 张学标
Original assignee: Shanghai Ennovabio Pharmaceuticals Co Ltd
Current assignee: Shanghai Ennovabio Pharmaceuticals Co Ltd
Priority date: 2021-06-04
Filing date: 2021-12-24
Publication date: 2022-12-06
Also published as: WO2022253327A1; CN117642392A

Abstract

The invention discloses a preparation method and application of a compound with an immunoregulation effect. Specifically, the invention discloses a compound with a structure shown as a formula I, wherein the definition of each group is described in the specification. The invention also provides the application of the compounds in the aspects of regulating immunity and inhibiting PD-1/PD-L1.

Description

Preparation and application of compound with immunoregulation function

Technical Field

The invention relates to the field of small molecule protein inhibitors, and particularly provides preparation and application of a compound with an immunoregulation function.

Background

The immune system has the functions of monitoring, defending, regulating and the like. Cellular immunity is involved mainly in the immune response to intracellular parasitic pathogenic microorganisms and to tumor cells, in the formation of delayed allergy and autoimmune diseases, in graft rejection and in the regulation of humoral immunity. Activation of T lymphocytes by antigen presenting cells is often regulated by two distinct signals. The primary signal is transduced by the presentation of foreign antigen peptides by the Major Histocompatibility Complex (MHC) on APC cells via the T Cell Receptor (TCR). Secondary signals, also known as co-stimulatory signals, are transmitted to T cells by binding of co-stimulatory molecules on APC cells to T cell surface receptors, regulating T cell proliferation, cytokine secretion and effector functions. The secondary signal comprises positive regulation and negative regulation, wherein the positive regulation signal promotes T cell activation, and the negative regulation signal induces T cell tolerance, so that the secondary signal is very important for human body to adapt and adjust the response of autoimmune cells to different external antigens.

Programmed death protein ligand 1 (PD-L1), also known as cluster of differentiation 274 (CD274) or B7 homologous protein 1 (B7 homolog1, B7-H1), belongs to the tumor necrosis factor superfamily, is a type I transmembrane glycoprotein consisting of 290 amino acid residues, contains an IgV-like region, an IgC-like region, a transmembrane hydrophobic region and an intracellular tail of 30 amino acids, and has a complete molecular weight of 40kDa. PD-L1 mRNA is present in almost all tissues, but PD-L1 protein is persistently expressed in only a small portion of tissues, including the liver, lungs, tonsils, and tissues privileged for immunity such as the eye, placenta, and the like. PD-L1 is also expressed on activated T cells, B cells, monocytes, dendritic cells, macrophages, and the like.

The receptor of PD-L1 is PD-1, and is mainly expressed on the surfaces of immune cells such as activated CD4+ T cells, CD8+ T cells, NK cells, B cells, activated monocytes and the like. Binding of PD-L1 to PD-1 can promote phosphorylation of tyrosine in ITSM (immunoreceptor tyrosine switch motif) structure in PD-1 cytoplasm, recruit SHP1/2, dephosphorylate downstream Syk and PI3K, and limit interaction of antigen presenting cells or dendritic cells with T cells. The combination can further inhibit the metabolism of T cells, reduce the secretion of effector cytokines IL-2 and IFN-gamma, and induce the exhaustion and apoptosis of the T cells, thereby reducing the immune response involved by the T cells and performing the negative regulation function.

T cells recognize antigen and, upon activation, secrete IFN- γ. T cell-derived IFN- γ expands and maintains T cell functions, such as upregulation of MHC molecules, enhancement of antigen processing and presentation by target cells, and promotion of T cell differentiation. IFN-gamma can also induce PD-L1 expression of tissues at immune inflammation positions, and damage to tissues caused by excessive immunity is prevented. PD-L1 is expressed on the surface of antigen presenting cells (DC cells, macrophages, etc.), as well as vascular endothelial cells, upon IFN- γ stimulation. IFN-gamma-induced interferon regulatory factor 1 (IRF-1) can also bind to interferon regulatory factor binding sites 200bp and 320bp before the transcription start site of PD-L1 to regulate PD-L1 from the transcription level. PD-L1 can combine with PD-1 on the surface of T cells to perform negative regulation function, thereby protecting inflammatory sites.

The negative regulation function of PD-L1 plays an important role in tumor immunity. In 2004, konishi et al first found expression of PD-L1 in tissue samples of patients with non-small cell lung cancer, and then PD-L1 was found to be expressed in tissues of patients with various tumors, including stomach cancer, lung cancer, liver cancer, intrahepatic bile duct cancer, colon cancer, pancreatic cancer, ovarian cancer, breast cancer, cervical cancer, squamous cell carcinoma of the head and neck, nasopharyngeal carcinoma, esophageal cancer, bladder cancer, renal cell carcinoma, skin cancer, oral squamous cell carcinoma, and the like. In the process of cell malignant transformation, new protein molecules can be generated due to gene mutation, exogenous gene (virus) expression or resting gene activation and the like, and after the proteins are degraded in cells, certain degraded peptide segments can be expressed on the surfaces of the cells to become tumor antigens. The immune system can recognize tumor antigens and eliminate tumor cells through immune surveillance, and the tumor cells can evade immune attack by using PD-L1.

Expression of PD-L1 at the tumor site can protect tumor cells from damage in a variety of ways. The secretion of IFN-gamma by Tumor Infiltrating Lymphocytes (TIL) can induce the expression of PD-L1 by tumor cells and peripheral stromal cells. And PD-L1 of the tumor cells can be combined with PD-1 on the TIL, inhibit the activation of the TIL cells and further cause the apoptosis of the TIL cells. In vitro experiments show that tumor cell associated PD-L1 can increase apoptosis of tumor specific T cells, and the PD-L1 monoclonal antibody can weaken the effect. The tumor-associated PD-L1 can promote T cells to express IL-10, and further inhibit immune response. PD-L1 is not only a ligand of PD-1, but also can be used as a receptor to transmit reverse signals to protect tumor cells from apoptosis induced by other anti-tumor pathways such as FAS-FASL.

At present, a plurality of marketed monoclonal antibody medicines targeting PD-1 or PD-L1 prove that the blocking agent of PD-1/PD-L1 can be used for clinical treatment of various tumors. However, antibody drugs have their own characteristics, such as high production cost, poor stability, need to be administered by injection, and easy generation of immunogenicity. The micromolecule drug has the advantages of good tissue permeability, convenient storage and transportation, lower production cost, no immunogenicity, generally oral administration and the like, so that the research and development of the micromolecule blocking agent of PD-1/PD-L1 have obvious application value and social value.

In view of the above, there is an urgent need in the art to develop small molecule PD-1/PD-L1 blockers.

Disclosure of Invention

The invention aims to provide a small-molecule PD-1/PD-L1 blocking agent.

In a first aspect of the present invention, there is provided a compound represented by the following formula I, a stereoisomer thereof or a tautomer thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof:

wherein the content of the first and second substances,

ring A is selected from the group consisting of: 5-12 membered heteroaryl (preferably 6 membered heteroaryl), C6-C10 aryl (preferably phenyl), 5-12 membered heterocycloalkyl (including saturated or partially unsaturated monocyclic, bicyclic, spiro or bridged rings), C3-C12 (preferably C5-C12) cycloalkyl, wherein said heterocycloalkyl has 1-3 heteroatoms selected from N, O, S;

R ₁ 、R ₄ each independently selected from the group consisting of: H. halogen, CN, C1-C3 alkyl, C1-C3 alkoxy;

R ₅ independently selected from the group consisting of: H. halogen, CN, C1-C6 alkyl, halogenated C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkoxy, C3-C6 cycloalkyl, halogenated C3-C6 cycloalkyl;

R ₂ independently selected from the group consisting of: C1-C4 alkyl,

n1 is 0, 1,2,3 or 4;

ra and Rb are each independently selected from the group consisting of: H. C1-C8 alkyl, C3-C8 cycloalkyl (including monocyclic, fused, spiro and bridged rings), 5-10 membered heterocyclyl (including monocyclic, fused, spiro and bridged rings) having 1-3 heteroatoms selected from N, S and O, or- (CH) ₂ ) _n2 -Rc; wherein Rc is a 5-7 membered nitrogen-containing saturated ring, a C3-C8 membered cycloalkyl group, and n2 is 0, 2,3 or 4; or Ra and Rb together with the N atom to which they are attached form a 5-to 10-membered saturated heterocyclic ring;

wherein Ra, rb, rc, or Ra and Rb together forming a ring may be further substituted with 1 to 3 groups selected from: halogen, COOH, C (R) ₆ R ₇ ) _m1 -COOH, C1-C3 alkyl, C1-C6 acylamino (-C (= O) -N (Rd) ₂ or-NH-C (= O) (Rd), rd being H or C1-C5 alkyl); wherein m1 is 1,2 or 3,R ₆ And R ₇ Each independently selected from H, halogen, C1-C3 alkyl;

R ₃ independently selected from the group consisting of: a 4-to 8-membered nitrogen-containing heterocycle,

n3 is 0, 1 or 2;

the 4-to 8-membered nitrogen-containing heterocyclic ring may be substituted by CH ₂ -Re substitution, wherein Re is a 5-7 membered nitrogen containing saturated ring;

ra 'and Rb' are each independently selected from the group consisting of: H. C1-C3 alkyl, or CH ₂ -Re; wherein Re is a 5-7 membered nitrogen-containing saturated ring;

wherein the carbon atoms on Ra ', rb' and Re may be optionally substituted with carbonyl groups;

n is 0, 1 or 2;

m is 0, 1,2 or 3;

each L is independently selected from the group consisting of: a bond, C2-C4 alkenylene, halogenated C2-C4 alkenylene;

or the said

Together form a structure shown by the following formula:

wherein X and Y are each independently selected from the group consisting of: o, S, N, CH, NH, CH ₂ (ii) a The dotted line is a single or double bond;

is the attachment site of the group;

each of which is

Each independently is a single or double bond;

with the proviso that the compounds of formula I are chemically stable structures.

In another preferred embodiment, R ¹ And R ⁴ Each independently selected from the group consisting of: halogen and methyl.

In another preferred embodiment, R ¹ And R ⁴ Each independently selected from the group consisting of: cl and Br.

In another preferred embodiment, R is ⁵ Comprises at least one group selected from the group consisting of: -OCHF ₂ 、-OCH ₂ F. Cyclopropyl, CN, ethyl, -CH ₂ CF ₃ 。

In another preferred embodiment, R is ² And R ³ Each independently selected from the group consisting of:

wherein the E ring is an aza 5-7 membered saturated ring, p, R and s are each independently 0, 1,2 or 3, and R is selected from the group consisting of: oxo (= O), carboxyl.

In another preferred embodiment, the compound has the structure shown as the following formula:

wherein, X ¹ And X ² Each independently is CH or N, and when X is ¹ Or X ² When is CH, the R is ⁵ Or R ⁵ ' can be located on CH (at this time corresponding to X) ¹ Or X ² Is C);

R ⁵ is a group selected from: unsubstituted or halogenated C2-C6 alkyl, halogenated C1-C6 alkoxy, cyano-substituted C1-C6 alkoxy, unsubstituted or halogenated C3-C6 cycloalkyl; preferably a halogenated C1-C6 alkoxy group, an unsubstituted or halogenated C2-C6 alkyl group;

R ⁵ ' is a group selected from: H. unsubstituted or halogenated C1-C6 alkyl, unsubstituted or halogenSubstituted C1-C6 alkoxy, unsubstituted or halogenated C3-C6 cycloalkyl; unsubstituted or halogenated C1-C6 alkyl, unsubstituted or halogenated C1-C6 alkoxy, unsubstituted or halogenated C3-C6 cycloalkyl are preferred. In another preferred embodiment, R is ₅ Comprises at least one group selected from the group consisting of: -OCHF ₂ 、-OCH ₂ F. Cyclopropyl, CN, ethyl, -CH ₂ CF ₃ 、-OCH ₂ CN。

In another preferred embodiment, ra and Rb are each independently selected from the group consisting of: H. C1-C8 alkyl, C3-C8 cycloalkyl (including monocyclic, fused, spiro and bridged rings), 5-10 membered heterocyclyl (including monocyclic, fused, spiro and bridged rings) having 1-3 heteroatoms selected from N, S and O; or Ra and Rb, together with the linking N atom, form a 5-to 10-membered saturated heterocyclic ring;

wherein Ra, rb, and the saturated heterocycle formed by Ra and Rb may be further substituted by 1 to 3 groups selected from: halogen, C1-C3 alkyl, C1-C6 acylamino, COOH, C (R) ₆ R ₇ ) _m1 -COOH; wherein m1 is 1,2 or 3,R ₆ And R ₇ Each independently selected from H, halogen, C1-C3 alkyl.

In another preferred embodiment, ra is selected from the group consisting of: H. a C1-C3 alkyl group;

the Rb is selected from the following groups: C3-C8 cycloalkyl (including monocyclic, fused, spiro and bridged rings), 5-10 membered heterocyclyl (including monocyclic, fused, spiro and bridged rings) having 1-3 heteroatoms selected from N, S and O;

or Ra and Rb together with the N atom to which they are attached form a 5-to 10-membered saturated heterocyclic ring;

wherein Ra, rb, or the saturated heterocyclic ring formed by Ra and Rb can be further substituted by 1 to 3 groups selected from the following group: halogen, C1-C3 alkyl, C1-C6 acylamino, COOH, C (R) ₆ R ₇ ) _m1 -COOH; wherein m1 is 1,2 or 3,R ₆ And R ₇ Each independently selected from H, halogen, C1-C3 alkyl.

the Rb is selected from the following groups: C1-C8 alkyl, or a group selected from:

or Ra and Rb together with the attached N atom form a saturated heterocyclic ring selected from the group consisting of:

and said Ra, rb, or a co-formed group thereof may be further substituted with 1 to 3 groups selected from: C1-C3 alkyl, COOH, C (R) ₆ R ₇ ) _m1 -COOH; wherein m1 is 1,2 or 3,R ₆ And R ₇ Each independently selected from H, halogen, C1-C3 alkyl.

In another preferred embodiment, the compound is selected from the group consisting of:

in another preferred embodiment, each L is independently selected from the group consisting of: C2-C4 alkenylene, halogenated C2-C4 alkenylene.

in another preferred embodiment, the compound has the structure shown as follows:

in another preferred embodiment, said X and Y are each independently selected from the group consisting of: o, N, CH, NH, CH ₂ 。

In another preferred embodiment, the

The ring is selected from the group consisting of:

in another preferred embodiment, the above

The ring is selected from the group consisting of:

in another preferred embodiment, ra, rb, or the nitrogen-containing heterocycle formed by Ra and Rb is selected from the group consisting of:

in a second aspect of the invention, there is provided a pharmaceutical composition comprising (1) a compound according to the first aspect of the invention or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof; (2) a pharmaceutically acceptable carrier.

In a third aspect of the present invention, there is provided a use of the compound according to the first aspect of the present invention or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or the pharmaceutical composition according to the second aspect of the present invention, for the preparation of a pharmaceutical composition for the prevention and/or treatment of a disease associated with an activity or an expression amount of PD-1/PD-L1; preferably, the disease is selected from the group consisting of: tumors, pathogen infections, autoimmune response related diseases.

In another preferred embodiment, the disease is selected from the group consisting of: tumors, pathogen infections, autoimmune response related diseases.

In another preferred embodiment, the pharmaceutical composition is for the treatment of a disease selected from the group consisting of: melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone refractory prostate adenocarcinoma), breast cancer, colon cancer, and lung cancer (e.g., non-small cell lung cancer). Bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, gastrointestinal, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, hodgkin's disease, non-hodgkin's lymphoma, esophageal cancer, small bowel cancer, cancer of the endocrine system, thyroid cancer, paralytic adenocarcinoma, adrenal cancer, chondro-sarcoma, urinary tract cancer, penile cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors in childhood, lymphocytic lymphoma, bladder cancer, renal or ureteral cancer, renal carcinoma, neoplasms/tumors of the Central Nervous System (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis (spinoaxys) tumors, brain stem glioma, pituitary adenoma, kaposi's (Kaposi's), kaposi's sarcoma, squamous cell carcinoma induced cancers, environmental cancers and combinations thereof. Metastatic cancer, particularly metastatic cancer expressing PD-Ll

In another preferred embodiment, the pharmaceutical composition is used in a combination regimen, preferably, the combination regimen comprises: in combination with tumor chemotherapy regimens, other tumor immunotherapeutics (small molecule compounds and antibodies, etc.), radiation therapy regimens, tumor targeting drugs, tumor vaccines (e.g., human Papilloma Virus (HPV), hepatitis virus (HBV and HCV), and kaposi's sarcoma virus (KHSV)).

