Preparation and use of a class of compounds with immunomodulatory function

Abstract

The application discloses a preparation method and application of a compound with an immunoregulatory effect. Specifically, the application discloses a compound with a structure as shown in formula I, wherein definitions of the groups are described in the specification. The application also provides application of the compound in regulating immunity and inhibiting PD-1 / PD-L1.

Description

Technical Field

[0001] This invention relates to the field of small molecule protein inhibitors. Specifically, this invention provides a method for preparing and applying a compound with immunomodulatory functions. Background Technology

[0002] The immune system plays a role in surveillance, defense, and regulation. Cellular immunity mainly participates in immune responses against intracellular parasitic pathogens and tumor cells, as well as in the formation of delayed-type hypersensitivity reactions and autoimmune diseases, transplant rejection, and the regulation of humoral immunity. The activation of T lymphocytes by antigen-presenting cells is typically regulated by two different signals. Primary signals are transmitted via T cell receptors (TCRs) through the major histocompatibility complex (MHC) on APC cells, presenting foreign antigenic peptides. Secondary signals, also known as co-stimulatory signals, are transmitted to T cells via the binding of co-stimulatory molecules on APC cells to T cell surface receptors, regulating T cell proliferation, cytokine secretion, and effector functions. Secondary signals include both positive and negative regulation. Positive regulation promotes T cell activation, while negative regulation induces T cell tolerance, which is crucial for the body's adaptation and adjustment of its immune cell responses to different external antigens.

[0003] Programmed death-ligand 1 (PD-L1), also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), belongs to the tumor necrosis factor superfamily. It is a type I transmembrane glycoprotein composed of 290 amino acid residues, including an IgV-like region, an IgC-like region, a transmembrane hydrophobic region, and a 30-amino acid intracellular tail, with a total molecular weight of 40 kDa. PD-L1 mRNA is present in almost all tissues, but the PD-L1 protein is persistently expressed only in a few tissues, including the liver, lungs, tonsils, and immune-specific tissues such as the eye and placenta. PD-L1 is also expressed in activated T cells, B cells, monocytes, dendritic cells, and macrophages.

[0004] The receptor for PD-L1 is PD-1, which is mainly expressed on the surface of activated CD4+ T cells, CD8+ T cells, NK cells, B cells, and activated monocytes. The binding of PD-L1 to PD-1 promotes phosphorylation of tyrosine residues in the ITSM (immunoreceptor tyrosine switching motif) structure within the PD-1 cytoplasm, recruits SHP1 / 2, and dephosphorylates downstream Syk and PI3K, thus limiting the interaction between antigen-presenting cells or dendritic cells and T cells. This binding can further inhibit T cell metabolism, reduce the secretion of effector cytokines IL-2 and IFN-γ, induce T cell exhaustion and apoptosis, thereby reducing the immune response involving T cells and exercising a negative regulatory function.

[0005] T cells, upon recognizing and activating antigens, secrete IFN-γ. T cell-derived IFN-γ amplifies and maintains T cell function, such as upregulating MHC molecules, enhancing antigen processing and presentation on target cells, and promoting T cell differentiation. IFN-γ also induces PD-L1 expression in sites of immune inflammation, preventing excessive immune response from damaging tissues. Under IFN-γ stimulation, PD-L1 is expressed on the surface of antigen-presenting cells (DCs, macrophages, etc.) and vascular endothelial cells. Interferon regulatory factor 1 (IRF-1), induced by IFN-γ, can also bind to the interferon regulatory factor binding sites 200 bp and 320 bp before the PD-L1 transcription start site, regulating PD-L1 at the transcriptional level. PD-L1 can bind to PD-1 on the surface of T cells, exercising a negative regulatory function, thereby protecting inflammatory sites.

[0006] PD-L1 plays a crucial role in tumor immunity through its negative regulatory function. In 2004, Konishi et al. first discovered PD-L1 expression in tissue samples from non-small cell lung cancer patients. Subsequently, PD-L1 was found to be expressed in tissues from various cancer patients, including gastric cancer, lung cancer, liver cancer, intrahepatic cholangiocarcinoma, colon cancer, pancreatic cancer, ovarian cancer, breast cancer, cervical cancer, head and neck squamous cell carcinoma, nasopharyngeal carcinoma, esophageal cancer, bladder cancer, renal cell carcinoma, skin cancer, and oral squamous cell carcinoma. During malignant transformation, new protein molecules are generated due to gene mutations, exogenous gene (virus) expression, or activation of dormant genes. After these proteins degrade within the cell, certain degraded peptides can be expressed on the cell surface, becoming tumor antigens. The immune system can recognize tumor antigens and eliminate tumor cells through immune surveillance, while tumor cells utilize PD-L1 to evade immune attack.

[0007] PD-L1 expression at the tumor site can protect tumor cells from harm through multiple pathways. Tumor-infiltrating lymphocytes (TILs) secrete IFN-γ, which induces PD-L1 expression in tumor cells and surrounding stromal cells. PD-L1 on tumor cells can bind to PD-1 on TILs, inhibiting TIL cell activation and further leading to apoptosis. In vitro experiments have shown that tumor-associated PD-L1 can increase tumor-specific T cell apoptosis, while PD-L1 monoclonal antibodies can attenuate this effect. Tumor-associated PD-L1 can promote T cell expression of IL-10, further suppressing the immune response. PD-L1 is not only a ligand for PD-1; it can also act as a receptor to transmit reverse signals, protecting tumor cells from apoptosis induced by other anti-tumor pathways such as FAS-FASL.

[0008] Currently, several marketed monoclonal antibody drugs targeting PD-1 or PD-L1 have demonstrated that PD-1 / PD-L1 blockers can be used in the clinical treatment of various tumors. However, antibody drugs have their own characteristics, such as high production costs, poor stability, the need for injection administration, and a tendency to induce immunogenicity. Small molecule drugs, on the other hand, have advantages such as good tissue penetration, convenient storage and transportation, lower production costs, no immunogenicity, and the ability to be administered orally. Therefore, the research and development of small molecule blockers for PD-1 / PD-L1 has significant application and social value.

[0009] In summary, there is an urgent need in this field to develop small molecule PD-1 / PD-L1 blockers. Summary of the Invention

[0010] The purpose of this invention is to provide a small molecule PD-1 / PD-L1 blocker.

[0011] A first aspect of the present invention provides a compound of formula I, a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof:

[0012]

[0013] in,

[0014] 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 heterocyclic alkyl (including saturated or partially unsaturated monocyclic, bicyclic, spirocyclic or bridged rings), and C3-C12 (preferably C5-C12) cycloalkyl, wherein the heterocyclic alkyl has 1-3 heteroatoms selected from N, O, and S;

[0015] R1 and R4 are each independently selected from the following group: H, halogen, CN, C1-C3 alkyl, C1-C3 alkoxy;

[0016] R5 is 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, and halogenated C3-C6 cycloalkyl.