In another preferred embodiment, the pharmaceutical compositions are for use in therapy, alone or in combination, for treating a patient exposed to a particular toxin or pathogen. Including but not limited to the treatment of various viruses, pathogenic bacteria, pathogenic fungi, pathogenic parasites, and the like. Established infections with pathogens such as HIV, hepatitis viruses (A, B, C), influenza, herpes, giardia, malaria, leishmania, staphylococcus aureus, pseudomonas aeruginosa, etc.

In another preferred embodiment, the pharmaceutical composition is for use in inducing a therapeutic autoimmune response.

In another preferred embodiment, said pharmaceutical composition is used for the treatment of patients with inappropriate accumulation of other autoantigens, such as amyloid deposits, including Α β in alzheimer's disease, cytokines such as TNFa and IgE.

In a fourth aspect of the invention, there is provided a PD-1/PD-L1 inhibitor, which comprises a compound according to the first aspect of the invention, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be repeated herein, depending on the space.

Detailed Description

The inventor designs and synthesizes a novel PD-1 small molecule inhibitor through long-term and intensive research. On this basis, the inventors have completed the present invention.

Term(s) for

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 5% from the recited value. For example, as used herein, the expression "about 100" includes all values between 95 and 105 and (e.g., 95.1, 95.2, 95.3, 95.4, etc.).

As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

Definition of

As used herein, the term "alkyl" includes straight or branched chain alkyl groups. E.g. C ₁ -C ₈ Alkyl represents a straight or branched chain alkyl group having 1 to 8 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, and the like.

As used herein, the term "alkenyl" includes straight or branched chain alkenyl groups. E.g. C ₂ -C ₆ Alkenyl means a straight or branched alkenyl group having 2 to 6 carbon atoms, such as vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, or the like.

As used herein, the term "alkynyl" includes straight or branched chain alkynyl groups. E.g. C ₂ -C ₆ Alkynyl means straight or branched chain alkynyl having 2 to 6 carbon atoms, such as ethynyl, propynyl, butynyl, or the like.

As used herein, the term "C ₃ -C ₈ Cycloalkyl "meansCycloalkyl having 3 to 8 carbon atoms. It may be a single ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or the like. It may also be in the form of a double ring, such as a bridged or spiro ring.

As used herein, the term "C ₁ -C ₈ Alkoxy "means a straight or branched chain alkoxy group having 1 to 8 carbon atoms; for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy and the like.

As used herein, the term "3-10 membered heterocycloalkyl having 1-3 heteroatoms selected from the group consisting of N, S and O" refers to a saturated or partially saturated cyclic group having 3-10 atoms and wherein 1-3 atoms are heteroatoms selected from the group consisting of N, S and O. It may be monocyclic or may be in the form of a double ring, for example a bridged or spiro ring. Specific examples may be oxetane, azetidine, tetrahydro-2H-pyranyl, piperidinyl, tetrahydrofuranyl, morpholinyl, pyrrolidinyl, and the like.

As used herein, the term "C ₆ -C ₁₀ Aryl "means an aryl group having 6 to 10 carbon atoms, for example, phenyl or naphthyl and the like.

As used herein, the term "5-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S and O" refers to a cyclic aromatic group having 5-10 atoms and wherein 1-3 atoms are heteroatoms selected from the group consisting of N, S and O. It may be a single ring or a condensed ring form. Specific examples may be pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrrolyl, pyrazolyl, imidazolyl, (1,2,3) -triazolyl and (1,2,4) -triazolyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl and the like.

Unless otherwise specified, the groups of the present invention may be substituted with a substituent selected from the group consisting of: halogen, nitrile group, nitro group, hydroxyl group, amino group, C ₁ -C ₆ Alkyl-amino, C ₁ -C ₆ Alkyl radical, C ₂ -C ₆ Alkenyl radical, C ₂ -C ₆ Alkynyl, C ₁ -C ₆ Alkoxy, halo C ₁ -C ₆ Alkyl, halo C ₂ -C ₆ Alkenyl, halo C ₂ -C ₆ Alkynyl, halo C ₁ -C ₆ Alkoxy, allyl, benzyl, C ₆ -C ₁₂ Aryl radical, C ₁ -C ₆ alkoxy-C ₁ -C ₆ Alkyl radical, C ₁ -C ₆ Alkoxy-carbonyl, phenoxycarbonyl, C ₂ -C ₆ Alkynyl-carbonyl, C ₂ -C ₆ Alkenyl-carbonyl, C ₃ -C ₆ Cycloalkyl-carbonyl, C ₁ -C ₆ Alkyl-sulfonyl, and the like.

As used herein, "halogen" or "halogen atom" refers to F, cl, br, and I. More preferably, the halogen or halogen atom is selected from F, cl and Br. "halogenated" means substituted with an atom selected from F, cl, br, and I.

Unless otherwise specified, the structural formulae depicted herein are intended to include all isomeric forms (e.g., enantiomers, diastereomers and geometric isomers (or conformational isomers)): such as R, S configuration containing an asymmetric center, (Z), (E) isomers of double bonds, and the like. Thus, individual stereochemical isomers of the compounds of the present invention or mixtures of enantiomers, diastereomers or geometric isomers (or conformers) thereof are within the scope of the present invention.

As used herein, the term "tautomer" means that structural isomers having different energies may exceed the low energy barrier, thereby converting each other. For example, proton tautomers (i.e., proton transmutations) include interconversion by proton shift, such as 1H-indazoles and 2H-indazoles. Valence tautomers include interconversion by recombination of some of the bonding electrons.

As used herein, the term "solvate" refers to a complex of a compound of the present invention coordinated to solvent molecules in a specific ratio.

Preparation of Compounds of formula I

The invention also provides a process for the preparation of a compound according to the first aspect of the invention, the synthesis of compound I-a being shown below, and other compounds of formula I may be similarly synthesized. The synthesis comprises the following steps: in a proper solvent, the compound 1 and the compound 2 react under the action of a palladium catalyst and in the presence of a base and a phosphine compound to obtain a compound 3, and simultaneously in a proper solvent, the compound 4 and the compound 5 react under the action of an acid to obtain a compound 6, and then in a proper solvent, the compound 3 and the compound 6 react under the action of a palladium catalyst and in the presence of a base and a phosphine compound to obtain a compound I-a.

Pharmaceutical compositions and methods of administration

The compound of the present invention and various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates thereof, and pharmaceutical compositions containing the compound of the present invention as a main active ingredient are useful for preventing and/or treating diseases (e.g., cancer) associated with the PD-1/PD-L1 signaling pathway, since the compound of the present invention has excellent inhibitory activity against PD-1.

The pharmaceutical compositions of the present invention comprise a safe and effective amount of a compound of the present invention in combination with a pharmaceutically 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. Typically, the pharmaceutical composition contains 1-2000mg of a compound of the invention per dose, more preferably, 10-200mg of a compound of the invention per dose. Preferably, said "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 be of sufficient purity and sufficiently low toxicity. By "compatible" is meant herein that the components of the composition are capable of intermixing with and with the compounds of the present invention without significantly diminishing the efficacy of the compounds. Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g. sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g. stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g. soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols, and the like(such as propylene glycol, glycerol, mannitol, sorbitol, etc.), and emulsifier (such as

) Wetting agents (e.g., sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.

The mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, parenteral (intravenous, intramuscular injection, inhalation or subcutaneous).

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, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, for example, glycerol; (d) Disintegrating agents, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) absorption accelerators, e.g., quaternary ammonium compounds; (g) Wetting agents, such as cetyl alcohol and glycerol monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coatings and shells such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be delayed in release in a certain part of the digestive tract. Examples of embedding components which can be used are polymeric substances and wax-like substances. If desired, the active compound may also be in microencapsulated form with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide, and oils, in particular, cottonseed, groundnut, corn germ, olive, castor, and sesame oils or mixtures of these materials and the like.

In addition to these inert diluents, the compositions can also contain 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 as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.

Compositions for parenteral injection may comprise 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 nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.

The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.

When administered in combination, the pharmaceutical composition further comprises one or more other pharmaceutically acceptable compounds. One or more of the other pharmaceutically acceptable compounds may be administered simultaneously, separately or sequentially with a compound of the invention.

When using pharmaceutical compositions, a safe and effective amount of a compound of the present invention is administered to a mammal (e.g., a human) in need of treatment, wherein the administration is a pharmaceutically acceptable and effective dose, and the daily dose for a human of 60kg body weight is usually 1 to 2000mg, preferably 20 to 500mg. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

In a preferred embodiment of the invention, the pharmaceutical composition may be used for:

(1) For the treatment of various tumors, including but not limited to melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone refractory prostate adenocarcinoma), breast cancer, colon cancer, and lung cancer (e.g., non-small cell lung cancer). Bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, gastrointestinal, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, hodgkin's disease, non-hodgkin's lymphoma, esophageal cancer, small bowel cancer, cancer of the endocrine system, thyroid cancer, paralytic adenocarcinoma, adrenal cancer, chondro-sarcoma, urinary tract cancer, penile cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors in childhood, lymphocytic lymphoma, bladder cancer, renal or ureteral cancer, renal carcinoma, neoplasms/tumors of the Central Nervous System (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis (spinoaxys) tumors, brain stem glioma, pituitary adenoma, kaposi's (Kaposi's), kaposi's sarcoma, squamous cell carcinoma induced cancers, environmental cancers and combinations thereof. Metastatic cancer, particularly metastatic cancer expressing PD-Ll

(2) For combined administration regimens, such as combined tumor chemotherapy regimens, other tumor immunotherapeutics (small molecule compounds and antibodies, etc.), radiation therapy regimens, tumor targeting drugs, tumor vaccines, etc., such as Human Papilloma Virus (HPV), hepatitis virus (HBV and HCV), and kaposi's sarcoma virus (KHSV). The agent may be administered before, after, or simultaneously with, or may be co-administered with other known therapies.

(3) For treatment of patients exposed to particular toxins or pathogens, either alone or in combination. Including but not limited to the treatment of various viruses, pathogenic bacteria, pathogenic fungi, pathogenic parasites, and the like. Established infections with pathogens such as HIV, hepatitis viruses (A, B, C), influenza, herpes, giardia, malaria, leishmania, staphylococcus aureus, pseudomonas aeruginosa, etc.

(4) For inducing a therapeutic autoimmune response to treat patients with inappropriate accumulation of other self-antigens, such as amyloid deposits, including Α β in alzheimer's disease, cytokines such as TNFa and IgE.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Percentages are by weight and the proportions of reagents used in column chromatography purification are by volume unless otherwise indicated.

The test materials and reagents used in the following examples are commercially available, unless otherwise specified, or may be synthesized by methods known in the art.

Synthesis of intermediate a:

n- (3-bromo-2-chlorophenyl) -2- (bromomethyl) thiazolo [4,5-c]Pyridin-4-amines

3-bromo-2-chloroaniline

3-bromo-2-chloronitrobenzene (18 g, 76.1 mmol) and iron (21.3 g, 380.6 mmol) were added to a solution of ammonium chloride (4 g, 76.1 mmol) in ethanol (50 ml)/water (25 ml) and stirred at 60 ℃ for 4 hours. The reaction solution was filtered and depressurizedConcentrated and the residue was purified by normal phase column chromatography (petroleum ether: ethanol = 20) to give the title compound, 3-bromo-2-chloroaniline (22 g, 84.6%) as a light yellow liquid. MS (ESI) m/z =207.9[ 2 ] M + H] ⁺ .

N- (3-bromo-2-chlorophenyl) -2- (methoxymethyl) thiazolo [4,5-c]Pyridin-4-amines

3-bromo-2-chloroaniline (30 g, 145.6 mmol), N- (3-bromo-2-chlorophenyl) -2- (methoxymethyl) thiazolo [4,5-c]A solution of pyridin-4-amine (29 g, 133.6 mmol) and p-toluenesulfonic acid (9 g, 52.3 mmol) in 2-pentanol (15 ml) was added sequentially to the tube and stirred at 100 ℃ for 24 h. The reaction mixture was extracted with water (400 ml), methylene chloride/methanol (110 ml × 3,v/v = 10/1), and the organic phase was washed successively with water, brine, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by normal phase column chromatography (petroleum ether: dichloromethane = 1:1) to give the title compound a-2 (23 g, 45%) as a yellow solid. MS (ESI) m/z =385.9[ 2 ] M + H] ⁺ .

N- (3-bromo-2-chlorophenyl) -2- (bromomethyl) thiazolo [4,5-c]Pyridin-4-amines

A-2 (23 g, 59.8 mmol) in hydrobromic acid in acetic acid (150 ml) was stirred at 80 ℃ for 15 h. The reaction solution was neutralized with potassium carbonate in an ice bath, and the precipitated solid was neutralized with petroleum ether: a mixed solution of ethyl acetate (100 ml: 10 ml) was slurried to give the title compound a (24 g, 93%) as a yellow solid. MS (ESI) m/z =433.8[ 2 ] M + H] ⁺ .

Synthesis of intermediate B:

2,2' - (2-chloro-1,3-phenylene) bis (4,4,5,5-tetramethyl-1,3,2-dioxaborolane)

2,2' - (2-chloro-1,3-phenylene) bis (4,4,5,5-tetramethyl-1,3,2-dioxaborolane)

Under nitrogen protection, 2-chloro-1,3-2 bromobenzene (25 g, 92.4 mmol), pinacol diboron (58.5 g, 231 mmol), [1,1' -bis (diphenylphosphino) ferrocene]Palladium dichloride dichloromethane (10 g, 12.2 mmol) and potassium acetate (36.3 g, 370 mmol) were mixed well, 1,4-dioxane (400 ml) was added, and the reaction mixture was stirred at 80 ℃ for 18 hours. The reaction solution was filtered, the filter cake was washed twice with methanol, the filtrate was concentrated, and the residue was purified by normal phase column chromatography (petroleum ether: ethyl acetate = 10) to give the title compound B (21 g, 62.3%) as a white solid. MS (ESI) m/z =365.2[ 2 ] M + H] ⁺ .

Synthesis of intermediate C:

6-chloro-2- (difluoromethoxy) -4-methylnicotinaldehyde

Methyl 2,6-dichloro-4-methyl nicotinate

To a solution of 2,6-dichloro-4-methyl nicotinic acid (90 g, 436.9 mmol) in N, N-dimethylformamide (0.5 l) was added potassium carbonate (90.4 g, 655.3 mmol), the reaction mixture was cooled to 0 ℃ with an ice bath, iodomethane (186 g, 1.31 mol) was slowly added dropwise, the ice bath was removed, allowed to spontaneously warm to room temperature and stirred for 13 hours. Water (1 liter) was added to the reaction solution,it was then extracted three times with ethyl acetate (1 l), and the organic phase was washed with saturated brine (800 ml. Times.4), dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound C-1 (105 g, 99%) as a crude white solid. MS (ESI) m/z =219.9[ 2 ], [ M + H ]] ⁺ .

¹ H NMR(400MHz,CDCl ₃ )δ7.16(d,J＝0.6Hz,1H),3.97(s,3H),2.35(d,J＝0.5Hz,3H).

Methyl 6-chloro-2-methoxy-4-methyl nicotinate

Sodium methoxide solid (21.5 g, 398.2 mmol) was added in portions to a solution of C-1 (87.6 g, 398.2 mmol) in dichloromethane (900 ml) under ice-bath, stirred, and the ice-bath was removed and reacted at room temperature for 6 hours. And (3) detecting that raw materials are remained by liquid phase mass spectrometry, supplementing sodium methoxide solid (4.3 g, 79.64 mmol) into the reaction solution, continuing stirring for 13 hours, supplementing sodium methoxide solid (4.3 g, 79.64 mmol) again, and reacting for 6 hours until the raw materials are completely disappeared by liquid phase mass spectrometry. The reaction was poured into aqueous sodium bicarbonate (500 ml), extracted three times with dichloromethane (1 l), and the combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound C-2 (84.4 g, 98.3%) as a white solid. MS (ESI) m/z =215.9[ 2 ] M + H] ⁺ .

¹ H NMR(400MHz,CDCl ₃ )δ6.81–6.78(m,1H),3.96(s,3H),3.91(s,3H),2.29(s,3H).

Methyl 6-chloro-2-hydroxy-4-methyl nicotinate

C-2 (87.4 g, 405.4 mmol) was added to a 33% hydrobromic acid in acetic acid (450 ml) and stirred at 60 ℃ for 30 minutes. Concentrating the reaction solution to remove part of hydrogen bromide and acetic acid, and treating the residue with carbonic acid in ice bathThe pH was adjusted to 4-5 with sodium bicarbonate solution, filtered, and the filter cake was purified by column chromatography (dichloromethane: methanol = 100) to give the title compound C-3 (57 g, 69.6%) as a white solid. MS (ESI) m/z =201.9[ 2 ] M + H] ⁺ .