[0017] R2 is independently selected from the following group: C1-C4 alkyl groups, n1 is 0, 1, 2, 3 or 4;

[0018] Ra and Rb are each independently selected from the following group: H, C1-C8 alkyl, C3-C8 cycloalkyl (including monocyclic, fused, spirocyclic and bridged rings), 5-10 membered heterocyclic groups (including monocyclic, fused, spirocyclic and bridged rings) having 1-3 heteroatoms selected from N, S and O, or -(CH2). n2 -Rc; wherein Rc is a 5-7 member nitrogen-containing saturated ring, a C3-C8 member cycloalkyl ring, and n2 is 0, 2, 3 or 4; or Ra and Rb together with the attached N atom form a 5-10 member saturated heterocycle;

[0019] The ring formed by Ra, Rb, Rc, or Ra and Rb may be further substituted by 1-3 groups selected from the following group: halogen, COOH, C(R6R7). m1 -COOH, C1-C3 alkyl, C1-C6 amide (-C(=O)-N(Rd)2 or -NH-C(=O)(Rd), where Rd is H or a C1-C5 alkyl group); wherein m1 is 1, 2 or 3, and R6 and R7 are each independently selected from H, halogen, C1-C3 alkyl;

[0020] R3 is independently selected from the following group: 4-8 membered nitrogen-containing heterocycles. n3 is 0, 1, or 2;

[0021] The 4-8 member nitrogen-containing heterocycle can be replaced by CH2-Re, wherein the Re is a 5-7 member nitrogen-containing saturated ring;

[0022] Ra' and Rb' are each independently selected from the group consisting of H, C1-C3 alkyl, or CH2-Re; wherein the Re is a 5-7 member nitrogen-containing saturated ring;

[0023] In this context, the carbon atoms on Ra', Rb', and Re can be optionally replaced by carbonyl groups;

[0024] n is 0, 1, or 2;

[0025] m can be 0, 1, 2, or 3;

[0026] Each L is independently selected from the following group: chemical bond, C2-C4 alkenyl group, halogenated C2-C4 alkenyl group;

[0027] or the aforementioned Together they form the structure shown in the following formula: In this context, X and Y are each independently selected from the following groups: O, S, N, CH, NH, CH2; the dashed lines represent single or double bonds.

[0028] This is the linking site of the functional group;

[0029] each Each can be a single bond or a double bond independently;

[0030] The additional condition is that the compound of formula I has a chemically stable structure.

[0031] In another preferred embodiment, R 1 and R 4 Each is independently selected from the following group: halogen, methyl.

[0032] In another preferred embodiment, R 1 and R 4 Each of them is independently selected from the following groups: Cl, Br.

[0033] In another preferred embodiment, the R 5 It includes at least one group selected from the group consisting of: -OCHF2, -OCH2F, cyclopropyl, CN, ethyl, -CH2CF3.

[0034] In another preferred embodiment, the R 2 and R 3 Each person independently selects from the following groups: Wherein, the E ring is a 5-7 member saturated ring of aza, p, r and s are each independently 0, 1, 2 or 3, and the R is selected from the following group: oxo (=O) and carboxyl.

[0035] In another preferred embodiment, the compound has the structure shown in the following formula:

[0036]

[0037] Among them, X 1 and X 2 Each is independently CH or N, and when X 1 or X 2 When it is CH, the R mentioned 5 Or R 5 'Can be located on CH (the corresponding X at this time) 1 or X 2 (C)

[0038] R 5The group is selected from the group consisting of: unsubstituted or halogenated C2-C6 alkyl, halogenated C1-C6 alkoxy, cyano-substituted C1-C6 alkoxy, and unsubstituted or halogenated C3-C6 cycloalkyl; preferably halogenated C1-C6 alkoxy or unsubstituted or halogenated C2-C6 alkyl.

[0039] R 5 The group is selected from the group consisting of: H, unsubstituted or halogenated C1-C6 alkyl, unsubstituted or halogenated C1-C6 alkoxy, and unsubstituted or halogenated C3-C6 cycloalkyl; preferably, unsubstituted or halogenated C1-C6 alkyl, unsubstituted or halogenated C1-C6 alkoxy, and unsubstituted or halogenated C3-C6 cycloalkyl. In another preferred embodiment, R5 includes at least one group selected from the group consisting of: -OCHF2, -OCH2F, cyclopropyl, CN, ethyl, -CH2CF3, and -OCH2CN.

[0040] 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, spirocyclic and bridged rings), and 5-10 member heterocyclic groups (including monocyclic, fused, spirocyclic and bridged rings) having 1-3 heteroatoms selected from N, S and O; or Ra and Rb together with the attached N atom to form a 5-10 member saturated heterocycle;

[0041] Ra, Rb, and the saturated heterocycle formed by Ra and Rb can be further substituted by 1-3 groups selected from the following groups: halogen, C1-C3 alkyl, C1-C6 amide, COOH, C(R6R7). m1 -COOH; where m1 is 1, 2 or 3, and R6 and R7 are each independently selected from H, halogen, C1-C3 alkyl.

[0042] In another preferred embodiment, Ra is selected from the group consisting of H, C1-C3 alkyl groups;

[0043] The Rb is selected from the following group: C3-C8 cycloalkyl groups (including monocyclic, fused, spirocyclic and bridged rings), and 5-10 member heterocyclic groups (including monocyclic, fused, spirocyclic and bridged rings) having 1-3 heteroatoms selected from N, S and O.

[0044] Alternatively, Ra and Rb together with the attached N atom can form a 5-10 member saturated heterocycle;

[0045] The saturated heterocycle formed by Ra, Rb, or Ra and Rb together can be further substituted by 1-3 groups selected from the following group: halogen, C1-C3 alkyl, C1-C6 amide, COOH, C(R6R7). m1 -COOH; wherein m1 is 1, 2 or 3, and R6 and R7 are each independently selected from H, halogen, C1-C3 alkyl.

[0046] In another preferred embodiment, Ra is selected from the group consisting of H, C1-C3 alkyl groups;

[0047] The Rb is selected from the group consisting of C1-C8 alkyl groups, or groups selected from the group consisting of:

[0048] Alternatively, Ra and Rb, together with the attached N atom, can form a saturated heterocycle selected from the following group:

[0049] Furthermore, the Ra, Rb, or groups formed therefrom may be further substituted by 1-3 groups selected from the following groupings: C1-C3 alkyl, COOH, C(R6R7). m1 -COOH; where m1 is 1, 2 or 3, and R6 and R7 are each independently selected from H, halogen, C1-C3 alkyl.

[0050] In another preferred embodiment, the compound is selected from the group consisting of:

[0051]

[0052]

[0053]

[0054]

[0055] In another preferred embodiment, each L is independently selected from the group consisting of: C2-C4 alkenyl groups and halogenated C2-C4 alkenyl groups.

[0056] In another preferred embodiment, the compound is selected from the group consisting of:

[0057]

[0058] In another preferred embodiment, the compound has the structure shown in the following formula:

[0059]

[0060] In another preferred embodiment, X and Y are each independently selected from the group consisting of: O, N, CH, NH, CH2.

[0061] In another preferred embodiment, the described The ring is selected from the following group:

[0062] In another preferred embodiment, the described The ring is selected from the following group:

[0063] In another preferred embodiment, the compound is selected from the group consisting of:

[0064]

[0065] In another preferred embodiment, the nitrogen-containing heterocycle formed by Ra, Rb, or Ra and Rb together is selected from the group consisting of:

[0066]

[0067] In another preferred embodiment, the compound is selected from the group consisting of:

[0068]

[0069] A second aspect of the present invention provides a pharmaceutical composition comprising (1) a compound as described in the first aspect of the present invention, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof; and (2) a pharmaceutically acceptable carrier.

[0070] A third aspect of the present invention provides the use of a compound as described in the first aspect of the present invention, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition as described in the second aspect of the present invention, for the preparation of a pharmaceutical composition for the prevention and / or treatment of diseases related to the activity or expression level of PD-1 / PD-L1; preferably, the diseases are selected from the group consisting of: tumors, pathogen infections, and diseases related to autoimmune responses.

[0071] In another preferred embodiment, the disease is selected from the group consisting of: tumors, pathogen infections, and diseases related to autoimmune responses.

[0072] In another preferred embodiment, the pharmaceutical composition is used to treat diseases selected from the group consisting of: melanoma (e.g., metastatic malignant melanoma), kidney cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone-insensitive prostate adenocarcinoma), breast cancer, colon cancer, and lung cancer (e.g., non-small cell lung cancer). Other cancers include: bone cancer, pancreatic cancer, skin cancer, head or neck cancer, malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastrointestinal cancer, 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, cancers of the endocrine system, thyroid cancer, parathyroid carcinoma, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, and chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia). Leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, vegetations / tumors of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers (including those induced by asbestos), and combinations thereof. Metastatic cancers, especially those expressing PD-L1.

[0073] In another preferred embodiment, the pharmaceutical composition is used in a combination therapy regimen, preferably including: a combination chemotherapy regimen, other tumor immunotherapy agents (small molecule compounds and antibodies, etc.), a radiotherapy regimen, a tumor-targeted drug, and a tumor vaccine (such as human papillomavirus (HPV), hepatitis virus (HBV and HCV) and Kaposi's herpes sarcoma virus (KHSV)).

[0074] In another preferred embodiment, the pharmaceutical composition is used alone or in combination for the treatment of patients exposed to specific toxins or pathogens. These include, but are not limited to, treatments for various viruses, pathogenic bacteria, pathogenic fungi, pathogenic parasites, etc., such as established infections caused by pathogens like HIV, hepatitis viruses (A, B, and C), influenza viruses, herpes viruses, Giardia lamblia, malaria, Leishmaniasis, Staphylococcus aureus, and Pseudomonas aeruginosa.

[0075] In another preferred embodiment, the pharmaceutical composition is used to induce a therapeutic autoimmune response.

[0076] In another preferred embodiment, the pharmaceutical composition is used to treat patients with inappropriate accumulations of other autoantigens, such as amyloid deposits, including Aβ in Alzheimer's disease, cytokines such as TNFα and IgE.

[0077] In a fourth aspect, the present invention provides a PD-1 / PD-L1 inhibitor comprising the compound described in the first aspect of the present invention, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof.

[0078] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Detailed Implementation

[0079] Through long-term and in-depth research, the inventors designed and synthesized a novel class of PD-1 small molecule inhibitors. Based on this, the inventors completed this invention.

[0080] the term

[0081] Unless otherwise defined, 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 pertains.

[0082] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 5% from the enumerated value. For example, as used herein, the expression “about 100” includes all values ​​between 95 and 105 (e.g., 95.1, 95.2, 95.3, 95.4, etc.).

[0083] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0084] definition

[0085] As used herein, the term "alkyl" includes straight-chain or branched alkyl groups. For example, C1-C8 alkyl groups refer to straight-chain or branched alkyl groups having 1-8 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, etc.

[0086] As used herein, the term "alkenyl" includes straight-chain or branched alkenyl groups. For example, C2-C6 alkenyl refers to straight-chain or branched alkenyl groups having 2-6 carbon atoms, such as vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, or similar groups.

[0087] As used herein, the term "alkynyl" includes straight-chain or branched alkynyl groups. For example, C2-C6 alkynyl refers to straight-chain or branched alkynyl groups having 2-6 carbon atoms, such as ethynyl, propynyl, butynyl, or similar groups.

[0088] As used herein, the term "C3-C8 cycloalkyl" refers to a cycloalkyl group having 3-8 carbon atoms. It can be monocyclic, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or similar groups. It can also be bicyclic, such as bridged or spirocyclic forms.

[0089] As used herein, the term “C1-C8 alkoxy” refers to a straight-chain or branched alkoxy group having 1 to 8 carbon atoms; for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, etc.

[0090] As used herein, the term "3-10 membered heterocyclic alkyl group 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, of which 1-3 atoms are heteroatoms selected from the group consisting of N, S, and O. It can be monocyclic or bicyclic, such as a bridged or spirocyclic form. Specific examples include oxetane, azabutane, tetrahydro-2H-pyranyl, piperidinyl, tetrahydrofuranyl, morpholinyl, and pyrrolidinyl, etc.

[0091] As used in this article, the term "C6-C" 10 "Aryl" refers to an aryl group having 6-10 carbon atoms, such as phenyl or naphthyl groups.

[0092] As used herein, the term "5-10 membered heteroaryl group having 1-3 heteroatoms selected from the group consisting of N, S, and O" refers to a cyclic aromatic group having 5-10 atoms, of which 1-3 atoms are heteroatoms selected from the group consisting of N, S, and O. It can be monocyclic or fused-ring. Specific examples include pyridinyl, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrroleyl, pyrazolyl, imidazoleyl, (1,2,3)-triazolyl and (1,2,4)-triazolyl, tetrazolyl, furanyl, thiopheneyl, isoxazolyl, thiazolyl, oxazolyl, etc.

[0093] Unless otherwise specified, the functional groups of this invention may be substituted by substituents selected from the group consisting of: halogen, nitrile, nitro, hydroxyl, amino, C1-C6 alkyl-amino, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, halo-C1-C6 alkyl, halo-C2-C6 alkenyl, halo-C2-C6 alkynyl, halo-C1-C6 alkoxy, allyl, benzyl, C6-C 12 Aryl, C1-C6 alkoxy-C1-C6 alkyl, C1-C6 alkoxy-carbonyl, phenoxycarbonyl, C2-C6 alkynyl-carbonyl, C2-C6 alkenyl-carbonyl, C3-C6 cycloalkyl-carbonyl, C1-C6 alkyl-sulfonyl, etc.

[0094] 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 by an atom selected from F, Cl, Br, and I.

[0095] Unless otherwise specified, the structural formulas described in this invention are intended to include all isomers (such as enantiomers, diastereomers, and geometric isomers (or conformational isomers)): for example, R and S configurations containing an asymmetric center, (Z) and (E) isomers with double bonds, etc. Therefore, any single stereochemical isomer of the compounds of this invention, or a mixture of its enantiomers, diastereomers, or geometric isomers (or conformational isomers), is within the scope of this invention.

[0096] As used herein, the term "tautomer" refers to structural isomers with different energies that can cross a low energy barrier and thus interconvert. For example, proton tautomers (i.e., proton shifts) include interconversion via proton migration, such as 1H-indazole and 2H-indazole. Valence tautomers include interconversion via some bonding electron recombination.

[0097] As used herein, the term "solvent complex" refers to a complex of the compound of the present invention coordinated with solvent molecules in a specific ratio.

[0098] Preparation of Compound I

[0099] The present invention also provides a method for preparing compounds as described in the first aspect of the invention. The synthesis of compound Ia is shown below, and other compounds of formula I can be synthesized similarly. The synthesis comprises the following steps: in a suitable solvent, compounds I-1 and I-2 react in the presence of a base and a phosphine compound under the action of a palladium catalyst to give compound I-3; simultaneously, in a suitable solvent, compounds I-4 and I-5 react in the presence of an acid to give compound I-6; then, in a suitable solvent, compounds I-3 and I-6 react in the presence of a base and a phosphine compound under the action of a palladium catalyst to give compound Ia.

[0100]

[0101] Pharmaceutical Compositions and Administration

[0102] Because the compounds of the present invention have excellent PD-1 inhibitory activity, the compounds of the present invention and their various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, and pharmaceutical compositions containing the compounds of the present invention as the main active ingredient can be used for the prevention and / or treatment of diseases (e.g., cancer) related to the PD-1 / PD-L1 signaling pathway.

[0103] The pharmaceutical compositions of the present invention comprise the compound of the present invention within a safe and effective range and a pharmaceutically acceptable excipient or carrier. "Safe and effective range" refers to an amount of the compound sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, more preferably, 10-200 mg of the compound of the present invention per dose. Preferably, "one dose" is one capsule or tablet.

[0104] "Pharmaceutically acceptable carriers" refer to one or more compatible solid or liquid fillers or gelling substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with and with the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), and emulsifiers (such as Tween). Wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0105] There are no particular limitations on the administration of the compounds or pharmaceutical compositions of the present invention. Representative administration methods include (but are not limited to): oral administration, parenteral administration (intravenous, intramuscular injection, inhalation, or subcutaneous administration).