Methyl 6-chloro-2- (difluoromethoxy) -4-methyl nicotinate

60% sodium hydride (13.4 g, 334.8 mmol) was added to acetonitrile (270 ml), and after stirring at room temperature for 10 minutes, C-3 (27 g, 133.9 mmol) was added, and after stirring for 30 minutes, 2-fluorosulfonyl difluoroacetic acid (40.5 g, 227.7 mmol) was rapidly injected into the reaction solution, whereupon the temperature of the reaction solution rapidly increased to 50 ℃ or higher. After the addition was completed, stirring was continued for one hour until the reaction solution was returned to room temperature. Liquid mass spectrometry detected 90% of the starting material disappeared. The reaction was quenched by slowly pouring into an ice-cooled saturated aqueous sodium bicarbonate solution (1.5 l) with stirring, extracted three times with ethyl acetate (1 l), the combined organic phases were dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by column chromatography (petroleum ether: ethyl acetate = 1:9) to give the title compound C-4 (31 g, 80% purity, 46% yield) as a yellow oil. MS (ESI) m/z =251.9[ 2 ] M + H] ⁺ .

(6-chloro-2- (difluoromethoxy) -4-methylpyridin-3-yl) methanol

A toluene solution of diisobutylaluminum hydride (1.5 mol/L, 159 ml, 238.5 mmol) was added dropwise to a solution of C-4 (30 g, 119.2 mmol) in methylene chloride (300 ml) at-60 deg.C, and the reaction was allowed to slowly warm to room temperature within two hours after the addition was completed, and the reaction was complete by liquid mass spectrometry. The reaction was quenched with aqueous potassium sodium tartrate (500 ml) and filtered through celite. Dissolving the solid in the upper layer of diatomite with dilute hydrochloric acid, and usingDichloromethane (300 ml) was extracted three times, and the organic phase was washed with aqueous sodium hydroxide (0.5 mol/l, 250 ml × 2), dried over anhydrous sodium sulfate and concentrated to give the title compound C-5 (19.3 g, 72.6%) as a red solid. MS (ESI) m/z =223.9[ 2 ] M + H] ⁺ .

6-chloro-2- (difluoromethoxy) -4-methylnicotinaldehyde

Dess-martin oxidizer (52 g, 122.76 mmol) was added to a solution of C-5 (18.3 g, 81.84 mmol) in dichloromethane (200 ml) while cooling on ice and stirred at room temperature for three hours. The reaction was quenched by pouring into aqueous sodium bicarbonate (300 ml), filtered through celite, washed with dichloromethane (200 ml), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The residue was purified by column chromatography (petroleum ether: ethyl acetate = 1:9) to give the title compound C (15.3 g, 84.6%) as a white solid.

¹ H NMR(400MHz,CDCl ₃ )δ10.40(d,J＝0.4Hz,1H),7.66(s,0.25H),7.48(s,0.5H),7.30(s,0.25H),6.99(s,1H),2.57(d,J＝0.5Hz,3H).

Synthesis of intermediate D:

(S) -1- ((3-chloro-5-formyl-6-methoxypyridine) -2-oxo) -2,3-dihydro-1H-indene-4-trifluoromethanesulfonate

4- (tert-butyldimethylsilyloxy) -2,3-dihydro-1H-inden-1-one

4-hydroxy-2,3-dihydro-1H-inden-1-one (20.54 g, 138.8 mmol), tert-butyldimethylChlorosilane (31.45 g, 208.7 mmol), imidazole (18.92 g, 278.2 mmol) were dissolved in dioxane (300 ml), and the reaction was stirred at 20 ℃ for 16 hours. After completion of the liquid mass spectrometric detection reaction, the reaction solution was adjusted to neutral pH with 1 mol/l hydrochloric acid, diluted with water (100 ml), extracted with ethyl acetate (50 ml × 3), and the organic phase was dried over anhydrous sodium sulfate and concentrated. The residue was purified by column chromatography (petroleum ether: ethyl acetate =50,>99%) as a colorless oil. MS (ESI) m/z =263.1[ 2 ] M + H] ⁺ .

(S) -4- (tert-butyldimethylsilyloxy) -2,3-dihydro-1H-inden-1-ol

(R) -2-methyl-CBS-oxazolylborane (1.61 g, 5.81 mmol) was dissolved in toluene (3 mL), borane dimethylsulfide (10 mol/L, 21 mL) was added to the above solution, the reaction solution was stirred at room temperature for 30 minutes, dichloromethane (40 mL) was added, then cooled to-20 ℃ and a solution of D-1 (10 g, 38.16 mmol) in dichloromethane (40 mL) was added dropwise. The reaction solution was kept stirring at-15 ℃ to-5 ℃ for 3 hours. After completion of the liquid mass spectrometric detection reaction, the reaction solution was quenched with methanol (40 ml), concentrated, and purified by column chromatography (petroleum ether: dichloromethane = 10) to obtain D-2 (8.29 g, 82%) as a white solid. MS (ESI) m/z =247.1[ M-OH ]] ⁺ .

¹ H NMR(CDCl ₃ ,400MHz)δ7.12(t,1H),7.02(d,J＝7.2Hz,1H),6.70(d,J＝7.6Hz,1H),5.21(s,1H),3.03-2.96(m,1H),2.76-2.68(m,1H),2.49-2.41(m,1H),1.94-1.84(m,2H),1.00(s,9H),0.20(s,6H).

(S) -6- (4-tert-Butyldimethylsilanyloxy) -2,3-dihydro-1H-indene-1-oxo-2-methoxynicotinaldehyde

The amount of D-2 (2.07 g,7.84 mmol) and 6-chloro-2-methoxynicotinaldehyde (2.72 g, 15.9 mmol) were dissolved in toluene (70 ml), argon was bubbled through the above solution for 10 minutes, and palladium acetate (0.36 g, 1.61 mmol), 2-di-tert-butylphosphino-2 ',4',6' -triisopropylbiphenyl (1.33 g, 3.14 mmol), cesium carbonate (10.21 g, 31.41 mmol) were added. The reaction mixture was stirred at 35 ℃ for 48 hours. The reaction was concentrated, and the residue was purified by column chromatography (petroleum ether: dichloromethane = 1:1) to give the title compound D-3 (3.01 g, 96%) as a white solid. MS (ESI) m/z =247.1[ M-152 ]] ⁺ .

¹ H NMR(CDCl ₃ ,400MHz):δ10.25(s,1H),8.07(d,J＝8.4Hz,1H),7.15(t,J＝7.6Hz,1H),7.08(d,J＝7.6Hz,1H),6.78(d,J＝7.6Hz,1H),6.61(dd,J＝6.8Hz,4.4Hz,1H),6.40(d,J＝8.4Hz,1H),4.12(s,3H),3.14-3.03(m,1H),2.93-2.86(m,1H),2.71-2.62(m,1H),2.27-2.19(m,1H),1.04(s,9H),0.25(s,6H).

(S) -6- (4-hydroxy) -2,3-dihydro-1H-indene-1-oxo-2-methoxynicotinaldehyde

D-3 (2.79 g, 6.99 mmol) was dissolved in tetrahydrofuran (50 ml), the reaction was cooled to-78 ℃ and a tetrahydrofuran solution of tetrabutylammonium fluoride (1 mol/L, 7.0 ml) was added dropwise to the solution, and the reaction was automatically and slowly returned to room temperature. After the reaction was completed, the reaction was quenched with acetic acid (460 mg), the organic phase was separated from an ethyl acetate/saturated citric acid mixed solution (30 ml/40 ml), extracted with ethyl acetate (50 ml × 3), the organic phase was dried over anhydrous sodium sulfate, concentrated, and the residue was purified by column chromatography (petroleum ether/ethyl acetate = 2/1) to obtain D-4 (1.78 g, 89%) as a white solid. MS (ESI) m/z =308.1[ 2 ] M +23] ⁺ .

¹ H NMR(CDCl ₃ ,400MHz)δ10.22(s,1H),8.05(d,J＝8.0Hz,1H),7.14(t,J＝8.0Hz,1H),7.04(d,J＝7.6Hz,1H),6.78(d,J＝8.0Hz,1H),6.60(dd,J＝6.8Hz,4.0Hz,1H),6.38(dd,J＝8.0Hz,0.8Hz,1H),5.30(s,1H),4.10(s,3H),3.15-3.07(m,1H),2.93-2.86(m,1H),2.74-2.64(m,1H),2.31-2.23(m,1H).

(S) -1- ((5-formyl-6-methoxypyridine) -2-oxo) -2,3-dihydro-1H-indene-4-trifluoromethanesulfonate

A solution of D-4 (1.78 g, 6.24 mmol), triethylamine (1.73 ml, 12.47 mmol), pyridine (1.0 ml, 12.41 mmol), 4-dimethylaminopyridine (77 g, 0.63 mmol) in dichloromethane (40 ml) was cooled to-78 ℃ and trifluoromethanesulfonic anhydride (2.03 g, 7.20 mmol) was added dropwise. The reaction was stirred for 15 minutes, then the dry ice bath was removed, allowed to warm to room temperature and stirred for 1 hour. After adding a saturated citric acid solution (50 ml) to the reaction solution, the aqueous layer was extracted with ethyl acetate (20 ml. Times.2), and the combined organic phases were dried over anhydrous sodium sulfate and concentrated. The residue was purified by column chromatography (petroleum ether: ethyl acetate = 8:1) to give the title compound D-5 (2.01 g, 77%) as a white solid. MS (ESI) m/z =440.1[ 2 ] M +23] ⁺ .

D-5 (200 mg, 0.48 mmol) was dissolved in a dichloromethane/N, N-dimethylformamide (4 ml/2 ml) mixed solution, and trifluoroacetic acid (11 mg, 0.10 mmol), and pamau chloride (Palau' chloride, CBMG) (82 mg, 0.39 mmol) were added to the above solution. The reaction solution was stirred at room temperature for 16 hours, and saturated sodium thiosulfate (2 ml) and sodium bicarbonate (4 ml) were added to quench the reaction, and extracted with dichloromethane (5 ml × 3), the organic phase was dried over anhydrous sodium sulfate, concentrated, and the residue was purified by column chromatography (petroleum ether/dichloromethane = 1/4) to give the title compound D (150 g, 70%) as a white solid. MS (ESI) m/z =474.1[ 2 ] M +23] ⁺ .

¹ H NMR(CDCl ₃ ,400MHz)δ10.19(s,1H),8.11(s,1H),7.51-7.48(d,J＝7.6Hz,1H),7.35(t,J＝7.6Hz,1H),7.24(s,1H),6.63-6.61(m,1H),4.10(s,3H),3.34-3.27(m,1H),3.12-3.04(m,1H),2.85-2.77(m,1H),2.39-2.30(m,1H).

Synthesis of intermediate E:

methyl 6-chloro-2- (fluoromethoxy) -4-methyl nicotinate

After C-3 (500 mg, 2.48 mmol) was dissolved in acetonitrile (5 ml) in a 20 ml microwave tube at room temperature, 60% sodium hydride (200 mg, 4.96 mmol) was added and the nitrogen was replaced after blocking, and after inserting a nitrogen balloon, the reaction mixture was stirred at room temperature for 30 minutes. Then, fluorobromomethane (0.32 ml, 4.96 mmol) was added, the reaction mixture was stirred at 40 ℃ for 24 hours and then the liquid phase detection feed conversion was 30%, after cooling to room temperature, fluorobromomethane (0.5 ml, 7.8 mmol) was added, and after reaction at 40 ℃ for 24 hours and then the liquid phase detection feed conversion was 80%. After cooling to room temperature, the reaction was poured into 30 ml of methanol, stirred well and filtered, washed with 20 ml of methanol, and the filtrate was concentrated to give 800 mg of crude E-1 (88% pure). MS (ESI) m/z =234.2, 236.2[ M + H ].

(6-chloro-2- (fluoromethoxy) -4-methylpyridin-3-yl) methanol

To a solution of E-1 (579.3 mg, 2.48 mmol) in dichloromethane (10 mL) was added dropwise a 1.5 mol/L toluene solution of diisobutylaluminum hydride (3.3 mL, 4.96 mmol) at-60 deg.CAnd after dripping at 60 ℃, slowly heating the reaction solution to room temperature within two hours, and detecting the reaction to be complete by a liquid phase. The reaction solution was quenched with 50 ml of a saturated aqueous solution of potassium sodium tartrate under ice-bath, 30 ml of dichloromethane was added, after stirring at room temperature for two hours, dichloromethane was extracted (30 ml × 2), the organic phase was concentrated, and the residue was subjected to column chromatography (petroleum ether: ethyl acetate =0 to 30%) to obtain the title compound E-2 (333 mg, purity: 90%, yield: 65.5%) as a white solid. MS (ESI) m/z =206.1, 208.1[ M + H ]] ⁺ 。

6-chloro-2- (fluoromethoxy) -4-methyl nicotinaldehyde

Dess-martin oxidant (1 g, 2.43 mmol) was added to a solution of E-2 (330 mg, 1.62 mmol) in dichloromethane (20 ml) while cooling on ice, the reaction mixture was stirred at room temperature for half an hour and the reaction was complete by TLC. The reaction solution was quenched by adding 50 ml of an aqueous sodium bicarbonate solution under ice bath, the reaction solution was adjusted to neutral, 50 ml of dichloromethane was extracted three times, the organic phase was dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and the concentrate was passed through a normal phase silica gel column and eluted with a solvent (petroleum ether: ethyl acetate =0 to 20%) to give the title compound E (280 mg, purity: 95%, yield: 85%) as a white solid. MS (ESI) m/z =204.1, 206.1[ M + H ]] ⁺ 。

¹ H NMR(400MHz,CDCl ₃ )δ10.51(s,1H),6.99(s,1H),6.22(s,1H),6.09(s,1H),2.62(s,3H).

And (3) synthesis of an intermediate F:

5-bromo-2-chloro-4-cyclopropylpyridine

5-bromo-2-chloro-4-iodopyridine (5 g, 15.7 mmol) and cyclopropylboronic acid (1.48 g, 17.2 mmol), sodium carbonate (4.99 g, 47.1 mmol) and Pd (dppf) Cl were added under nitrogen blanket ₂ (574 mg, 0.785 mmol) were mixed well, 1,4-dioxane (40 ml) and water (10 ml) were added, the mixture was warmed to 100 ℃ and stirred for 16 hours, LCMS detected completion of the reaction, the reaction was cooled to room temperature and filtered, the filtrate was concentrated and purified by flash silica gel column separation (petroleum ether: ethyl acetate = 0-20%) to give the product as a white solid (1.3 g, 35.6% yield). MS (ESI) m/z =233.9[ 2 ] M + H] ⁺ 。

2-chloro-4-cyclopropyl-5-vinylpyridine

Compound F-1 (1.1 g, 4.73 mmol) was dissolved in 1,4-dioxane (10 mL) and water (2 mL) in a microwave tube, followed by the addition of potassium vinyltrifluoroborate (760 mg, 5.67 mmol), potassium carbonate (1.63 g, 11.8 mmol) and Pd (dppf) Cl ₂ (173 mg, 0.236 mmol), the mixture was warmed to 80 ℃ under nitrogen blanket and stirred for 16 hours, the reaction was cooled to room temperature and filtered, and the filtrate was concentrated and purified by flash silica gel column separation (petroleum ether: ethyl acetate = 0-2%) to give the product as a white oil (180 mg, 21.1% yield). MS (ESI) m/z =179.9[ 2 ] M + H] ⁺ 。

6-chloro-4-cyclopropyl nicotinaldehyde

Compound F-2 (180 mg, 1.00 mmol) was dissolved in tetrahydrofuran (2 mL) and water (0.5 mL), potassium osmate dihydrate (18 mg, 0.05 mmol) was added, sodium periodate (643 mg, 3.00 mmol) was added under nitrogen after cooling to 0 ℃ on ice,after stirring the mixture at 0 ℃ for half an hour and then warming to room temperature for 2 hours, LCMS detected reaction completion, water (30 ml) was added to the reaction, extracted with ethyl acetate (30 ml × 2), the combined organic phases were dried over anhydrous sodium sulfate, filtered and concentrated to give a yellow oil (199 mg, 100% yield). MS (ESI) m/z =182.1[ 2 ] M + H] ⁺ 。

¹ H NMR(400MHz,CDCl ₃ )δ10.37(s,1H),8.68-8.65(m,1H),6.89(s,1H),2.95(m,1H),1.28-1.25(m,2H),0.93-0.88(m,2H)。

Synthesis of intermediate G:

6-chloro-2-ethyl nicotinaldehyde

2,6-dichloronicotinaldehyde (10 g, 56.8 mmol), potassium carbonate (15.7 g, 113.6 mmol), ethylboronic acid (4.6 g, 62.4 mmol) and PdCl were combined under nitrogen blanket ₂ dppf (2 g, 2.84 mmol) was mixed well, 1,4-dioxane (50 ml) and water (10 ml) were added, the mixture was reacted at 90 ℃ for 5 hours, and the reaction was complete as detected by liquid phase. After the reaction solution was concentrated, 150 ml of water was added, 200 ml of ethyl acetate was extracted three times, the mixture was dried over anhydrous sodium sulfate, filtered and concentrated, and the crude product was subjected to column chromatography using petroleum ether/ethyl acetate (0 to 8%) to obtain 500mg of a colorless oily product (purity: 90%, yield: 10.3%). MS (ESI) m/z =169.9[ 2 ] M + H] ⁺ 。