[0106] 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 components: (a) fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol, and silica; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain complex silicates, and sodium carbonate; (e) slowing agents, such as paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. Buffers may also be included in capsules, tablets, and pills.

[0107] Solid dosage forms such as tablets, sugar pills, capsules, pellets, and granules can be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opacifying agents, and the release of the active compound or compound from such compositions can be delayed in a portion of the digestive tract. Examples of encapsulating components that can be used are polymeric substances and waxes. If necessary, the active compound may also be formed into microcapsules with one or more of the excipients described above.

[0108] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, e.g., ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures of these substances.

[0109] In addition to these inert diluents, the composition may also contain auxiliaries such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and fragrances.

[0110] In addition to the active compound, the suspension may contain suspending agents such as ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitol esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0111] 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 non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0112] The compounds of this invention can be administered alone or in combination with other pharmaceutically acceptable compounds.

[0113] When administered in combination, the pharmaceutical composition further includes one or more other pharmaceutically acceptable compounds. One or more of these other pharmaceutically acceptable compounds may be administered simultaneously, separately, or sequentially with the compounds of the present invention.

[0114] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to the mammal (such as a human) requiring treatment. The dosage administered is the pharmaceutically considered effective dose. For a person weighing 60 kg, the daily dose is typically 1–2000 mg, preferably 20–500 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skills of a skilled physician.

[0115] In a preferred embodiment of the present invention, the pharmaceutical composition can be used for:

[0116] (1) Used to treat various tumors, including but not limited to melanoma (e.g., metastatic malignant melanoma), kidney cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone-insensitive prostate adenocarcinoma), breast cancer, colon cancer, and lung cancer (e.g., non-small cell lung cancer). Bone cancer, pancreatic cancer, skin cancer, head or neck cancer, malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastrointestinal cancer, 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, cancers of the endocrine system, thyroid cancer, parathyroid carcinoma, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia). Leukemia, chronic lymphocytic leukemia, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, vegetations / tumors of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers (including those induced by asbestos), and combinations thereof. Metastatic cancers, especially those expressing PD-L1.

[0117] (2) For use in combination therapy regimens, such as combination chemotherapy regimens, other tumor immunotherapies (small molecule compounds and antibodies, etc.), radiotherapy regimens, tumor targeted drugs, tumor vaccines, etc., such as human papillomavirus (HPV), hepatitis viruses (HBV and HCV) and Kaposi's herpes sarcoma virus (KHSV). It can be administered before, after or simultaneously with the said drugs, or it can be administered in combination with other known therapies.

[0118] (3) Used alone or in combination for the treatment of patients exposed to specific toxins or pathogens. This includes, but is not limited to, the treatment of various viruses, pathogenic bacteria, pathogenic fungi, pathogenic parasites, etc., such as established infections caused by pathogens such as HIV, hepatitis viruses (A, B, C), influenza virus, herpesvirus, Giardia, malaria, Leishmania, Staphylococcus aureus, Pseudomonas aeruginosa, etc.

[0119] (4) Used to induce a therapeutic autoimmune response to treat patients with inappropriate accumulation of other autoantigens, such as amyloid deposits, including Aβ in Alzheimer's disease, cytokines such as TNFα and IgE.

[0120] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages are by weight, and reagent ratios used in column chromatography purification are by volume.

[0121] Unless otherwise specified, all experimental materials and reagents used in the following examples are available from commercial sources or can be synthesized using methods known in the art.

[0122] Synthesis of intermediate A:

[0123] N-(3-Bromo-2-chlorophenyl)-2-(bromomethyl)thiazo[4,5-c]pyridine-4-amine

[0124]

[0125] 3-Bromo-2-chloroaniline

[0126]

[0127] 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 °C for 4 hours. The reaction mixture was filtered and concentrated under reduced pressure. The residue was purified by normal-phase column chromatography (petroleum ether:ethanol = 20:1) to give the title compound 3-bromo-2-chloroaniline (22 g, 84.6%) as a pale yellow liquid. MS (ESI): m / z = 207.9 [M+H] + .

[0128] N-(3-Bromo-2-chlorophenyl)-2-(methoxymethyl)thiazo[4,5-c]pyridine-4-amine

[0129]

[0130] A solution of 3-bromo-2-chloroaniline (30 g, 145.6 mmol), N-(3-bromo-2-chlorophenyl)-2-(methoxymethyl)thiazo[4,5-c]pyridine-4-amine (29 g, 133.6 mmol), and p-toluenesulfonic acid (9 g, 52.3 mmol) in 2-pentanol (15 mL) was added to a sealed tube and stirred at 100 °C for 24 hours. The reaction mixture was then added to water (400 mL), and extracted three times with dichloromethane / methanol (110 mL, v / v = 10 / 1). The organic phase was washed successively with water and 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 [M+H]+ .

[0131] N-(3-Bromo-2-chlorophenyl)-2-(bromomethyl)thiazo[4,5-c]pyridine-4-amine

[0132]

[0133] A solution (23 g, 59.8 mmol) of hydrobromic acid in acetic acid (150 mL) was stirred at 80 °C for 15 hours. The reaction solution was neutralized with potassium carbonate in an ice bath, and the precipitated solid was slurried with a mixture of petroleum ether and ethyl acetate (100 mL: 10 mL) to give title compound A (24 g, 93%) as a yellow solid. MS (ESI): m / z = 433.8 [M+H] + .

[0134] Synthesis of intermediate B:

[0135] 2,2'-(2-chloro-1,3-phenylene)bis(4,4,5,5-tetramethyl-1,3,2-dioxoboronylcyclohexane)

[0136]

[0137] 2,2'-(2-chloro-1,3-phenylene)bis(4,4,5,5-tetramethyl-1,3,2-dioxoboronylcyclohexane)

[0138]

[0139] Under nitrogen protection, 2-chloro-1,3-2-bromobenzene (25 g, 92.4 mmol), pinacol diboronate (58.5 g, 231 mmol), [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride·dichloromethane (10 g, 12.2 mmol), and potassium acetate (36.3 g, 370 mmol) were mixed thoroughly, and 1,4-dioxane (400 mL) was added. The reaction mixture was stirred at 80 °C 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 = 10:1) to give title compound B (21 g, 62.3%) as a white solid. MS (ESI): m / z = 365.2 [M+H] + .

[0140] Synthesis of intermediate C:

[0141] 6-Chloro-2-(difluoromethoxy)-4-methylnicotine aldehyde

[0142]

[0143] Methyl 2,6-dichloro-4-methylnicotinic acid ester

[0144]

[0145] Potassium carbonate (90.4 g, 655.3 mmol) was added to a solution of 2,6-dichloro-4-methylnicotine (90 g, 436.9 mmol) in N,N-dimethylformamide (0.5 L). The reaction mixture was cooled to 0 °C in an ice bath, and iodomethane (186 g, 1.31 mol) was slowly added dropwise. The ice bath was removed, and the mixture was allowed to warm to room temperature naturally before stirring for 13 hours. Water (1 L) was added to the reaction mixture, followed by extraction three times with ethyl acetate (1 L). The organic phase was washed four times (800 mL) with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give crude compound C-1 (105 g, 99%) as a white solid. MS (ESI): m / z = 219.9 [M+H] + .

[0146] 1 H NMR (400MHz, CDCl3) δ7.16 (d, J = 0.6Hz, 1H), 3.97 (s, 3H), 2.35 (d, J = 0.5Hz, 3H).