¹ H NMR(400MHz,CDCl ₃ )δ10.31(s,1H),8.07(dd,J＝0.7,8.1Hz,1H),7.33(d,J＝8.2Hz,1H),3.19(q,J＝7.5Hz,2H),1.36-1.32(m,3H)。

Synthesis of intermediate H:

ethyl 6-chloro-4-methoxy-2-methyl nicotinate

To a solution of ethyl 4,6-dichloro-2-methyl nicotinate (5 g, 21.36 mmol) in tetrahydrofuran (50 ml) were added sodium methoxide (1.15 g, 21.36 mmol), cuprous iodide (419 mg, 2.2 mmol), and the reaction mixture was reacted at 70 ℃ for 3 hours under nitrogen substitution to complete the reaction as detected by liquid phase detection. The reaction mixture was concentrated and then subjected to column chromatography using petroleum ether/ethyl acetate (0 to 10%) to obtain 3 g of a colorless oily intermediate H-1 (purity: 90%, yield: 61.1%). MS (ESI) m/z =230.1[ 2 ] M + H] ⁺ 。

(6-chloro-4-methoxy-2-methylpyridin-3-yl) methanol

Intermediate H-1 (2 g, 8.73 mmol) was dissolved in dichloromethane (100 ml), the temperature was reduced to-70 ℃ after nitrogen substitution, a toluene solution of diisobutylaluminum hydride (11.7 ml, 17.5 mmol) was added dropwise, the reaction was slowly warmed to room temperature over two hours, and the starting material was completely reacted by liquid mass spectrometry. A saturated solution of potassium sodium tartrate (50 ml) was added to the reaction solution and stirred for one hour, followed by extraction with dichloromethane (100 ml) three times, drying of the organic phase with anhydrous sodium sulfate, filtration, concentration of the filtrate and column chromatography with petroleum ether/ethyl acetate (0 to 30%) to give 1.5 g of intermediate H-2 as a white solid (purity: 90%, yield: 91.6%). MS (ESI) m/z =187.9[ 2 ] M + H] ⁺ 。

6-chloro-4-methoxy-2-methyl nicotinaldehyde

The dess-martin reagent (5.1 g, 12 mmol) was added to a solution of H-2 (1.5 g, 8 mmol) in dichloromethane (30 ml) while cooling on ice, the reaction was stirred at room temperature for 30 minutes and the starting material was monitored by spotting. The reaction solution was adjusted to a weakly basic pH with sodium bicarbonate solution (50 ml), extracted three times with dichloromethane (80 ml), dried, filtered, and the organic phase was passed through a normal phase silica gel column (petroleum ether: ethyl acetate = 0-20%) to obtain 1.12 g of a white solid product (purity: 90%, yield: 75.4%). MS (ESI) m/z =186.2[ 2 ], [ M + H ]] ⁺ 。

¹ H NMR(400MHz,CDCl ₃ )δ10.53(d,J＝1.1Hz,1H),6.85(s,1H),3.98(d,J＝1.1Hz,3H),2.73(s,3H)。

Synthesis of intermediate K:

ethyl 4-methyl-2-vinyl nicotinate

Ethyl 2-bromo-4-methyl nicotinate (5 g, 21.7 mmol) and potassium vinyltrifluoroborate (5.82 g, 43.4 mmol) were dissolved in 1,4-dioxane (50 ml) and water (10 ml), followed by the addition of Pd (dppf) Cl ₂ (794 mg, 1.08 mmol) and potassium carbonate (7.5 g, 54.2 mmol), stirring at microwave 90 ℃ for 90 minutes after replacing nitrogen blanket, cooling the reaction to room temperature, filtering, concentrating the filtrate, and purifying by flash silica gel column separation (petroleum ether: ethyl acetate = 0-15%) to obtain the product K-1 as colorless oil (4.17 g, 100% yield). MS (ESI) m/z =192.0[ m + H ], [ m ], [] ⁺ 。

Ethyl 2-ethyl-4-methyl nicotinate

Compound K-1 (4.17 g, 21.8 mmol) was dissolved in methanol (40 mL) and then Pd/C (400 mg) was added and the mixture was washed with 15psi of H ₂ After stirring at room temperature for 2 hours under ambient conditions and detection of reaction completion by LCMS, the reaction was filtered through celite and the filtrate was concentrated to give product K-2 as a colorless oil (4.13 g, 98.0% yield).

MS(ESI):m/z＝194.0[M+H] ⁺ 。

3- (carbethoxy) radical<Ethoxycarbonyl group>) -2-ethyl-4-methylpyridine 1-oxide

Compound K-2 (4.13 g, 21.3 mmol) was dissolved in DCM (40 ml) and m-CPBA (9.22 g, 53.4 mmol) was added, the mixture was stirred at rt for 16 h, LCMS detected completion of the reaction, and the reaction was concentrated and purified by flash silica gel column separation (dichloromethane: methanol = 0-10%) to give product K-3 as a colorless oil (3.78 g, 84.8% yield). MS (ESI) m/z =210.0[ m + H ]] ⁺ 。

Ethyl 6-chloro-2-ethyl-4-methyl nicotinate

Dissolving compound K-3 (400 mg, 1.91 mmol) in phosphorus oxychloride (2 ml), adding DIEA (0.33 ml, 1.91 mmol), heating the mixture to 120 ℃, stirring for 2 hours, detecting the completion of the reaction by LCMS, cooling the reaction solution to room temperature, concentrating to remove phosphorus oxychloride, and separating and purifying by using a flash silica gel column (petroleum ether: ethyl acetate)

= 0-15%) product K-4 was obtained as a yellow oil (120 mg, 52.7% yield). MS (ESI) m/z =228.7[ 2 ] M + H] ⁺ 。

(6-chloro-2-ethyl-4-methylpyridin-3-yl) methanol

After dissolving compound K-4 (190 mg, 0.834 mmol) in dichloromethane (2 ml), the temperature was reduced to-60 ℃, then a toluene solution of diisobutylaluminum hydride (1.5 mol/l, 1.7 ml, 2.50 mmol) was slowly added under nitrogen protection, the mixture was stirred at-60 ℃ for 1 hour, LCMS detected reaction completion, potassium sodium tartrate solution (50 ml) was added to the reaction, stirred at room temperature for 1 hour, extracted with dichloromethane (30 ml × 2), the combined organic phases were dried over anhydrous sodium sulfate, filtered and concentrated to give product K-5 (154 mg, 100% yield) as colorless oil. MS (ESI) m/z =185.9[ 2 ] M + H] ⁺ 。

6-chloro-2-ethyl-4-methyl nicotinaldehyde

After compound K-5 (154 mg, 0.836 mmol) was dissolved in dichloromethane (3 ml), dess-martin reagent (530 mg, 1.25 mmol) was added, the reaction was stirred at room temperature for 1 hour, LCMS checked for completion of the reaction, saturated sodium sulfite solution (50 ml) was added to the reaction, extracted with dichloromethane (30 ml × 2), the combined organic phases were washed with saturated sodium bicarbonate solution (50 ml) and dried over anhydrous sodium sulfate, filtered and concentrated to give crude product, which was purified by flash silica gel column separation (petroleum ether: ethyl acetate = 0-10%) to give product K as a yellow oil (140 mg, 91.4% yield).

MS(ESI):m/z＝184.2[M+H] ⁺ 。

¹ H NMR(400MHz,CDCl ₃ )δ10.55(s,1H),7.10(s,1H),3.12(q,J＝7.6Hz,2H),2.58(s,3H),1.32(t,J＝7.5Hz,3H)。

Synthesis of intermediate J:

methyl 6-chloro-4-hydroxynicotinate

Methyl 6-chloro-4-methoxynicotinate (3.7 g, 18.35 mmol) was dissolved in 200 ml of toluene, anhydrous aluminum trichloride (7.34 g, 55.1 mmol) was added, the reaction solution was stirred at 100 ℃ for 1 hour, and liquid phase detection showed completion of the reaction. The reaction solution was concentrated, the residue was cooled on an ice bath, the pH was adjusted to weak alkalinity with a sodium bicarbonate solution, 150 ml of dichloromethane was extracted, dried over anhydrous sodium sulfate and filtered, the filtrate was concentrated and purified with a normal phase silica gel column, and eluted with a solvent (petroleum ether: ethyl acetate =0 to 20%) to give 1.8 g of red solid J-1 (purity: 90%, yield: 52.3%). MS (ESI) m/z =187.9[ 2 ] M + H] ⁺ 。

Methyl 6-chloro-4- (difluoromethoxy) nicotinate

Intermediate J-1 (2.25 g, 12 mmol), sodium difluorochloroacetate (2.74 g, 18 mmol), and potassium carbonate (1.66 g, 12 mmol) were dissolved in N, N-dimethylformamide (50 ml), and the reaction solution was stirred at 90 ℃ for 1 hour after nitrogen substitution, and liquid phase detection indicated completion of the reaction. The reaction solution was quenched with 50 ml of water after cooling, extracted with 100 ml of ethyl acetate, washed with saturated brine (80 ml × 3), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by a normal phase silica gel column chromatography, and eluted with a solvent (petroleum ether: ethyl acetate =0 to 20%) to give 1.1 g of red solid intermediate J-2 (purity: 90%, yield: 38.6%). MS (ESI) m/z =237.9[ 2 ] M + H] ⁺ 。

(6-chloro-4- (difluoromethoxy) pyridin-3-yl) methanol

1.5 mol/l diisobutylaluminum hydride solution in toluene (7.1 ml, 11.5 mmol) was added dropwise to J-2 (1.1 g, 4.63 mmol) in dichloromethane (50 ml) at-60 deg.C, and after completion of the addition at 60 deg.C, the reaction mixture was slowly warmed to room temperature over two hours, and the completion of the reaction was detected by liquid phase detection. The reaction solution was quenched with 50 ml of an aqueous solution of potassium sodium tartrate, stirred at room temperature for one hour, and then extracted three times with dichloromethane (100 ml). The organic phase was dried over anhydrous sodium sulfate and concentrated, and the crude product was chromatographed on normal phase silica gel using a solvent (petroleum ether: ethyl acetate = 0-40%) to give 670 mg (purity: 90%, yield: 69%) of intermediate J-3 as a red oil. MS (ESI) m/z =210.1[ 2 ] M + H] ⁺ 。

6-chloro-4- (difluoromethoxy) nicotinaldehyde

Dess-martin oxidant (2 g, 4.8 mmol) was added to a solution of J-3 (670 mg, 3.2 mmol) in dichloromethane (10 ml) while cooling on ice, the reaction mixture was stirred at room temperature for 1 hour and the reaction was complete by TLC. After the reaction solution was quenched with 50 ml of a saturated sodium bicarbonate solution, 40 ml of dichloromethane and 50 ml of a saturated sodium sulfite solution were added and washed twice, and after the organic phase was dried over anhydrous sodium sulfate, it was filtered and concentrated to obtain 500mg of a white solid as a final product (purity: 90%, yield: 75.2%). ¹ H NMR(400MHz,CDCl ₃ )δ10.37(s,1H),8.86(s,1H),7.23(s,1H),6.98-6.60(m,1H)。

Example 1 (trans) -4- (((4- ((2,2 '-dichloro-3' - (6- (difluoromethoxy) -4-methyl-5-) (((((S) -5-Carbonylpyrrolidin-2-yl) methyl) amino) methyl) pyridin-2-yl) - [1,1' -Biphenyl]-3-yl) amino group Thiazolo [4,5-c]Pyridin-2-yl) methyl) amino) cyclohexane-1-carboxylic acid

Methyl (1r, 4r) -4- (((4- ((3-bromo-2-chlorophenyl) amino) thiazolo [4,5-c)]Pyridin-2-yl) methyl group) Amino) cyclohexane-1-carboxylic acid esters

Cesium carbonate (76.65 g, 235.26 mmol) was added to a mixed solvent of methyl (1r, 4r) -4-aminocyclohexane-1-carboxylate hydrochloride (30.38 g, 193.67 mmol) and tetrahydrofuran/acetonitrile (120 ml, 3/1), and stirred at normal temperature for one hour. Then N- (3-bromo-2-chlorophenyl) -2- (bromomethyl) thiazolo [4,5-c]Pyridin-4-amine (34.00 g, 78.42 mmol) was added and stirring was continued at ambient temperature for 3 hours until the starting material completely disappeared. The reaction solution was diluted with dichloromethane (100 ml) and water (100 ml), separated, and the organic phase was washed with saturated ammonium chloride and brine in this order, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by slurrying with a mixed solvent of ethyl acetate and petroleum ether (1:5) to give the title compound methyl (1r, 4r) -4- (((4- ((3-bromo-2-chlorophenyl) amino) thiazolo [4,5-c)]Pyridin-2-yl) methyl) amino) cyclohexane-1-carboxylate (35.00 g, 88%) as a pale yellow solid. MS (ESI) m/z =509.1[ 2 ] M + H] ⁺ .

Methyl (1r, 4r) -4- (((4- ((2,2 '-dichloro-3' - (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-) - [1,1' -Biphenyl ]]-3-yl) amino) thiazolo [4,5-c]Pyridin-2-yl) methyl) amino) cyclohexane-1-carboxylic acid ester

Methyl (1r, 4r) -4- (((4- ((3-bromo-2-chlorophenyl) amino) thiazolo [4,5-c) under nitrogen blanket]Pyridin-2-yl) methyl) amino) cyclohexane-1-carboxylate (8.02 g, 15.73 mmol), 2,2' - (2-chloro-1,3-phenylene) bis (4,4,5)5-tetramethyl-1,3,2-dioxaborolane) (10.03 g, 27.53 mmol), tetratriphenylphosphine palladium (0.91 g, 0.79 mmol) and anhydrous potassium carbonate (5.43 g, 39.33 mmol) were mixed well, 1,4 dioxane (150 ml) and water (15 ml) were added and stirred at 80 ℃ for 4 hours. After the reaction, the reaction mixture was filtered and concentrated. The residue was purified by normal phase column chromatography (ethyl acetate: petroleum ether = 40) to give the title compound methyl (1r, 4r) -4- (((4- ((2,2 ' -dichloro-3 ' - (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) - [1,1' -biphenyl-l-2-yl)]-3-yl) amino) thiazolo [4,5-c]Pyridin-2-yl) methyl) amino) cyclohexane-1-carboxylate (7.00 g, 59%) was a pale yellow solid. MS (ESI) m/z =667.2[ m + H ]] ⁺ .

Methyl (1r, 4r) -4- (((4- ((2,2 '-dichloro-3' - (6- (difluoromethoxy) -5-formyl-4-methylpyridine) Pyridin-2-yl) - [1,1' -Biphenyl]-3-yl) amino) thiazolo [4,5-c]Pyridin-2-yl) methyl) amino) cyclohexane-1-carboxylic acid An acid ester.

Methyl (1r, 4r) -4- (((4- ((2,2 ' -dichloro-3 ' - (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) - [1,1' -biphenyl ] under nitrogen blanket]-3-yl) amino) thiazolo [4,5-c]Pyridin-2-yl) methyl) amino) cyclohexane-1-carboxylate (6941 mg, 10.40 mmol), 6-chloro-2- (difluoromethoxy) -4-methyl nicotinaldehyde (1773 mg, 8.00 mmol), tetrakistriphenylphosphine palladium (462 mg, 0.40 mmol) and anhydrous potassium carbonate (2764 mg, 20.00 mmol) were mixed well, added to 1,4 dioxane (15 ml) and water (1.5 ml) and stirred at 90 ℃ for 2 hours. The reaction mixture was diluted with ethyl acetate (50 ml), filtered, the filtrate was washed with brine and water, and the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by normal phase column chromatography (ethyl acetate: petroleum ether = 42) to give the title compound methyl (1r, 4r) -4- (((4- ((2,2 ' -dichloro-3 ' - (6- (difluoromethoxy) -5-formyl-4-methylpyridin-2-yl) - [1,1' -biphenyl-2-yl) -]-3-yl) amino) thiazolo [4,5-c]Pyridin-2-yl) methyl) amino) cyclohexylAlkane-1-carboxylic acid ester (5235 mg, 83%) was a light yellow solid. MS (ESI) m/z =726.1[ 2 ] M + H] ⁺ .

Methyl (1r, 4r) -4- (((4- ((2,2 '-dichloro-3' - (6- (difluoromethoxy) -4-methyl-5- (((((((S) -5-) Carbonylpyrrolidin-2-yl) methyl) amino) methyl) pyridin-2-yl) - [1,1' -biphenyl]-3-yl) amino) thiazolo [4,5- c]Pyridin-2-yl) methyl) amino) cyclohexane-1-carboxylic acid ester

Methyl (1r, 4r) -4- (((4- ((2,2 ' -dichloro-3 ' - (6- (difluoromethoxy) -5-formyl-4-methylpyridin-2-yl) - [1,1' -biphenyl)]-3-yl) amino) thiazolo [4,5-c]Pyridin-2-yl) methyl) amino) cyclohexane-1-carboxylate (5086 mg, 7.00 mmol) and (S) -5- (aminomethyl) pyrrolidin-2-one hydrochloride (2108 mg, 14.00 mmol) were added to a solution of N-ethyl-N-isopropylpropan-2-amine (904 mg, 7.00 mmol) in dry dichloromethane (14 ml) and stirred at room temperature for 3 hours. Sodium triacetoxyborohydride (2968 mg, 14.00 mmol) was then added to the reaction solution in portions and stirred at room temperature for 1 hour. The reaction was quenched with saturated sodium bicarbonate solution and diluted with ethyl acetate (500 ml). Filtering, and washing the filtrate with brine and water in sequence. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by normal phase column chromatography (methanol: dichloromethane = 5; 95) and reverse phase column chromatography (90% acetonitrile in aqueous ammonium bicarbonate) to give the title compound methyl (1r, 4r) -4- (((4- ((2,2 ' -dichloro-3 ' - (6- (difluoromethoxy) -4-methyl-5- ((((((S) -5-carbonylpyrrolidin-2-yl) methyl) amino) methyl) pyridin-2-yl) - [1,1' -biphenyl 3242 ' -methyl) amino) pyridin-2-yl) - [1,1' -biphenyl]-3-yl) amino) thiazolo [4,5-c]Pyridin-2-yl) methyl) amino) cyclohexane-1-carboxylate (3400 mg, 58%) was a light yellow solid. MS (ESI) m/z =824.2[ 2 ] M + H] ⁺ .

(1r, 4r) -4- (((4- ((2,2 '-dichloro-3' - (6- (difluoromethoxy) -4-methyl-5- ((((((S) -5-carbonyl) Pyrrolidin-2-yl) methyl) amino) methyl) pyridin-2-yl) - [1,1' -biphenylBase of]-3-yl) amino) thiazolo [4,5-c]Pyridine (II) Pyridin-2-yl) methyl) amino) cyclohexane-1-carboxylic acid

Methyl (1r, 4r) -4- (((4- ((2,2 ' -dichloro-3 ' - (6- (difluoromethoxy) -4-methyl-5- ((((((S) -5-carbonylpyrrolidin-2-yl) methyl) amino) methyl) pyridin-2-yl) - [1,1' -biphenylyl)]-3-yl) amino) thiazolo [4,5-c]Pyridin-2-yl) methyl) amino) cyclohexane-1-carboxylate (730 mg, 0.89 mmol), lithium hydroxide monohydrate (149 mg, 3.56 mmol) were added to a mixed solution of methanol (2 ml), tetrahydrofuran (1 ml) and water (0.5 ml), and stirred at room temperature for 4 hours. The reaction mixture was neutralized with dilute hydrochloric acid (1 mol/l), filtered and concentrated. The residue was purified by reverse phase flash column chromatography to give the title compound (1r, 4r) -4- (((4- ((2,2 ' -dichloro-3 ' - (6- (difluoromethoxy) -4-methyl-5- ((((((S) -5-carbonylpyrrolidin-2-yl) methyl) amino) methyl) pyridin-2-yl) - [1,1' -biphenylyl)]-3-yl) amino) thiazolo [4,5-c]Pyridin-2-yl) methyl) amino) cyclohexane-1-carboxylic acid (400 mg, 55%) as a white solid. MS (ESI) m/z =810.2[ 2 ] M + H] ⁺ .

¹ H NMR(400MHz,DMSO-d ₆ )δ8.90(d,J＝8.3Hz,1H),8.64(s,1H),8.11(d,J＝5.6Hz,1H),7.72(t,J＝73Hz,1H),7.67(dd,J＝7.7,1.5Hz,1H),7.65(s,1H),7.59(s,1H),7.56(dd,J＝11.6,3.9Hz,2H),7.50–7.42(m,3H),7.05(dd,J＝7.5,1.1Hz,1H),4.19(s,2H),3.77(s,2H),3.66–3.58(m,1H),2.61–2.55(m,2H),2.51(s,3H),2.47–2.42(m,1H),2.20–2.02(m,4H),1.96(d,J＝10.6Hz,2H),1.88(d,J＝11.5Hz,2H),1.74–1.62(m,1H),1.39–1.19(m,2H),1.18–1.03(m,2H)。

Example 2 (1r, 4r) -4- (((4- ((2,2 '-dichloro-3' - (6-methoxy-4-methyl-5- ((((5-carbonylpiridon-e)) Pyrrolidin-2-yl) methyl) amino) methyl) pyridin-2-yl) - [1,1' -biphenyl]-3-yl) amino) thiazolo [4,5-c]Pyridine (II) Pyridin-2-yl) methyl) amino) cyclohexane-1-carboxylic acid

Methyl (1r, 4r) -4- (((4- ((2,2 '-dichloro-3' - (5-formyl-6-methoxy-4-methylpyridine-2-) - [1,1' -Biphenyl ]]-3-yl) amino) thiazolo [4,5-c]Pyridin-2-yl) methyl) amino) cyclohexane-1-carboxylic acid ester

Methyl (1r, 4r) -4- (((4- ((3-bromo-2-chlorophenyl) amino) thiazolo [4,5-c) under nitrogen blanket]Pyridin-2-yl) methyl) amino) cyclohexane-1-carboxylate (2000 mg, 3.92 mmol), 6- (2-chloro-3- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) phenyl) -2-methoxy-4-methylnicotinaldehyde (2279 mg, 5.88 mmol), tetratriphenylphosphine palladium (231 mg, 0.20 mmol) and potassium carbonate (1355 mg, 9.80 mmol) were mixed well, 1,4 dioxane (20 ml) and water (2 ml) were added, followed by stirring at 90 ℃ for 2 hours. After completion of the reaction, the reaction solution was diluted with ethyl acetate (50 ml), filtered, the filtrate was washed with brine and water, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was dissolved in ethyl acetate (3 ml), and then slowly added dropwise to stirred petroleum ether (30 ml), filtered, the filter cake was washed with petroleum ether, the crude product obtained by drying the filter cake was slurried with methyl tert-butyl ether to give the title compound methyl (1r, 4r) -4- (((4- ((2,2 ' -dichloro-3 ' - (5-formyl-6-methoxy-4-methylpyridin-2-yl) - [1,1' -biphenyl ] ether]-3-yl) amino) thiazolo [4,5-c]Pyridin-2-yl) methyl) amino) cyclohexane-1-carboxylate (1260 mg, 46%) was a grey solid. MS (ESI) m/z =690.2[ 2 ] M + H] ⁺ .

Methyl (1r, 4r) -4- (((4- ((2,2 '-dichloro-3' - (6-methoxy-4-methyl-5- ((((5-carbonylpyrrole) Alk-2-yl) methyl) amino) methyl) pyridin-2-yl) - [1,1' -biphenyl]-3-yl) amino) thiazolo [4,5-c]Pyridine- 2-yl) methyl) amino) cyclohexane-1-carboxylic acid ester

Methyl (1r, 4r) -4- (((4- ((2,2 ' -dichloro-3 ' - (5-formyl-6-methoxy-4-methylpyridin-2-yl) - [1,1' -biphenyl)]-3-yl) amino) thiazolo [4,5-c]Pyridin-2-yl) methyl) amino) cyclohexane-1-carboxylate (180 mg, 0.26 mmol) and 5- (aminomethyl) pyrrolidin-2-one (59 mg, 0.52 mmol) were added to a solution of two drops of glacial acetic acid in dry dichloromethane (3 ml) and stirred at room temperature for 4 hours. Sodium triacetoxyborohydride (66 mg, 0.31 mmol) was then added to the reaction in portions and stirred at room temperature for 2 hours. The reaction was quenched with saturated sodium bicarbonate solution (10 ml), stirred for 30 minutes, extracted, the organic phase washed twice with water, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue purified by normal phase column chromatography (methanol: dichloromethane = 6) to give the title compound methyl (1r, 4r) -4- (((4- ((2,2 ' -dichloro-3 ' - (6-methoxy-4-methyl-5- ((((5-carbonylpyrrolidin-2-yl) methyl) amino) methyl) pyridin-2-yl) - [1,1' -biphenyl 3242 —]-3-yl) amino) thiazolo [4,5-c]Pyridin-2-yl) methyl) amino) cyclohexane-1-carboxylate (145 mg, 71%) was a light yellow solid. MS (ESI) m/z =788.1[ m + H ], [ solution of calcium ] and [ sic ], [ solution of calcium ] in (ESI)] ⁺ .

(1r, 4r) -4- (((4- ((2,2 '-dichloro-3' - (6-methoxy-4-methyl) -5- ((((5-carbonylpyrrolidine-2- Yl) methyl) amino) methyl) pyridin-2-yl) - [1,1' -biphenyl]-3-yl) amino) thiazolo [4,5-c]Pyridin-2-yl) Methyl) amino) cyclohexane-1-carboxylic acid

Methyl (1r, 4r) -4- (((4- ((2,2 ' -dichloro-3 ' - (6-methoxy-4-methyl-5- ((((5-carbonylpyrrolidin-2-yl) methyl) amino) methyl) pyridin-2-yl) - [1,1' -biphenylyl)]-3-yl) amino) thiazolo [4,5-c]Pyridin-2-yl) methyl) amino) cyclohexane-1-carboxylate (145 mg, 0.18 mmol)) Lithium hydroxide monohydrate (30 mg, 0.72 mmol) was added to a mixed solution of methanol (2 ml), tetrahydrofuran (2 ml) and water (0.5 ml), and stirred at room temperature for 16 hours. The reaction mixture was neutralized with dilute hydrochloric acid (1 mol/l) and concentrated. The residue was purified by reverse phase column chromatography (85% methanol in 0.1% aqueous ammonium bicarbonate) to give the title compound (1r, 4r) -4- (((4- ((2,2 ' -dichloro-3 ' - (6-methoxy-4-methyl-5- (((((5-carbonylpyrrolidin-2-yl) methyl) amino) methyl) pyridin-2-yl) - [1,1' -biphenylyl) amino) methyl]-3-yl) amino) thiazolo [4,5-c]Pyridin-2-yl) methyl) amino) cyclohexane-1-carboxylic acid (85 mg, 61%) as a white solid. MS (ESI) m/z =774.1[ M + H ]] ⁺ .

¹ H NMR(400MHz,DMSO-d ₆ )δ8.87(dd,J＝8.3,1.4Hz,1H),8.61(s,1H),8.08(d,J＝5.6Hz,1H),7.64–7.59(m,2H),7.55(d,J＝5.6Hz,1H),7.51(t,J＝7.6Hz,1H),7.45(t,J＝8.0Hz,1H),7.40(dd,J＝7.5,1.7Hz,1H),7.10(s,1H),7.02(dd,J＝7.6,1.5Hz,1H),4.16(s,2H),3.87(s,3H),3.72(s,2H),3.63–3.56(m,1H),2.60–2.52(m,2H),2.45–2.41(m,1H),2.38(s,3H),2.17–2.00(m,4H),1.99–1.90(m,2H),1.90–1.80(m,2H),1.71–1.61(m,1H),1.35–1.20(m,2H),1.15–1.01(m,2H).

Example 3 (S) -5- (((5- (2,2 '-dichloro-3' - ((2-methylthiooxazolo [4,5-c)]Pyridin-4-yl) amino - [1,1' -Biphenyl ]]-3-yl) -3- (difluoromethoxy) pyrazin-2-yl) methyl) amino) methyl) pyrrolidin-2-one

6- (2-chloro-3- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) phenyl) -2- (difluoromethoxy) 4-methyl nicotinaldehyde

Under the protection of nitrogen, 6-bromo-2- (difluoromethoxy) -4-methyl nicotinaldehyde(500 mg, 1.88 mmol), 2,2' - (2-chloro-1,3-phenylene) bis (4,4,5,5-tetramethyl-1,3,2-dioxaborolan) (1.37 g, 3.76 mmol), tetrakis (triphenylphosphine) palladium (77 mg, 0.066 mmol) and potassium acetate (461 mg, 4.70 mmol) were mixed well, 1,4-dioxane (10 ml) was added and stirred at 80 ℃ for 5 hours. The reaction solution was filtered and concentrated. The residue was purified by normal phase column chromatography (petroleum ether: ethyl acetate = 100) to give the title compound 6- (2-chloro-3- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) phenyl) -2- (difluoromethoxy) -4-methyl nicotinaldehyde (900 mg, 62%, purity 66%) as a white solid. MS (ESI) m/z =424.0[ m + H ]] ⁺ .

N- (3-bromo-2-chlorophenyl) -2-methylthiooxazolo [4,5-c]Pyridin-4-amines

A solution of N- (3-bromo-2-chlorophenyl) -2- (bromomethyl) thiazolo [4,5-c ] pyridin-4-amine (500 mg, 1.15 mmol) in dimethyl sulfoxide (20 mL) was stirred at 0 deg.C and sodium borohydride (430 mg, 11.5 mmol) was added and stirred at room temperature for 2 hours. The reaction mixture was diluted with dichloromethane (100 ml), washed with brine and then with water. The organic phase is concentrated. The residue was purified by normal phase column chromatography (petroleum ether: ethyl acetate = 60) to give the title compound N- (3-bromo-2-chlorophenyl) -2-methylthiooxazolo [4,5-c ] pyridin-4-amine (160 mg, 39%) as a yellow solid. MS (ESI): m/z =356.0

[M+H] ⁺ .

6- (2,2 '-dichloro-3' - ((2-methylthiooxazolo [4,5-c)]Pyridin-4-yl) amino) - [1,1' -biphenyl]-3- 2- (difluoromethoxy) -4-methylnicotinaldehyde

Under the protection of nitrogen, 6- (2-chloro-3- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) phenyl) -2- (difluoroMethoxy) -4-methyl nicotinaldehyde (248 mg, 0.59 mmol), N- (3-bromo-2-chlorophenyl) -2-methylthiooxazolo [4,5-c]Pyridin-4-amine (160 mg, 0.45 mmol), tetrakis (triphenylphosphine) palladium (52 mg, 0.045 mmol) and potassium carbonate (155 mg, 1.12 mmol) were mixed well, 1,4-dioxane (5 ml) and water (1 ml) were added, followed by stirring at 90 ℃ for 2 hours, and the reaction was filtered and concentrated. The residue was purified by normal phase column chromatography (petroleum ether: ethyl acetate = 84) to obtain the objective product 6- (2,2 '-dichloro-3' - ((2-methylthiopyrazolo [4,5-c)]Pyridin-4-yl) amino) - [1,1' -biphenyl]-3-yl) -2- (difluoromethoxy) -4-methyl nicotinaldehyde (140 mg, 54%) as a yellow solid. MS (ESI) m/z =571.1[ 2 ] M + H] ⁺ .

(S) -5- (((5- (2,2 '-dichloro-3' - ((2-methylthio-oxazolo [4,5-c)]Pyridin-4-yl) amino) - [1,1- Biphenyl radical]-3-yl) -3- (difluoromethoxy) pyrazin-2-yl) methyl) amino) methyl) pyrrolidin-2-one

The mixture of 6- (2,2 '-dichloro-3' - ((2-methylthio-oxazolo [4,5-c)]Pyridin-4-yl) amino) - [1,1' -biphenyl]A mixture of-3-yl) -2- (difluoromethoxy) -4-methyl nicotinaldehyde (140 mg, 0.25 mmol) and (S) -5- (aminomethyl) pyrrolidin-2-one (56 mg, 0.49 mmol), acetic acid (0.1 ml), anhydrous dichloromethane (2 ml) was stirred at room temperature for 16 h. Sodium triacetoxyborohydride (104 mg, 0.49 mmol) was then added in portions and the reaction was stirred at room temperature for an additional 2 hours. The reaction was dissolved in dichloromethane, washed with brine, washed with water and concentrated. The residue was purified by preparative high performance liquid column chromatography to give the title compound (S) -5- ((((5- (2,2 '-dichloro-3' - ((2-methylthiooxazolo [4,5-c)]Pyridin-4-yl) amino) - [1,1' -biphenyl]-3-yl) -3- (difluoromethoxy) pyrazin-2-yl) methyl) amino) methyl) pyrrolidin-2-one (11.4 mg, 7%) as a white solid. MS (ESI): m/z =669.2[ m ] +H] ⁺ .