[0147] Methyl 6-chloro-2-methoxy-4-methylnicotinic acid ester

[0148]

[0149] Under ice bath conditions, 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 900 mL of dichloromethane. The mixture was stirred, and the reaction was allowed to proceed at room temperature for 6 hours after removing the ice bath. Liquid chromatography-mass spectrometry (LC-MS) detected residual starting material. Sodium methoxide solid (4.3 g, 79.64 mmol) was added to the reaction solution, and stirring continued for 13 hours. Sodium methoxide solid (4.3 g, 79.64 mmol) was added again, and the reaction proceeded for another 6 hours. LC-MS detected complete disappearance of the starting material. The reaction solution was poured into a 500 mL aqueous solution of sodium bicarbonate and extracted three times with 1 L of dichloromethane. 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 [M+H] + .

[0150] 1 H NMR (400MHz, CDCl3) δ6.81-6.78(m,1H),3.96(s,3H),3.91(s,3H),2.29(s,3H).

[0151] Methyl 6-chloro-2-hydroxy-4-methylnicotinic acid ester

[0152]

[0153] C-2 (87.4 g, 405.4 mmol) was added to a 33% hydrobromic acid solution in acetic acid (450 mL) and stirred at 60 °C for 30 min. The reaction solution was concentrated to remove some of the hydrogen bromide and acetic acid. The residue was adjusted to pH 4-5 with sodium bicarbonate solution in an ice bath, filtered, and the filter cake was purified by column chromatography (dichloromethane:methanol = 100:1) to give the title compound C-3 (57 g, 69.6%) as a white solid. MS (ESI): m / z = 201.9 [M+H] + .

[0154] Methyl 6-chloro-2-(difluoromethoxy)-4-methylnicotinic acid ester

[0155]

[0156] 60% sodium hydride (13.4 g, 334.8 mmol) was added to acetonitrile (270 mL), and stirred at room temperature for 10 minutes. Then, C-3 (27 g, 133.9 mmol) was added, and stirring continued for 30 minutes. 2-Fluorosulfonyl difluoroacetic acid (40.5 g, 227.7 mmol) was rapidly injected into the reaction solution, causing the temperature to rise rapidly to above 50°C. After the addition was complete, stirring continued for one hour until the reaction solution returned to room temperature. Liquid chromatography-mass spectrometry (LC-MS) detected the disappearance of 90% of the starting material. The reaction solution was slowly poured into an ice-bath-cooled saturated sodium bicarbonate aqueous solution (1.5 L) for quenching, while stirring. Extraction was performed three times with ethyl acetate (1 L). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated. 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), a yellow oil. MS(ESI): m / z = 251.9 [M+H] + .

[0157] (6-chloro-2-(difluoromethoxy)-4-methylpyridin-3-yl)methanol

[0158]

[0159] At -60°C, a toluene solution of diisobutylaluminum hydride (1.5 mol / L, 159 mL, 238.5 mol) was added dropwise to a solution of C-4 (30 g, 119.2 mmol) in dichloromethane (300 mL). After the addition was complete, the reaction mixture was slowly heated to room temperature over two hours, and the reaction was confirmed to be complete by liquid chromatography-mass spectrometry. The reaction mixture was quenched with an aqueous solution of potassium sodium tartrate (500 mL) and filtered through diatomaceous earth. The solid on the diatomaceous earth was dissolved in dilute hydrochloric acid and extracted three times with dichloromethane (300 mL). The organic phase was washed twice with an aqueous solution of sodium hydroxide (0.5 mol / L, 250 mL), 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 [M+H] +.

[0160] 6-Chloro-2-(difluoromethoxy)-4-methylnicotine aldehyde

[0161]

[0162] Add Dys-Martin oxidant (52 g, 122.76 mmol) to a solution of C-5 (18.3 g, 81.84 mmol) in 200 mL of dichloromethane under ice bath conditions, and stir at room temperature for three hours. Quench the reaction mixture in an aqueous solution of sodium bicarbonate (300 mL), filter with diatomaceous earth, wash with 200 mL of dichloromethane, dry the organic phase with anhydrous sodium sulfate, filter, and concentrate the filtrate. Purify the residue by column chromatography (petroleum ether:ethyl acetate = 1:9) to give title compound C (15.3 g, 84.6%) as a white solid.

[0163] 1 H NMR (400MHz, CDCl3) 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).

[0164] Synthesis of intermediate D:

[0165] (S)-1-((3-chloro-5-aldehyde-6-methoxypyridine)-2-oxo)-2,3-dihydro-1H-inden-4-trifluoromethanesulfonate

[0166]

[0167] 4-(tert-butyldimethylsiloxy)-2,3-dihydro-1H-inden-1-one

[0168]

[0169] 4-Hydroxy-2,3-dihydro-1H-inden-1-one (20.54 g, 138.8 mmol), tert-butyldimethylchlorosilane (31.45 g, 208.7 mmol), and imidazole (18.92 g, 278.2 mmol) were dissolved in 1,4-dioxane (300 mL). The reaction mixture was stirred at 20 °C for 16 hours. After the reaction was detected by liquid chromatography-mass spectrometry, the pH of the reaction mixture was adjusted to neutral with 1 mol / L hydrochloric acid and diluted with water (100 mL). The mixture was extracted three times with ethyl acetate (50 mL), and the organic phase was dried over anhydrous sodium sulfate and concentrated. The residue was purified by column chromatography (petroleum ether:ethyl acetate = 50:1) to give the title compound D-1 (35 g, >99%) as a colorless oil. MS (ESI): m / z = 263.1 [M+H] + .

[0170] (S)-4-(tert-butyldimethylsiloxy)-2,3-dihydro-1H-inden-1-ol

[0171]

[0172] (R)-2-methyl-CBS-oxazolamide (1.61 g, 5.81 mmol) was dissolved in toluene (3 mL). Borane dimethyl sulfide (10 mol / L, 21 mL) was added to the above solution. The reaction mixture was stirred at room temperature for 30 minutes, then dichloromethane (40 mL) was added, and the mixture was cooled to -20 °C. A dichloromethane (40 mL) solution of D-1 (10 g, 38.16 mmol) was added dropwise. The reaction mixture was stirred between -15 °C and -5 °C for 3 hours. After the reaction was completed, the reaction mixture was quenched with methanol (40 mL), concentrated, and purified by column chromatography (petroleum ether:dichloromethane = 10:1) to give D-2 (8.29 g, 82%) as a white solid. MS (ESI): m / z = 247.1 [M-OH] + .

[0173] 1 H NMR(CDCl3,400MHz)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).

[0174] (S)-6-(4-tert-butyldimethylsiloxy)-2,3-dihydro-1H-inden-1-oxo-2-methoxynicotinaldehyde

[0175]

[0176] 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 gas was continuously bubbled into the solution for 10 minutes. Palladium acetate (0.36 g, 1.61 mmol), 2-di-tert-butylphosphino-2',4',6'-triisopropylbiphenyl (1.33 g, 3.14 mmol), and cesium carbonate (10.21 g, 31.41 mmol) were added. The reaction mixture was stirred at 35 °C for 48 hours. The reaction mixture 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] + .

[0177] 1H NMR (CDCl3, 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).

[0178] (S)-6-(4-hydroxy)-2,3-dihydro-1H-inden-1-oxo-2-methoxynicotinaldehyde

[0179]

[0180] D-3 (2.79 g, 6.99 mmol) was dissolved in tetrahydrofuran (50 mL). The reaction solution was cooled to -78 °C, and a tetrahydrofuran solution of tetrabutylammonium fluoride (1 mol / L, 7.0 mL) was added dropwise. The reaction solution was allowed to slowly return to room temperature. After the reaction was complete, the reaction was quenched with acetic acid (460 mg). The organic phase was separated in a mixture of ethyl acetate / saturated citric acid (30 mL / 40 mL), and then extracted three times with ethyl acetate (50 mL). 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 give D-4 (1.78 g, 89%) as a white solid. MS (ESI): m / z = 308.1 [M+23] + .