¹ H NMR(400MHz,MeOD)δ8.87(dd,J＝8.3,1.4Hz,1H),8.09(d,J＝5.7Hz,1H),7.65(s,1H),7.63(dd,J＝7.7,1.6Hz,1H),7.52(t,J＝7.6Hz,1H),7.48–7.38(m,4H),7.02(dd,J＝7.5,1.4Hz,1H),3.96(s,1H),3.87(t,J＝12.2,1H),2.89(s,3H),2.77(qd,J＝12.1,6.0Hz,2H),2.55(s,3H),2.39–2.26(m,3H),1.90–1.80(m,1H).

Example 4 (1r, 4r) -4- (((4- ((2,2 '-dichloro-3' - ((E) -5- ((((((S) -5-carbonylpyrrolidine-2-) Yl) methyl) amino) methyl) -2- (trifluoromethyl) styryl) - [1,1' -biphenyl]-3-yl) amino) thiazolo [4,5- c]Pyridin-2-yl) methyl) amino) cyclohexane-1-carboxylic acid

(S, E) -5- (((3- (3-bromo-2-chlorostyryl) -4- (trifluoromethyl) benzyl) amino) methyl) pyrrolidine- 2-ketones

A mixture of (E) -3- (3-bromo-2-chlorostyryl) -4- (trifluoromethyl) benzaldehyde (1000 mg, 1.75 mmol, 68%) and (S) -5- (aminomethyl) pyrrolidin-2-one hydrochloride (527 mg, 3.50 mmol) was added to a solution of N-ethyl-N-isopropylpropan-2-amine (226 mg, 1.75 mmol) in anhydrous dichloromethane (3 ml) and methanol (9 ml) and stirred at room temperature for 16 h. Sodium triacetoxyborohydride (1133 mg, 5.25 mmol) was then added portionwise and stirring continued at room temperature for 4 hours. The reaction was quenched with saturated sodium bicarbonate solution and stirred at room temperature for half an hour, extracted three times with dichloromethane, and the organic phase was washed twice with saturated brine solution, dried, and concentrated. The residue was purified by normal phase column chromatography (methanol: dichloromethane = 3) to give the title compound (S, E) -5- (((3- (3-bromo-2-chlorostyryl) -4- (trifluoromethyl) benzyl) amino) methyl) pyrrolidin-2-one (750 mg, 78%) as a light yellow solid. MS (ESI) m/z =487.0[ M + H ]] ⁺ .

(S, E) - (2-chloro-3- (5- ((((5-carbonylpyrrolidin-2-yl) methyl) amino) methyl) -2- (trifluoromethyl) benzene Vinyl) phenyl) boronic acid

(S, E) -5- (((3- (3-bromo-2-chlorostyryl) -4- (trifluoromethyl) benzyl) amino) methyl) pyrrolidin-2-one (488 mg, 1.00 mmol), 4,4,4',4',5,5,5',5' -octamethyl-2,2 '-bis (1,3,2-dioxaborolane) (381 mg, 1.50 mmol), (1,1' -bis (diphenylphosphino) ferrocene) was placed under nitrogen blanket]Palladium dichloride (41 mg, 0.05 mmol) and anhydrous potassium acetate (245 mg, 2.50 mmol) were mixed well and 1,4 dioxane (5 ml) was added, followed by stirring at 100 ℃ for 10 hours. The reaction solution was diluted with ethyl acetate (50 ml), filtered, the filtrate was washed with brine and water, and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by normal phase column chromatography (methanol: dichloromethane =3 = 97) to give the title compound (S, E) - (2-chloro-3- (5- ((((5-carbonylpyrrolidin-2-yl) methyl) amino) methyl) -2- (trifluoromethyl) styryl) phenyl) boronic acid (113 mg, 25%) as a white solid. MS (ESI) m/z =535.1[ 2 ] M + H] ⁺ .

(1r, 4r) -4- (((4- ((2,2 '-dichloro-3' - ((E) -5- (((((S) -5-carbonylpyrrolidin-2-yl) methyl) Amino) methyl) -2- (trifluoromethyl) styryl) - [1,1' -biphenyl]-3-yl) amino) thiazolo [4,5-c]Pyridine-2- Yl) methyl) amino) cyclohexane-1-carboxylic acid

(1r, 4r) -4- (((4- ((3-bromo-2-chlorophenyl) amino) thiazolo [4,5-c) under nitrogen protection]Pyridin-2-yl) methyl) amino) cyclohexane-1-carboxylic acid (65 mg, 0.13 mmol), (S, E) -5- (((3- (2-chloro-3- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) styryl) -4- (trifluoromethyl) benzyl) amino) methyl) pyrroleAlk-2-one (77 mg, 0.17 mmol), tetrakistriphenylphosphine palladium (8 mg, 0.0065 mmol) and anhydrous potassium carbonate (46 mg, 0.33 mmol) were mixed well, 1,4 dioxane (3 ml) and water (0.5 ml) were added, and then stirred at 90 ℃ for 2 hours. The reaction solution was diluted with ethyl acetate (50 ml), filtered, and the filtrate was washed with brine and water. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by normal phase column chromatography (methanol: dichloromethane =8 = 92) and reverse phase column chromatography (60% acetonitrile in ammonium bicarbonate aqueous solution) to give the title compound (1r, 4r) -4- (((4- ((2,2 ' -dichloro-3 ' - ((E) -5- ((((((S) -5-carbonylpyrrolidin-2-yl) methyl) amino) methyl) -2- (trifluoromethyl) styryl) - [1,1' -biphenyl ] yl]-3-yl) amino) thiazolo [4,5-c]Pyridin-2-yl) methyl) amino) cyclohexane-1-carboxylic acid (35 mg, 33%) as a white solid. MS (ESI) m/z =823.3[ 2 ] M + H] ⁺ .

¹ H NMR(400MHz,DMSO-d ₆ )δ8.85(d,J＝7.1Hz,1H),8.59(s,1H),8.07(d,J＝5.6Hz,1H),7.92(s,1H),7.83(d,J＝6.8Hz,1H),7.69(d,J＝8.1Hz,1H),7.62(s,1H),7.59–7.51(m,2H),7.50–7.38(m,3H),7.34(d,J＝6.3Hz,1H),6.98(d,J＝6.3Hz,1H),4.15(s,2H),3.80(q,J＝14.8Hz,2H),3.62–3.55(m,1H),2.52–2.47(m,2H),2.43–2.38(m,1H),2.15–1.98(m,4H),1.92(d,J＝10.2Hz,2H),1.84(d,J＝13.2Hz,2H),1.69–1.62(m,1H),1.32–1.19(m,2H),1.13–0.99(m,2H).

Example 5 (S) -1- (5-chloro-4- (((S) -4- (2-chloro-3- ((E) -5- ((((((S) -5-carbonylpyrrolidine-2-) Yl) methyl) amino) methyl) -2- (trifluoromethyl) styryl) phenyl) -2,3-dihydro-1H-inden-1-yl) oxo) -2- ((5-cyanopyridin-3-yl) methoxy) benzyl) piperidine-3-carboxylic acid

(S) -1- (4- (((S) -4-bromo-2,3-dihydro-1H-inden-1-yl) oxo) -5-chloro-2- ((5-cyanopyridine-3- Yl) methoxy) benzyl) piperidine-3-carboxylic acid

A mixture of (S) -5- ((5- ((4-bromo-2,3-dihydro-1H-inden-1-yl) oxo) -4-chloro-2-formylphenoxy) methyl) nicotinonitrile (300 mg, 0.62 mmol), (S) -piperidine-3-carboxylic acid (240 mg, 1.86 mmol) and a catalytic amount of glacial acetic acid, dry dichloromethane (3 ml) was stirred at room temperature for 16H. Sodium triacetoxyborohydride (262 mg, 1.24 mmol) was then added portionwise and the reaction was allowed to continue stirring at room temperature for 2 hours. The reaction was quenched with saturated sodium bicarbonate solution, diluted with ethyl acetate (50 ml), filtered, and the filtrate washed with brine, water. The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by normal phase column chromatography (ethyl acetate: petroleum ether = 60) to give the title compound ((S) -1- (4- (((S) -4-bromo-2,3-dihydro-1H-inden-1-yl) oxo) -5-chloro-2- ((5-cyanopyridin-3-yl) methoxy) benzyl) piperidine-3-carboxylic acid (110 mg, 30%) as a white solid MS (ESI): m/z =596.1[ m + H ]] ⁺ .

(S) -1- (5-chloro-4- (((S) -4- (2-chloro-3- ((E) -5- ((((((S) -5-carbonylpyrrolidin-2-yl) methyl) Amino) methyl) -2- (trifluoromethyl) styryl) phenyl) -2,3-dihydro-1H-inden-1-yl) oxo) -2- ((5-cyanopyrazine Pyridin-3-yl) methoxy) benzyl) piperidine-3-carboxylic acid

(S) -1- (4- (((S) -4-bromo-2,3-dihydro-1H-inden-1-yl) oxo) -5-chloro-2- ((5-cyanopyridin-3-yl) methoxy) benzyl) piperidine-3-carboxylic acid (110 mg, 0.18 mmol), (S, E) - (2-chloro-3- (5- ((((5-carbonylpyrrolidin-2-yl) methyl) amino) methyl) -2- (trifluoromethyl) styryl) phenyl) boronic acid (109 mg, 0.24 mmol), tetratriphenylphosphine palladium (46 mg, 0.04 mmol) and anhydrous potassium carbonate (62 mg, 0.45 mmol) were mixed well under nitrogen and 1,4 dioxane (3 mL) and water (0) were added.5 ml) and then stirred at 90 ℃ for 2 hours. The reaction solution was diluted with ethyl acetate (50 ml), filtered, and the filtrate was washed with brine and water. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by normal phase column chromatography (methanol: dichloromethane = 13) and Prep-HPLC to give the title compound (S) -1- (5-chloro-4- (((S) -4- (2-chloro-3- ((E) -5- (((((((((S) -5-carbonylpyrrolidin-2-yl) methyl) amino) methyl) -2- (trifluoromethyl) styryl) phenyl) -2,3-dihydro-1H-inden-1-yl) oxo) -2- ((5-cyanopyridin-3-yl) methoxy) benzyl) piperidine-3-carboxylic acid (45 mg, 27%) as a white solid. MS (ESI) m/z =924.3[ 2 ] M + H] ⁺ .

¹ H NMR(400MHz,DMSO-d ₆ )δ9.01–8.95(m,2H),8.39(s,1H),7.92(s,1H),7.79(d,J＝6.5Hz,1H),7.69(d,J＝8.2Hz,1H),7.62(s,1H),7.56(d,J＝15.7Hz,1H),7.51–7.40(m,3H),7.40–7.30(m,3H),7.29(s,1H),7.24–7.20(m,1H),7.09(s,1H),6.09–5.95(m,2H),5.38–5.28(m,2H),3.80(q,J＝14.9Hz,2H),3.61–3.55(m,1H),3.47–3.39(m,2H),2.91–2.72(m,3H),2.71–2.52(m,4H),2.42–2.33(m,1H),2.19–1.91(m,6H),1.82–1.71(m,1H),1.72–1.55(m,3H),1.50–1.40(m,1H),1.37–1.28(m,1H).

Example 6 (1r, 4r) -4- (((4- ((2-chloro-3- (2- ((((((S) -5-carbonylpyrrolidin-2-yl) methyl) amino) Yl) methyl) benzo [ d]Oxazol-5-yl) phenyl) amino) thiazolo [4,5-c]Pyridin-2-yl) methyl) amino) cyclohexane-1- Carboxylic acids

5-bromo-2- (chloromethyl) benzo [ d]Oxazole (I)

A solution of 2-amino-4-bromophenol (2 g, 10.64 mmol), 2-chloro-1,1,1-trimethoxyethane (1.64 g, 10.64 mmol) in ethanol (10 mL) was heated to60 ℃ and stirred for 3 hours. The reaction solution was concentrated, and the residue was purified by normal phase column chromatography (petroleum ether: ethanol = 100) to give the title compound 5-bromo-2- (chloromethyl) benzo [ d]Oxazole (2.3 g, 88%) as a light yellow solid. MS (ESI) m/z =247.9[ 2 ] M + H] ⁺ .

5- ((((5-bromobenzo [ d))]Oxazol-2-yl) methyl) amino) methyl) pyrrolidin-2-one

Reacting 5-bromo-2- (chloromethyl) benzo [ d]A mixture of oxazole (1 g, 4.00 mmol), 5- (aminomethyl) pyrrolidin-2-one (2.8 g, 24.4 mmol), potassium carbonate (1.12 g, 8.1 mmol), tetrahydrofuran (5 ml) and acetonitrile (ml) was heated to 55 ℃ and stirred for 3.5 hours. The reaction solution was filtered, the filter cake was washed twice with methanol, the filtrate was concentrated, and the residue was purified by normal phase column chromatography (petroleum ether: ethyl acetate = 3:1) to give the title compound 5- (((5-bromobenzo [ d ])]Oxazol-2-yl) methyl) amino) methyl) pyrrolidin-2-one (780 mg, 59%) as a light yellow solid. MS (ESI) m/z =324.0[ m + H ]] ⁺ .

5- ((((5- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) benzo [ d]Oxazol-2-yl) methyl) Amino) methyl) pyrrolidin-2-one

Under the protection of nitrogen, 5- (((5-bromobenzo [ d ]) is reacted]Oxazol-2-yl) methyl) amino) methyl) pyrrolidin-2-one (700 mg, 2.16 mmol), pinacol ester diboron (1.10 g, 4.32 mmol), [1,1' -bis (di-phenylphosphino) ferrocene]Palladium (II) chloride in dichloromethane complex (1:1) (106 mg, 0.13 mmol) and potassium acetate (424 mg, 3.07 mmol) were mixed well, 1,4-dioxane (6 ml) was added, and then stirred at 80 ℃ for 5 hours. The reaction solution was concentrated, and the residue was purified by normal phase column chromatography (petroleum ether: ethyl acetate = 82)The compound 5- ((((5- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) benzo [ d]Oxazol-2-yl) methyl) amino) methyl) pyrrolidin-2-one (350 mg, 43%) as a light yellow solid. MS (ESI) m/z =372.2[ 2 ] M + H] ⁺ .

(1r, 4r) -4- (((4- ((3-bromo-2-chlorophenyl) amino) thiazolo [4,5-c)]Pyridin-2-yl) methyl) amino) Cyclohexane-1-carboxylic acid

Methyl (1r, 4r) -4- (((4- ((3-bromo-2-chlorophenyl) amino) thiazolo [4,5-c)]Pyridin-2-yl) methyl) amino) cyclohexane-1-carboxylate (400 mg, 0.78 mmol) and lithium hydroxide hydrate (128 mg, 3.2 mmol) were added to a mixed solution of tetrahydrofuran (0.4 ml), methanol (2 ml) and water (0.4 ml), and stirred at room temperature for 2 hours. The reaction solution was neutralized with 2 mol/l hydrochloric acid, the solvent was dried by spinning, and the residue was purified by normal phase column chromatography (dichloromethane: methanol = 100) to give the title compound (1r, 4r) -4- (((4- ((3-bromo-2-chlorophenyl) amino) thiazolo [4,5-c]Pyridin-2-yl) methyl) amino) cyclohexane-1-carboxylic acid (300 mg, 77%) as a white solid. MS (ESI) m/z =496.9[ 2 ] M + H] ⁺ .

(1r, 4r) -4- (((4- ((2-chloro-3- (2- (((((((S) 5-carbonylpyrrolidin-2-yl) methyl) amino) methyl) benzene) And [ d ]]Oxazol-5-yl) phenyl) amino) thiazolo [4,5-c]Pyridin-2-yl) methyl) amino) cyclohexane-1-carboxylic acid

5- ((((5- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) benzo [ d ] in nitrogen]Oxazol-2-yl) methyl) amino) methyl) pyrrolidin-2-one (350 mg, 0.94 mmol), (1r, 4r) -4- (((4- ((3-bromo-2-chlorophenyl) amino) thiazolo [4,5-c)]Pyridin-2-yl) methyl) amino) cyclohexane-1-carboxylic acid (437 mg, 0.88 mmol), tetrakis (triphenylphosphine) palladium (100 mg)0.086 mmol) and potassium carbonate (237 mg, 1.72 mmol) were mixed well, 1,4-dioxane (5 ml) and water (1 ml) were added, and then stirred at 80 ℃ for 2 hours. The reaction solution was filtered, the filter cake was washed twice with methanol, the filtrate was concentrated, and the residue was purified by preparative high performance liquid chromatography to obtain the objective product (1r, 4r) -4- (((4- ((2-chloro-3- (2- ((((((5-carbonylpyrrolidin-2-yl) methyl) amino) methyl) benzo [ d ] d]Oxazol-5-yl) phenyl) amino) thiazolo [4,5-c]Pyridin-2-yl) methyl) amino) cyclohexane-1-carboxylic acid (24.0 mg, 4%) as a white solid. MS (ESI) m/z =660.2[ 2 ], [ M + H ]] ⁺ .

¹ H NMR(400MHz,DMSO-d ₆ )δ8.80(dd,J＝8.3,1.4Hz,1H),8.67(s,1H),8.07(d,J＝5.6Hz,1H),7.76(d,J＝8.4Hz,1H),7.74(d,J＝1.4Hz,1H),7.62(s,1H),7.53(d,J＝5.6Hz,1H),7.46–7.38(m,2H),7.07(dd,J＝7.6,1.5Hz,1H),4.15(s,2H),4.02(q,J＝15.8Hz,3H),3.58(s,1H),2.65–2.55(m,3H),2.50(s,1H),2.46-2.36(m,1H),2.10-2.04(m,4H),1.93(d,J＝9.9Hz,2H),1.85(d,J＝10.6Hz,2H),1.70–1.62(m,1H),1.26(dd,J＝25.1,12.2Hz,2H),1.07(dd,J＝23.2,10.5Hz,2H).

Example 7 (S) -1- ((5-chloro-2-methoxy-6- (((S) -4- (2- (1- (((S) -5-carbonylpyrrolidine-2-) Yl) methyl) piperidin-4-yl) benzo [ d]Oxazol-5-yl) -2,3-dihydro-1H-inden-1-yl) oxo) pyridin-3-yl) methyl) Piperidine-3-carboxylic acid

5-bromo-2- (piperidin-4-yl) benzo [ d]Oxazole (oxazole)

Polyphosphoric acid (30 g), 2-amino-4-bromophenol (1.00 g, 5.32 mmol) and piperidine-4-carboxylic acid (0.69 g, 5.32 mmol) were added sequentially in a sealed tube and stirred at 190 ℃ for 3.5 hours. Cooling the reaction solution to normal temperature, diluting with water under ice bathAfter adjusting the pH to 9 with sodium hydroxide, it is extracted three times with ethyl acetate, dried with organic phase and concentrated to give the title compound 5-bromo-2- (piperidin-4-yl) benzo [ d]Oxazole (1.10 g, 74%) as a light yellow solid. MS (ESI) m/z =283.0[ m + H ]] ⁺ .

(S) -5- ((4- (5-bromobenzo [ d ]]Oxazol-2-yl) piperidin-1-yl) methyl) pyrrolidin-2-one

Reacting 5-bromo-2- (piperidin-4-yl) benzo [ d]Oxazole (500 mg, 1.78 mmol), (S) - (5-carbonylpyrrolidin-2-yl) methyl 4-methylbenzenesulfonate (719 mg, 2.67 mmol), and a solution of anhydrous potassium carbonate (495 mg, 3.58 mmol) in acetonitrile (5 ml) were sequentially added to the tube and stirred at 100 ℃ for 20 hours. The reaction solution was diluted with ethyl acetate, washed with water and brine, the organic phase was dried over anhydrous sodium sulfate, concentrated, and the residue was purified by normal phase column chromatography (methanol: dichloromethane =3]Oxazol-2-yl) piperidin-1-yl) methyl) pyrrolidin-2-one (490 mg, 73%) as a light yellow solid. MS (ESI) m/z =378.0[ M + H ]] ⁺ .

(S) -5- ((4- (5- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) benzo [ d]Oxazol-2-yl) Piperidin-1-yl) methyl) pyrrolidin-2-one.

Under the protection of nitrogen, (S) -5- ((4- (5-bromobenzo [ d)]Oxazol-2-yl) piperidin-1-yl) methyl) pyrrolidin-2-one (490 mg, 1.30 mmol), 4,4,4',4',5,5,5',5' -octamethyl-2,2 ' -bis (1,3,2-dioxaborolan) (495 mg, 1.95 mmol), tetratriphenylphosphine palladium (75 mg, 0.065 mmol) and anhydrous potassium acetate (319 mg, 3.25 mmol) were mixed well, 1,4 dioxane (4 ml) and water (0.5 ml) were added, followed by stirring at 80 ℃ for 6 hours. Filtering the reaction solution, and concentratingThe residue was purified by normal phase column chromatography (ethyl acetate: petroleum ether = 60) to give the title compound (S) -5- ((4- (5- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) benzo [ d]Oxazol-2-yl) piperidin-1-yl) methyl) pyrrolidin-2-one (445 mg, 81%) was a yellow solid. MS (ESI) m/z =426.2[ 2 ], [ M + H ]] ⁺ .

5-chloro-2-methoxy-6- (((S) -4- (2- (1- (((S) -5-carbonylpyrrolidin-2-yl) methyl) piperidin-4-yl) Benzo [ d ] carbonyl]Oxazol-5-yl) -2,3-dihydro-1H-inden-1-yl) oxo) nicotinaldehyde

Under the protection of nitrogen, (S) -5- ((4- (5- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) benzo [ d]Oxazol-2-yl) piperidin-1-yl) methyl) pyrrolidin-2-one (98 mg, 0.23 mmol), (S) -1- ((3-chloro-5-formyl-6-methoxypyridin-2-yl) oxo) -2,3-dihydro-1H-inden-4-yl 4-methylbenzenesulfonate (70 mg, 0.15 mmol), tetratriphenylphosphine palladium (8 mg, 0.0075 mmol) and anhydrous potassium carbonate (62 mg, 0.45 mmol) were mixed well, 1,4 dioxane (4 ml) and water (0.5 ml) were added, followed by stirring at 90 ℃ for 2 hours. The reaction solution was diluted with ethyl acetate, filtered, and the filtrate was washed with water and brine, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by normal phase column chromatography (methanol: dichloromethane =3 97) to give the title compound 5-chloro-2-methoxy-6- (((S) -4- (2- (1- (((S) -5-carbonylpyrrolidin-2-yl) methyl) piperidin-4-yl) benzo [ d]Oxazol-5-yl) -2,3-dihydro-1H-inden-1-yl) oxo) nicotinaldehyde (85 mg, 83%) as a light yellow solid. MS (ESI) m/z =690.2[ 2 ] M + H] ⁺ .

(S) -1- ((5-chloro-2-methoxy-6- (((S) -4- (2- (1- (((S) -5-carbonylpyrrolidin-2-yl) methyl) piper-ine) Pyridin-4-yl) benzo [ d]Oxazol-5-yl) -2,3-dihydro-1H-inden-1-yl) oxo) pyridin-3-yl) methyl) piperidine-3-carboxylic acid

Reacting 5-chloro-2-methoxy-6- (((S) -4- (2- (1- (((S) -5-carbonylpyrrolidin-2-yl) methyl) piperidin-4-yl) benzo [ d]Oxazol-5-yl) -2,3-dihydro-1H-inden-1-yl) oxo) nicotinaldehyde (40 mg, 0.067 mmol), a mixture of (S) -piperidine-3-carboxylic acid (43 mg, 0.335 mmol) was added to a catalytic amount of glacial acetic acid in anhydrous dichloromethane (1 ml) and methanol (0.5 ml) and stirred at ambient temperature for 16 hours. Sodium triacetoxyborohydride (28 mg, 0.13 mmol) was then added to the reaction and stirring continued at room temperature for 2 hours. The reaction was quenched with saturated sodium bicarbonate solution, concentrated, and the residue was purified by Prep-HPLC to give the title compound (S) -1- ((5-chloro-2-methoxy-6- (((S) -4- (2- (1- (((S) -5-carbonylpyrrolidin-2-yl) methyl) piperidin-4-yl) benzo [ d ] c]Oxazol-5-yl) -2,3-dihydro-1H-inden-1-yl) oxo) pyridin-3-yl) methyl) piperidine-3-carboxylic acid (15 mg, 31%) was a white solid. MS (ESI) m/z =714.1[ 2 ] M + H] ⁺ .

¹ H NMR(400MHz,DMSO-d ₆ )δ7.76(s,1H),7.71(d,J＝8.4Hz,1H),7.68(s,1H),7.53(s,1H),7.44(d,J＝8.5Hz,1H),7.41(d,J＝6.9Hz,1H),7.39–7.30(m,2H),6.54–6.47(m,1H),3.91(s,3H),3.70–3.64(m,1H),3.36(s,2H),3.11–3.05(m,1H),3.01–2.94(m,2H),2.92–2.82(m,2H),2.74(d,J＝10.9Hz,1H),2.65–2.56(m,2H),2.43–2.29(m,3H),2.28–2.00(m,11H),1.93–1.79(m,3H),1.79–1.54(m,4H),1.47–1.31(m,3H).

Examples 8-15 were prepared in analogy to examples 1-7:

example 16- (1- ((4- ((2,2 '-dichloro-3' - (6- (difluoromethoxy) -5- (((((S) -5-carbonylpyrrole) Alk-2-yl) methyl) amino) methyl) Pyrazin-2-yl) - [1,1' -biphenyl]-3-yl) amino) thiazolo [4,5-c]Pyridine- 2-yl) methyl) pyrrolidin-3-yl) acetic acid

Ethyl 2- (1- ((4- ((2,2 ' -dichloro-3 ' - (6- (difluoromethoxy) -5- (((((S) -5-carbonylpyrrolidin-2-yl) methyl) amino) methyl) pyrazin-2-yl) - [1,1' -biphenyl ] -3-yl) amino) thiazolo [4,5-c ] pyridin-2-yl) methyl) pyrrolidin-3-yl) acetate

According to the synthesis method of the example 1, the compound can be synthesized by replacing corresponding raw materials. MS (ESI) m/z =811.3[ 2 ] M + H] ⁺ .

Ethyl 2- (1- ((4- ((2,2 ' -dichloro-3 ' - (6- (difluoromethoxy) -5- ((((((S) -5-carbonylpyrrolidin-2-yl) methyl) amino) methyl) pyrazin-2-yl) - [1,1' -biphenylyl)]-3-yl) amino) thiazolo [4,5-c]Pyridin-2-yl) methyl) pyrrolidin-3-yl) acetate (50 mg, 0.06 mmol) and hydrochloric acid (1 mol/l, 2 ml) were heated to 40 ℃ and stirred for 18 hours. Neutralizing the reaction solution with saturated sodium bicarbonate, spin-drying, and subjecting the residue to preparative high performance liquid column chromatography to obtain the target product 2- (1- ((4- ((2,2 ' -dichloro-3 ' - (6- (difluoromethoxy) -5- (((((S) -5-carbonylpyrrolidine-2-yl) methyl) amino) methyl) pyrazine-2-yl) - [1,1' -biphenyl]-3-yl) amino) thiazolo [4,5-c]Pyridin-2-yl) methyl) pyrrolidin-3-yl) acetic acid (19.5 mg, 40%) as whiteA colored solid. MS (ESI) m/z =783.1[ m + H ]] ⁺ .

¹ H NMR(400MHz,DMSO-d ₆ )δ8.87(d,J＝7.3Hz,1H),8.81(s,1H),8.67(s,1H),8.13(d,J＝5.6Hz,1H),7.80(s,1H),7.78(dd,J＝7.7,1.6Hz,1H),7.70–7.58(m,3H),7.55(dd,J＝7.6,1.5Hz,1H),7.50(dd,J＝8.0，8.0Hz,1H),7.07(dd,J＝7.5,1.3Hz,1H),4.14–4.10(m,2H),3.96(s,2H),3.67-2.60(m,1H),2.88(m,1H),2.73(m,2H),2.69–2.62(m,2H),2.48-2.43(m,1H),2.43–2.37(m,1H),2.27(m,2H),2.15–1.95(m,4H),1.76-1.67(m,1H),1.48-1.38(m,1H).

Example 17- ((4- ((2,2 '-dichloro-3' - (6- (difluoromethoxy) -4-methyl-5- ((((((S) -5-carbonyl)) methyl Pyrrolidin-2-yl) methyl) amino) methyl) pyridin-2-yl) - [1,1' -biphenyl]-3-yl) amino) thiazolo [4,5-c]Pyridine (II) Pyridin-2-yl) methyl) -8-azabicyclo [3.2.1]Octane-3-carboxylic acid

N- (2,2 ' -dichloro-3 ' - (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) - [1,1' -biphenyl]- 3-yl) -2- (methoxymethyl) thiazolo [4,5-c]Pyridin-4-amines

Compound a-2 (1 g, 2.6 mmol), compound B (1.66 g, 4.5 mmol), tetratriphenylphosphine palladium (300 mg, 0.26 mmol) and anhydrous potassium carbonate (0.9 g, 6.5 mmol) were added to 1,4 dioxane (16 ml) and water (3 ml) under nitrogen blanket, mixed well and then stirred at 80 ℃ for 4 hours. After the reaction was completed, the reaction solution was filtered, the filtrate was concentrated, and the residue was purified by a normal phase silica gel column (petroleum ether: ethyl acetate =0 to 30%) to obtain the objective compound (470 mg, 31%) as a yellow solid. MS (ESI) m/z =542.0[ m + H ], [ m ], [ phi ]] ⁺ 。

6-(2,2 '-dichloro-3' - ((2- (methoxymethyl) thiazolo [4,5-c)]Pyridin-4-yl) amino) - [1,1' -bi Phenyl radical]-3-yl) -2- (difluoromethoxy) -4-methylnicotinaldehyde

Compound 17-1 (11.0 g, 20.3 mmol), compound C (4.08 g, 18.4 mmol), tetratriphenylphosphine palladium (1.06 g, 0.92 mmol) and anhydrous potassium carbonate (6.37 g, 46.08 mmol) were added to 1,4 dioxane (80 mL) and water (20 mL) under nitrogen blanket, mixed well and then stirred at 90 deg.C for 2 hours. After completion of the reaction, the reaction mixture was filtered, the filtrate was concentrated, and the residue was purified with a normal-phase silica gel column (ethyl acetate: petroleum ether = 1:2) to give the title compound 6- (2,2 '-dichloro-3' - ((2- (methoxymethyl) thiazolo [4,5-c)]Pyridin-4-yl) amino) - [1,1' -biphenyl]-3-yl) -2- (difluoromethoxy) -4-methyl nicotinaldehyde (8.00 g, 72%) was a light yellow solid. MS (ESI) m/z =601.2[ m + H ]] ⁺ 。

(S) -5- (((6- (2,2 '-dichloro-3' - ((2- (methoxymethyl) thiazolo [4,5-c)]Pyridin-4-yl) amino - [1,1' -biphenyl]-3-yl) -2- (difluoromethoxy) -4-methylpyridin-3-yl) methyl) amino) methyl) pyrrolidinone- 2-ketones

A solution of compound 17-2 (8.0 g, 13.3 mmol) and (S) -5- (aminomethyl) pyrrolidin-2-one hydrochloride (6.0 g, 39.9 mmol) and N, N-diisopropylethylamine (2.58 g, 19.95 mmol) in dry dichloromethane (40 ml) was stirred at room temperature for 12 hours. Sodium triacetoxyborohydride (8.46 g, 39.90 mmol) was then added in portions and the reaction was continued at room temperature for 3 hours with stirring. After the reaction is finished, quenching saturated ammonium bicarbonate solution, extracting dichloromethane, drying extract liquor by using anhydrous sodium sulfate, concentrating, and making residue pass through normal phasePurification on silica gel (methanol: dichloromethane = 6) gave the title compound (8.76 g, 94%) as a light yellow solid. MS (ESI) m/z =699.3[ m + H ], [ solution of calcium ] and [ solution of calcium ], [ solution of calcium ] in (ESI) ]] ⁺ 。

(S) -5- (((6- (3' - ((2- (bromomethyl) thiazolo [4,5-c)]Pyridin-4-yl) amino) -2,2' -dichloro- [1,1' -biphenyl]-3-yl) -2- (difluoromethoxy) -4-methylpyridin-3-yl) methyl) amino) methyl) pyrrolidin-2-one

Compound 17-3 (2.00 g, 2.86 mmol) was added to a solution of hydrobromic acid (20 ml) in acetic acid and the mixture was heated to 60 ℃ and stirred for 10 hours. After completion of the reaction, the reaction solution was slowly added dropwise to a saturated aqueous solution of sodium hydrogencarbonate in an ice bath, extracted twice with dichloromethane (50 ml), and the combined organic phases were washed with brine, dried over anhydrous sodium sulfate, and concentrated to obtain the objective compound (1.75 g, 57%) as a yellow solid. MS (ESI) m/z =747.1[ 2 ] M + H] ⁺ 。

Methyl (1R, 5S) -8- ((4- ((2,2 '-dichloro-3' - (6- (difluoromethoxy) -4-methyl-5- ((((((S) -5-) Carbonylpyrrolidin-2-yl) methyl) amino) methyl) pyridin-2-yl) - [1,1' -biphenyl]-3-yl) amino) thiazolo [4,5- c]Pyridin-2-yl) methyl) -8-azabicyclo [3.2.1]Octane-3-carboxylic acid esters

Cesium carbonate (391 mg, 1.20 mmol) and methyl (1R, 5S) -8-azabicyclo [3.2.1]Octane-3-carboxylate hydrochloride (165 mg, 0.80 mmol) was dissolved in tetrahydrofuran (3 ml) and acetonitrile (1 ml), and stirred at room temperature for one hour. Then compound 17-4 (300 mg, 0.40 mmol) was added thereto and stirring at room temperature was continued until the starting material completely disappeared. The reaction mixture was diluted with dichloromethane (50 ml), washed successively with water and saturated brineWashing, drying, concentration and purification of the residue on a reverse phase flash column (85% acetonitrile in ammonium bicarbonate (0.1%) in water) afforded the title compound (122 mg, 38%) as a pale yellow solid. MS (ESI) m/z =836.4[ m + H ])] ⁺ 。

8- ((4- ((2,2 '-dichloro-3' - (6- (difluoromethoxy) -4-methyl-5- ((((((S) -5-carbonylpyrrolidine-2-) Yl) methyl) amino) methyl) pyridin-2-yl) - [1,1' -biphenyl]-3-yl) amino) thiazolo [4,5-c]Pyridin-2-yl) Methyl) -8-azabicyclo [3.2.1]Octane-3-carboxylic acid

Compound 17-5 (122 mg, 0.15 mmol) and lithium hydroxide monohydrate (25 mg, 0.60 mmol) were dissolved in a mixed solution of methanol (1.5 ml), tetrahydrofuran (0.5 ml) and water (0.5 ml), and the reaction solution was stirred at room temperature overnight. After completion of the reaction, it was neutralized with dilute hydrochloric acid, concentrated, and the residue was purified by preparative high performance liquid chromatography to obtain the objective product (95 mg, 80%) as a white solid. MS (ESI) m/z =822.3[ 2 ] M + H] ⁺ 。

¹ H NMR(400MHz,DMSO-d ₆ )δ8.93(d,J＝8.3Hz,1H),8.63(s,1H),8.13(d,J＝5.6Hz,1H),7.72(t,J＝73Hz,,1H),7.70–7.65(m,1H),7.65–7.55(m,3H),7.48(m,3H),7.05(d,J＝7.5Hz,1H),3.98(s,2H),3.78(s,2H),3.67–3.59(m,1H),3.38–3.33(m,2H),2.62–2.57(m,2H),2.56–2.53(m,1H),2.49(s,3H),2.18–2.05(m,3H),2.04–1.92(m,2H),1.82(m,2H),1.74–1.58(m,5H)。

The following compounds were synthesized according to the synthesis method of example 17, substituting the corresponding starting materials.