[0181] 1 H NMR (CDCl3, 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),3.1 5-3.07(m,1H),2.93-2.86(m,1H),2.74-2.64(m,1H),2.31-2.23(m,1H).

[0182] (S)-1-((5-aldehyde-6-methoxypyridine)-2-oxo)-2,3-dihydro-1H-inden-4-trifluoromethanesulfonate

[0183]

[0184] 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), and 4-dimethylaminopyridine (77 g, 0.63 mmol) in dichloromethane (40 mL) was cooled to -78 °C, and trifluoromethanesulfonic anhydride (2.03 g, 7.20 mmol) was added dropwise. The reaction mixture was stirred for 15 minutes, then removed from the dry ice bath and allowed to warm to room temperature with stirring for 1 hour. A saturated citric acid solution (50 mL) was added to the reaction mixture, and the layers were separated. The aqueous layer was extracted twice with ethyl acetate (20 mL). 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 [M+23] + .

[0185] (S)-1-((3-chloro-5-aldehyde-6-methoxypyridine)-2-oxo)-2,3-dihydro-1H-inden-4-trifluoromethanesulfonate

[0186]

[0187] D-5 (200 mg, 0.48 mmol) was dissolved in a mixture of dichloromethane / N,N-dimethylformamide (4 mL / 2 mL). Trifluoroacetic acid (11 mg, 0.10 mmol) and Palau'chlor (CBMG) (82 mg, 0.39 mmol) were added to the solution. The reaction mixture was stirred at room temperature for 16 hours. The reaction was quenched by adding saturated sodium thiosulfate (2 mL) and sodium bicarbonate (4 mL), and extracted three times with dichloromethane (5 mL). The organic phase was dried over anhydrous sodium sulfate, concentrated, and 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 [M+23] + .

[0188] 1 H NMR(CDCl3,400MHz)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).

[0189] Synthesis of intermediate E:

[0190]

[0191] Methyl 6-chloro-2-(fluoromethoxy)-4-methylnicotinic acid ester

[0192]

[0193] At room temperature, C-3 (500 mg, 2.48 mmol) was dissolved in acetonitrile (5 mL) in a 20 mL microwave-safe tube. 60% sodium hydride (200 mg, 4.96 mmol) was added, the tube was sealed, and nitrogen gas was purged. A nitrogen balloon was inserted, and the reaction mixture was stirred at room temperature for 30 minutes. Then, fluorobromomethane (0.32 mL, 4.96 mmol) was added, and the reaction mixture was stirred at 40 °C for 24 hours. Liquid chromatography showed a 30% conversion of the starting material. After cooling to room temperature, fluorobromomethane (0.5 mL, 7.8 mmol) was added, and the reaction was continued at 40 °C for another 24 hours. Liquid chromatography showed an 80% conversion of the starting material. After cooling to room temperature, the reaction mixture was poured into 30 mL of methanol, stirred thoroughly, filtered, washed with 20 mL of methanol, and the filtrate was concentrated to give 800 mg of crude E-1 (88% purity). MS (ESI): m / z = 234.2, 236.2 [M+H].

[0194] (6-chloro-2-(fluoromethoxy)-4-methylpyridin-3-yl)methanol

[0195]

[0196] At -60°C, a 1.5 mol / L toluene solution of diisobutylaluminum hydride (3.3 mL, 4.96 mmol) was added dropwise to a 10 mL solution of dichloromethane containing E-1 (579.3 mg, 2.48 mmol). After the addition was complete at -60°C, the reaction mixture was slowly heated to room temperature over two hours, and the reaction was confirmed to be complete by liquid chromatography. The reaction mixture was quenched with 50 mL of saturated sodium potassium tartrate solution in an ice bath, and 30 mL of dichloromethane was added. After stirring at room temperature for two hours, the mixture was extracted twice with dichloromethane (30 mL). The organic phase was concentrated, and the residue was column chromatography (ethyl acetate: petroleum ether = 0–30%) to give 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] + .

[0197] 6-Chloro-2-(fluoromethoxy)-4-methylnicotine aldehyde

[0198]

[0199] Under ice bath conditions, a solution of E-2 (330 mg, 1.62 mmol) in 20 mL of dichloromethane was added with Dys-Martin oxidant (1 g, 2.43 mmol). The reaction mixture was stirred at room temperature for half an hour, and the reaction was monitored by TLC until complete. Under ice bath conditions, the reaction mixture was quenched with 50 mL of sodium bicarbonate aqueous solution, adjusted to neutral, and extracted three times with 50 mL of dichloromethane. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The concentrate was passed through a normal-phase silica gel column and eluted with a solvent (ethyl acetate: petroleum ether = 0–20%) 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] + .

[0200] 1 H NMR (400MHz, CDCl3) δ10.51(s,1H),6.99(s,1H),6.22(s,1H),6.09(s,1H),2.62(s,3H).

[0201] Synthesis of intermediate F:

[0202]

[0203] 5-Bromo-2-chloro-4-cyclopropylpyridine

[0204]

[0205] Under nitrogen protection, 5-bromo-2-chloro-4-iodopyridine (5 g, 15.7 mmol), cyclopropylboronic acid (1.48 g, 17.2 mmol), sodium carbonate (4.99 g, 47.1 mmol), and Pd(dppf)Cl2 (574 mg, 0.785 mmol) were mixed thoroughly, followed by the addition of 1,4-dioxane (40 mL) and water (10 mL). The mixture was heated to 100 °C and stirred for 16 hours. The reaction was confirmed by LC-MS. The reactants were cooled to room temperature and filtered. The filtrate was concentrated and purified by rapid silica gel column chromatography (ethyl acetate: petroleum ether = 0–20%) to give a white solid product (1.3 g, yield 35.6%). MS (ESI): m / z = 233.9 [M+H] + .

[0206] 2-Chloro-4-cyclopropyl-5-vinylpyridine

[0207]

[0208] Compound F-1 (1.1 g, 4.73 mmol) was dissolved in a microwave tube in 1,4-dioxane (10 mL) and water (2 mL). Then, potassium vinyltrifluoroborate (760 mg, 5.67 mmol), potassium carbonate (1.63 g, 11.8 mmol), and Pd(dppf)Cl2 (173 mg, 0.236 mmol) were added. The mixture was then purged under nitrogen protection and heated to 80 °C with stirring for 16 hours. The reaction mixture was cooled to room temperature and filtered. The filtrate was concentrated and purified by rapid silica gel column chromatography (ethyl acetate: petroleum ether = 0–2%) to give a white oily product (180 mg, yield 21.1%). MS (ESI): m / z = 179.9 [M+H] + .

[0209] 6-Chloro-4-cyclopropylnicotinaldehyde

[0210]

[0211] Compound F-2 (180 mg, 1.00 mmol) was dissolved in tetrahydrofuran (2 mL) and water (0.5 mL), followed by the addition of potassium osmium tetroxide dihydrate (18 mg, 0.05 mmol). The mixture was cooled to 0 °C in an ice bath, and then sodium periodate (643 mg, 3.00 mmol) was added under nitrogen protection. The mixture was stirred at 0 °C for half an hour, then heated to room temperature and stirred for another 2 hours. LC-MS analysis confirmed the reaction was complete. Water (30 mL) was added to the reactants, and the mixture was extracted twice with ethyl acetate (30 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to give a yellow oily product (199 mg, 100% yield). MS (ESI): m / z = 182.1 [M+H] + .

[0212] 1 H NMR (400MHz, CDCl3) δ10.37(s,1H),8.68-8.65(m,1H),6.89(s,1H),2.95-2.93(m,1H),1.28-1.25(m,2H),0.93-0.88(m,2H).