Test example 1 detection of inhibitory Effect of Compounds on mutual binding of PD-1/PD-L1 proteins

PD-1/PD-L1 homogeneous Time-Resolved Fluorescence (HTRF) detection technology is adopted to detect the activity of the compound for inhibiting the binding of PD-1 and PD-L1 in vitro.

Selecting a PD-1/PD-L1 binding assay kit (Cisbio, cat #63ADK000 CPDECP), wherein the kit comprises two proteins of Tag 1-PD-L1 and Tag 2-PD-1, and Anti-Tag1-Eu ³⁺ And Anti-Tag2-XL 665. The detection principle is as follows: anti-tag1-Eu ³⁺ As a donor of HTRF, anti-Tag2-XL 665 is used as an acceptor of HTRF, when Tag 1-PD-L1 and Tag 2-PD-1 interact, the added HTRF donor and acceptor are close to each other, and after the donor receives excitation energy, partial energy is transferred to the acceptor, and 665nm emission light is generated. When the addition of the compound blocked the PD-1/PD-L1 interaction, only 620nm emitted light was produced. The inhibitory effect of the compounds was determined by comparing the ratio 665nm/620 nm. Tag 1-PD-L1 was diluted with Diluent buffer (cat #62 DLBDDF) to a working concentration of 10nM, tag 2-PD-1 was diluted with Diluent buffer to a working concentration of 500nM, anti-Tag1-Eu ³⁺ Diluted with detection buffer (cat #62DB1 FDG) at 1Dilute to 10X final concentration. mu.L of the compound was added to each well of a 384-well plate, followed by 4. Mu.L of Tag 1-PD-L1 and 4. Mu.L of Tag 2-PD-1, respectively, and incubated at room temperature for 15 minutes. Adding 5 mu L of Anti-Tag1-Eu ³⁺ And 5. Mu.L of Anti-Tag2-XL 665, incubated overnight at room temperature, using BioTek Synergy ^TM Detecting by a Neo2 multifunctional microplate reader to obtain a ratio of 665nm/620 nm. The IC50 curve was fitted with Prism.

TABLE 1 IC of part of the Compounds of the invention in test example 1 ₅₀ Value of

Compound number	PD-L1 IC ₅₀
		1	A
2	A
		3	A
4	A
		5	A
6	A
		7	A
8	B
		9	A
10	A
		11	A
12	A
		13	A
14	A

Letter A stands for IC ₅₀ Less than 5nM;

letter B stands for IC ₅₀ 5nM to 1. Mu.M;

letter C denotes IC ₅₀ Is greater than 1 μ M;

the results show that the compound of the invention can effectively inhibit the binding of PD-1/PD-L1 at different concentrations. Therefore, the compound can be used for treating diseases related to the mutual combination of PD-1/PD-L1.

Test example 2: cytology NFAT reporter Gene experiments

The cytological experiment of PD-1/PD-L1 requires two cells, PD-1 effector cells and PD-L1 aAPC/CHO-K1 cells, wherein the PD-1 effector cells express human PD-1 protein and luciferase reporter gene driven by NFAT and the PD-L1 aAPC/CHO-K1 cells express PD-L1 protein and anti-CD3 antibody. When these two cells were co-cultured, the interaction of PD-1/PD-L1 inhibited TCR signaling to NFAT-RE, interrupting NFAT-RE mediated fluorescence signaling. When the inhibitor of PD-1 or PD-L1 is added, the interaction of PD-1/PD-L1 is blocked, the signal inhibition of TCR to NFAT-RE pathway is released, the fluorescence signal is enhanced, and the blocking effect of the inhibitor is judged by the strength of the fluorescence signal.

The first day of the experiment, the recovered PD-L1 aAPC/CHO-K1 cells were digested, centrifuged and adjusted to a concentration of 2.0X 10 using medium (90% ham's F-12/10% FBS) ⁵ 40. Mu.L/well, 8X 10/mL ³ The amount of cells was plated in 384-well plates and placed in an incubator overnight. The next day, the test compound was first diluted with test buffer (99% RPMI1640/1% FBS) in a gradient of 2 times the desired test concentration, and PD-1 cells were centrifuged and then diluted with test buffer to a concentration of 6.25X 10 ⁵ and/mL. After removing the medium from overnight-cultured 384-well plates, 20. Mu.L of compound dilution and 20. Mu.L of diluted PD-1 effector cells were added to each well, and after 6 hours of incubation in a cell incubator, 20. Mu.L of Bio-Glo reagent (Promega, cat # G7940) was added to each well, and after about 10 minutes, the plates were read with a multifunctional microplate reader. Negative controls (cells only, no compound) and blank controls (detection buffer only) were set for each plate. The inhibitory activity of the compounds was analyzed by Prism on the basis of fluorescence values.

Table 2 EC of some compounds of the invention in test example 2 ₅₀ Value of

Compound numbering	NFAT EC ₅₀
		1	A
2	A
		3	B
4	B
		5	A
6	B
		7	B
9	A
		10	A
11	A
		12	A
13	A
		14	A
15	B
		16	A

Letter A stands for IC ₅₀ Less than 100nM;

letter B stands for IC ₅₀ 100nM to 1 μ M;

letter C stands for IC ₅₀ Is greater than 1 μ M;

the results show that the compound of the invention can effectively block the interaction of PD-1/PD-L1, and the half inhibitory concentration is equal to or better than that of the PD-1/PD-L1 inhibitor in clinical stage.

Test example 3: human PBMC functional assay

SEB (Toxin Technology, cat # BT 202) induces T cell activation and secretion to produce IL-2. The addition of anti-PD-1/PD-L1 antibody or small molecule inhibitor in the reaction system can promote T cell activation and increase IL-2 secretion. The activity of the compounds was assessed by measuring the amount of IL-2 in the cell supernatant using an IL-2ELISA kit.

On the first day of experiment, test compound, antibody solution and control solution were prepared, 50. Mu.L of the test compound, antibody or control solution was added to each well, human PBMC cells were revived as required by the instructions, and the cell concentration was adjusted to 3X 10 using medium (90% RPMI1640 10% FBS) ⁶ Per mL, at 100. Mu.L per well, 3X 10 ⁵ The amount of cells was plated on a 96-well cell culture plate (Corning-3599), cultured at 37 ℃ for 4 days in an incubator, centrifuged at 300g for 5 minutes to collect cell supernatants, and then subjected to IL-2ELISA kit (R)&D-DY 202-05) detecting a change in the cytokine IL-2.

Table 3 concentration of a portion of the compounds of the invention effective in test example 3

Compound numbering	Concentration of onset of action
		2	B
10	B

Letter a represents an onset of less than 1nM;

letter B represents onset concentration of 1-10nM;

letter C represents an onset concentration of 10-100nM;

the results show that the compounds of the invention are capable of increasing IL-2 secretion, and that IL-2 secretion is dose-related to the concentration of the compound, which is comparable to the control antibody at high concentrations.

Test example 4: in-vivo efficacy experiment of small molecule inhibitor for treating tumor

Mouse models of subcutaneously transplanted tumors were established to examine the in vivo inhibitory effect of these compounds on tumor growth. The method comprises the following steps: digesting the cultured specific tumor cells, centrifuging, collecting the cells, washing the cells twice by using sterile normal saline, counting the cells, adjusting the cell concentration to the required concentration by using the normal saline, and inoculating 0.2mL of cell suspension to hPD-L1C57BL/6 or Balb/C immune healthy mice subcutaneously. After inoculation, the tumor growth is observed to reach a specific size, animals are randomly grouped, 6-15 animals are in each group, the animals are weighed and then dosed, and the compound to be tested is dosed 1 or 2 times a day, wherein the grouping comprises: a vector group, a control anti-PD-1/PD-L1 antibody group and a test compound group. Mice were monitored weekly for tumor growth for approximately 6 weeks, and after tumor volume reached the tumor endpoint, mice were weighed and euthanized. Tumor tissue, spleen tissue and blood samples were removed. Calculating the tumor inhibition rate, detecting the inhibition rate and immune cell composition of hPD-L1 in tumor, spleen and blood samples, and calculating the immunoregulatory activity of the compound to be detected.

The results show that the compound can effectively inhibit the tumor growth in tumor-bearing mice, and the inhibition effect is equivalent to or better than that of a PD-1/PD-L1 inhibitor in a clinical stage.

Test example 5: pharmacokinetic experiments of the small molecule inhibitor of the invention on mice, rats and monkeys

Single Intravenous (IV) and oral (PO) administrations of ICR mouse, rat and monkey test compounds, respectively, blood samples were taken at different time points, LC-MS/MS determined concentrations of the test substance in the plasma of the animals andand calculating the related parameters. The method comprises the following specific steps: taking the required amount of test sample, in 20% PEG400+80% (20% SBE-. Beta. -CD at 100mM citrate buffer pH 3.0), make up the solution of the required concentration for intravenous or oral administration. Establishing a biological sample analysis method and a sample detection method. Calculating pharmacokinetic parameters, such as AUC, of blood concentration data at different time points by using Phoenix WinNonlin 7.0 software _(0-t) ，AUC _(0-∞) ，T _1/2 ，C _max ，T _max F%, MRT, etc.

TABLE 4 mouse pharmacokinetic parameters of partial compounds of the invention

TABLE 5 rat pharmacokinetic parameters for a part of the Compounds of the invention

TABLE 6 Macaca fascicularis pharmacokinetic parameters of partial compounds of the invention

The results show that the compounds of the invention have excellent pharmacokinetic properties.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A compound of formula I, a stereoisomer thereof or a tautomer thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof:

wherein the content of the first and second substances,

R ₂ independently selected from the group consisting of: C1-C4 alkyl,

n1 is 0, 1,2,3 or 4;

ra and Rb are each independently selected from the group consisting of: H. C1-C8 alkyl, C3-C8 cycloalkyl (including monocyclic, fused, spiro and bridged rings), 5-10 membered heterocyclyl (including monocyclic, fused, spiro and bridged rings) having 1-3 heteroatoms selected from N, S and O, or- (CH) ₂ ) _n2 -Rc; wherein Rc is a 5-7 membered nitrogen-containing saturated ring or C3-C8 membered cycloalkyl, and n2 is 0, 2,3 or 4; or Ra and Rb together with the N atom to which they are attached form a 5-to 10-membered saturated heterocyclic ring;

wherein Ra, rb, rc, or Ra and Rb together forming a ring may be further substituted with 1 to 3 groups selected from: halogen, COOH, C (R) ₆ R ₇ ) _m1 -COOH, C1-C3 alkyl, C1-C6 amido: (-C(＝O)-N(Rd) ₂ or-NH-C (= O) (Rd), rd being H or C1-C5 alkyl); wherein m1 is 1,2 or 3,R ₆ And R ₇ Each independently selected from H, halogen, C1-C3 alkyl;

n3 is 0, 1, 2;

the 4-to 8-membered nitrogen-containing heterocycle may be substituted by-CH ₂ -Re substitution, wherein Re is a 5-7 membered nitrogen containing saturated ring;

n is 0, 1 or 2;

m is 0, 1,2 or 3;

or the said

Together form a structure shown by the following formula:

is the attachment site of the group;

each one of

Each independently is a single or double bond;

2. The compound of claim 1, a stereoisomer or tautomer thereof, or pharmaceutically acceptable salt, hydrate or solvate thereof, wherein R is ₅ Comprises at least one group selected from the group consisting of: -OCHF ₂ 、-OCH ₂ F. Cyclopropyl, CN, ethyl, -CH ₂ CF ₃ 、-OCH ₂ CN。

3. The compound of claim 1, a stereoisomer or tautomer thereof, or pharmaceutically acceptable salt, hydrate or solvate thereof, wherein Ra and Rb are each independently selected from the group consisting of: H. C1-C8 alkyl, C3-C8 cycloalkyl (including monocyclic, fused, spiro and bridged rings), 5-10 membered heterocyclyl (including monocyclic, fused, spiro and bridged rings) having 1-3 heteroatoms selected from N, S and O; or Ra and Rb together with the N atom to which they are attached form a 5-to 10-membered saturated heterocyclic ring;

4. The compound of claim 3, a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof,

the Ra is selected from the following group: H. a C1-C3 alkyl group;

wherein Ra, rb, or Ra and RbThe saturated heterocyclic ring formed by Rb together can be further substituted by 1 to 3 groups selected from the group consisting of: halogen, C1-C3 alkyl, C1-C6 acylamino, COOH, C (R) ₆ R ₇ ) _m1 -COOH; wherein m1 is 1,2 or 3,R ₆ And R ₇ Each independently selected from H, halogen, C1-C3 alkyl.

5. The compound of claim 1, a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof,

the Ra is selected from the following group: H. a C1-C3 alkyl group;

6. The compound of claim 1, wherein the compound has the formula:

R ⁵ ' is a group selected from: H. unsubstituted or halogenated C1-C6 alkyl, unsubstituted or halogenated C1-C6 alkoxy, unsubstituted or halogenated C3-C6 cycloalkyl; unsubstituted or halogenated C1-C6 alkyl, unsubstituted or halogenated C1-C6 alkoxy, unsubstituted or halogenated C3-C6 cycloalkyl are preferred.

7. The compound of any one of claims 1 to 6, a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein the compound is selected from the group consisting of:

8. the compound of claim 1, a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, wherein each L is independently selected from the group consisting of: C2-C4 alkenylene, halogenated C2-C4 alkenylene.

9. The compound of claim 8, a stereoisomer or tautomer thereof, or pharmaceutically acceptable salt, hydrate or solvate thereof, selected from the group consisting of:

10. the compound of claim 1, a stereoisomer or tautomer thereof, or pharmaceutically acceptable salt, hydrate or solvate thereof, wherein said compound has the structure shown in the formula:

11. the compound of claim 10, a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein said compound is selected from the group consisting of:

12. the compound of claim 1, a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein Ra, rb, or the nitrogen-containing heterocycle formed by Ra and Rb is selected from the group consisting of:

13. the compound of claim 12, a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein said compound is selected from the group consisting of:

14. a pharmaceutical composition comprising (1) a compound of claim 1 or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof; (2) a pharmaceutically acceptable carrier.

15. Use of a compound according to claim 1 or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition according to claim 12, for the preparation of a pharmaceutical composition for the prophylaxis and/or treatment of diseases associated with the activity or expression of PD-1/PD-L1; preferably, the disease is selected from the group consisting of: tumors, pathogen infections, autoimmune response related diseases.