[0213] Synthesis of intermediate G:

[0214]

[0215] 6-Chloro-2-ethylnicotinaldehyde

[0216]

[0217] Under nitrogen protection, 2,6-dichloronicotinaldehyde (10 g, 56.8 mmol), potassium carbonate (15.7 g, 113.6 mmol), ethylboric acid (4.6 g, 62.4 mmol), and PdCl₂dppf (2 g, 2.84 mmol) were mixed thoroughly. 1,4-dioxane (50 mL) and water (10 mL) were added, and the mixture was reacted at 90 °C for 5 hours. Liquid chromatography analysis showed the reaction was complete. The reaction solution was concentrated, and 150 mL of water was added. Extraction was performed three times with 200 mL of ethyl acetate. The extract was dried over anhydrous sodium sulfate, filtered, and concentrated. The crude product was purified by column chromatography with petroleum ether / ethyl acetate (0–8%) to give 500 mg of a colorless oily product (purity: 90%, yield: 10.3%). MS (ESI): m / z = 169.9 [M+H] + .

[0218] 1 H NMR (400MHz, CDCl3) 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).

[0219] Synthesis of intermediate H:

[0220]

[0221] Ethyl 6-chloro-4-methoxy-2-methylnicotinic acid ester

[0222]

[0223] Sodium methoxide (1.15 g, 21.36 mmol) and cuprous iodide (419 mg, 2.2 mmol) were added to a tetrahydrofuran solution (50 mL) of ethyl 4,6-dichloro-2-methylnicotinic acid ester (5 g, 21.36 mmol). After purging with nitrogen, the reaction mixture was reacted at 70 °C for 3 h, and the reaction was confirmed to be complete by liquid chromatography. The reaction solution was concentrated and purified by column chromatography with petroleum ether / ethyl acetate (0–10%) to give 3 g of a colorless oily intermediate H-1 (purity: 90%, yield: 61.1%). MS (ESI): m / z = 230.1 [M+H] + .

[0224] (6-chloro-4-methoxy-2-methylpyridin-3-yl)methanol

[0225]

[0226] Intermediate H-1 (2 g, 8.73 mmol) was dissolved in dichloromethane (100 mL), purged with nitrogen, and cooled to -70 °C. A toluene solution of diisobutylaluminum hydride (11.7 mL, 17.5 mmol) was added dropwise. The reaction was allowed to slowly rise to room temperature over two hours, and liquid chromatography-mass spectrometry (LC-MS) confirmed the reaction was complete. A saturated potassium sodium tartrate solution (50 mL) was added to the reaction mixture and stirred for one hour. The mixture was then extracted three times with dichloromethane (100 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and column-sected with petroleum ether / ethyl acetate (0–30%) to give 1.5 g of a white solid intermediate H-2 (purity: 90%, yield: 91.6%). MS (ESI): m / z = 187.9 [M+H] + .

[0227] 6-Chloro-4-methoxy-2-methylnicotine aldehyde

[0228]

[0229] Under ice bath conditions, 5.1 g (12 mmol) of Desmond reagent was added to a solution of 1.5 g (8 mmol) of H₂ in 30 mL of dichloromethane. The reaction mixture was stirred at room temperature for 30 minutes, and the reaction was monitored by TLC to ensure complete reaction of the starting material. The pH of the reaction mixture was adjusted to weakly alkaline with 50 mL of sodium bicarbonate solution. After three extractions with 80 mL of dichloromethane, the mixture was dried and filtered. The organic phase was purified by normal-phase silica gel chromatography (petroleum ether: ethyl acetate = 0–20%) to give 1.12 g of a white solid product (purity: 90%, yield: 75.4%). MS (ESI): m / z = 186.2 [M+H] + .

[0230] 1 H NMR (400MHz, CDCl3) δ 10.53 (d, J = 1.1 Hz, 1H), 6.85 (s, 1H), 3.98 (d, J = 1.1 Hz, 3H), 2.73 (s, 3H).

[0231] Synthesis of intermediate K:

[0232]

[0233] Ethyl 4-methyl-2-vinylnicotinic acid ester

[0234]

[0235] Ethyl 2-bromo-4-methylnicotinic acid ester (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). Then, Pd(dppf)Cl2 (794 mg, 1.08 mmol) and potassium carbonate (7.5 g, 54.2 mmol) were added. The mixture was then microwaved at 90°C for 90 minutes under nitrogen protection. The reaction mixture was cooled to room temperature and filtered. The filtrate was concentrated and purified by rapid silica gel column chromatography (ethyl acetate: petroleum ether = 0–15%) to give a colorless oily product K-1 (4.17 g, 100% yield). MS (ESI): m / z = 192.0 [M+H] + .

[0236] Ethyl 2-ethyl-4-methylnicotinic acid ester

[0237]

[0238] Compound K-1 (4.17 g, 21.8 mmol) was dissolved in methanol (40 mL), followed by the addition of Pd / C (400 mg). The mixture was stirred at room temperature for 2 hours under a 15 psi H2 atmosphere. The reaction was detected as complete by LC-MS. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated to give a colorless oily product K-2 (4.13 g, 98.0% yield). MS (ESI): m / z = 194.0 [M+H] + .

[0239] 3-(ethyl ester <ethoxycarbonyl>)-2-ethyl-4-methylpyridine 1-oxide

[0240]

[0241] 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 room temperature for 16 hours, and the reaction was confirmed by LC-MS. The reactants were concentrated and purified by rapid silica gel column chromatography (methanol:dichloromethane = 0–10%) to give a colorless oily product K-3 (3.78 g, yield 84.8%). MS (ESI): m / z = 210.0 [M+H] + .

[0242] Ethyl 6-chloro-2-ethyl-4-methylnicotinic acid ester

[0243]

[0244] Compound K-3 (400 mg, 1.91 mmol) was dissolved in phosphorus oxychloride (2 mL), and then DIEA (0.33 mL, 1.91 mmol) was added. The mixture was heated to 120 °C and stirred for 2 hours. The reaction was confirmed to be complete by LCMS. After cooling the reaction solution to room temperature, the phosphorus oxychloride was removed by concentration. ， The product was then purified by rapid silica gel column chromatography (ethyl acetate: petroleum ether = 0–15%) to give a yellow oily product K-4 (120 mg, yield 52.7%). MS (ESI): m / z = 228.7 [M+H] + .

[0245] (6-Chloro-2-ethyl-4-methylpyridin-3-yl)methanol

[0246]

[0247] Compound K-4 (190 mg, 0.834 mmol) was dissolved in dichloromethane (2 mL) and cooled to -60 °C. Then, under nitrogen protection, a toluene solution of diisobutylaluminum hydride (1.5 mol / L, 1.7 mL, 2.50 mmol) was slowly added. The mixture was stirred at -60 °C for 1 hour. LC-MS analysis confirmed the reaction was complete. Potassium sodium tartrate solution (50 mL) was added to the reactants, and the mixture was stirred at room temperature for 1 hour. The mixture was extracted twice with dichloromethane (30 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to obtain a colorless oily product, K-5 (154 mg, 100% yield). MS (ESI): m / z = 185.9 [M+H] + .

[0248] 6-Chloro-2-ethyl-4-methylnicotine aldehyde

[0249]

[0250] Compound K-5 (154 mg, 0.836 mmol) was dissolved in dichloromethane (3 mL), and then Dysmart reagent (530 mg, 1.25 mmol) was added. The reaction mixture was stirred at room temperature for 1 hour, and the reaction was detected as complete by LC-MS. Saturated sodium sulfite solution (50 mL) was added to the reactants, and the mixture was extracted twice with dichloromethane (30 mL). The combined organic phases were washed with saturated sodium bicarbonate solution (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by rapid silica gel column chromatography (ethyl acetate: petroleum ether = 0–10%) to give a yellow oily product K (140 mg, yield 91.4%). MS (ESI): m / z = 184.2 [M+H] + .

[0251] 1H NMR (400MHz, CDCl3) 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).

[0252] Synthesis of intermediate J:

[0253]

[0254] Methyl 6-chloro-4-hydroxynicotinic acid ester

[0255]

[0256] Methyl 6-chloro-4-methoxynicotinic acid ester (3.7 g, 18.35 mmol) was dissolved in 200 mL of toluene, and anhydrous aluminum trichloride (7.34 g, 55.1 mmol) was added. The reaction mixture was stirred at 100 °C for 1 hour, and liquid chromatography showed that the reaction was complete. The reaction mixture was concentrated, and the residue was cooled in an ice bath and the pH was adjusted to weakly alkaline with sodium bicarbonate solution. It was extracted with 150 mL of dichloromethane, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by normal-phase silica gel column chromatography. The elution with solvent (ethyl acetate: petroleum ether = 0–20%) gave 1.8 g of red solid J-1 (purity: 90%, yield: 52.3%). MS (ESI): m / z = 187.9 [M+H] + .

[0257] Methyl 6-chloro-4-(difluoromethoxy)nicotinic acid ester

[0258]

[0259] 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). After nitrogen purging, the reaction mixture was stirred at 90 °C for 1 hour, and liquid chromatography showed that the reaction was complete. The reaction mixture was cooled and quenched with 50 mL of water, extracted with 100 mL of ethyl acetate, washed three times with saturated brine (80 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated and purified by normal-phase silica gel chromatography. Elution with a solvent (ethyl acetate: petroleum ether = 0–20%) yielded 1.1 g of red solid intermediate J-2 (purity: 90%, yield: 38.6%). MS (ESI): m / z = 237.9 [M+H] + .

[0260] (6-chloro-4-(difluoromethoxy)pyridin-3-yl)methanol

[0261]

[0262] At -60°C, a 1.5 mol / L toluene solution of diisobutylaluminum hydride (7.1 mL, 11.5 mmol) was added dropwise to a 50 mL solution of dichloromethane containing J-2 (1.1 g, 4.63 mmol). After the addition was complete at -60°C, the reaction mixture was slowly heated to room temperature over two hours, and the reaction was confirmed to be complete by liquid chromatography. The reaction mixture was quenched with 50 mL of potassium sodium tartrate aqueous solution, stirred at room temperature for one hour, and then extracted three times with 100 mL of dichloromethane. The organic phase was dried over anhydrous sodium sulfate and concentrated. The crude product was eluted with a normal-phase silica gel column using a solvent (ethyl acetate: petroleum ether = 0–40%) to give 670 mg of red oily intermediate J-3 (purity: 90%, yield: 69%). MS (ESI): m / z = 210.1 [M+H] + .

[0263] 6-Chloro-4-(difluoromethoxy)nicotinaldehyde

[0264]

[0265] Under ice bath conditions, 2 g of Des Martin oxidant (4.8 mmol) was added to a solution of J-3 (670 mg, 3.2 mmol) in 10 mL of dichloromethane. The reaction mixture was stirred at room temperature for 1 hour, and the reaction was monitored by TLC until complete. The reaction solution was quenched with 50 mL of saturated sodium bicarbonate solution, washed twice with 40 mL of dichloromethane and 50 mL of saturated sodium sulfite solution, and the organic phase was dried over anhydrous sodium sulfate. The mixture was then filtered and concentrated to give 500 mg of a white solid final product (purity: 90%, yield: 75.2%). 1 H NMR (400MHz, CDCl3) δ10.37(s,1H),8.86(s,1H),7.23(s,1H),6.98-6.60(m,1H).

[0266] 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) Thiazolo[4,5-c]pyridin-2-yl)methyl)amino)cyclohexane-1-carboxylic acid

[0267]

[0268] methyl(1r,4r)-4-(((4-(((3-bromo-2-chlorophenyl)amino)thiazo[4,5-c]pyridin-2-yl)methyl) (amino)cyclohexane-1-carboxylic acid ester

[0269]

[0270] Cesium carbonate (76.65 g, 235.26 mmol) was added to a mixture of tetrahydrofuran and acetonitrile (120 mL, 3 / 1) of methyl (1r,4r)-4-aminocyclohexane-1-carboxylic acid hydrochloride (30.38 g, 193.67 mmol) and stirred at room temperature for one hour. Then, compound A (34.00 g, 78.42 mmol) was added, and stirring continued at room temperature for 3 hours until the starting material was completely eliminated. The reaction mixture was diluted with dichloromethane (100 mL) and water (100 mL), separated, and the organic phase was washed successively with saturated ammonium chloride and brine, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by slurrying with a mixture of ethyl acetate and petroleum ether (1:5) to give the title compound 1-1 (35.00 g, 88%) as a pale yellow solid. MS (ESI): m / z = 509.1 [M+H] + .

[0271] Methyl(1r,4r)-4-(((4-((2,2'-dichloro-3'-(4,4,5,5-tetramethyl-1,3,2-dioxoboropentane-2- )-[1,1'-biphenyl]-3-yl)amino)thiazo[4,5-c]pyridin-2-yl)methyl)amino)cyclohexane-1-carboxylic acid ester

[0272]

[0273] Under nitrogen protection, 1-1 (8.02 g, 15.73 mmol), intermediate B (10.03 g, 27.53 mmol), tetraphenylphosphine palladium (0.91 g, 0.79 mmol), and anhydrous potassium carbonate (5.43 g, 39.33 mmol) were mixed thoroughly, and 1,4-dioxane (150 mL) and water (15 mL) were added. The mixture was stirred at 80 °C for 4 hours. After the reaction was complete, the reaction solution was filtered and concentrated. The residue was purified by normal-phase column chromatography (ethyl acetate: petroleum ether = 40:60) to give title compound 1-2 (7.00 g, 59%) as a pale yellow solid. MS (ESI): m / z = 667.2 [M+H] + .

[0274] Methyl(1r,4r)-4-(((4-((2,2'-dichloro-3'-(6-(difluoromethoxy)-5-formyl-4-methylpyridine) (P-2-yl)-[1,1'-biphenyl]-3-yl)amino)thiazo[4,5-c]pyridin-2-yl)methyl)amino)cyclohexane-1-carboxylic acid Ester acid acid.

[0275]

[0276] Under nitrogen protection, compounds 1-2 (6941 mg, 10.40 mmol), intermediate C (1773 mg, 8.00 mmol), tetraphenylphosphine palladium (462 mg, 0.40 mmol), and anhydrous potassium carbonate (2764 mg, 20.00 mmol) were mixed thoroughly. Then, 1,4-dioxane (15 mL) and water (1.5 mL) were added, and the mixture was stirred at 90 °C 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 (ethyl acetate: petroleum ether = 42:58) to give title compounds 1-3 (5235 mg, 83%) as a pale yellow solid. MS (ESI): m / z = 726.1 [M+H] + .

[0277] Methyl(1r,4r)-4-(((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)thiazo[4,5- c]pyridin-2-yl)methyl)amino)cyclohexane-1-carboxylic acid ester

[0278]

[0279] A mixture of 1–3 (5086 mg, 7.00 mmol) and (S)-5-(aminomethyl)pyrrolidine-2-one hydrochloride (2108 mg, 14.00 mmol) was added to a solution of N-ethyl-N-isopropylpropane-2-amine (904 mg, 7.00 mmol) in anhydrous dichloromethane (14 mL), and stirred at room temperature for 3 hours. Then, sodium triacetoxyborohydride (2968 mg, 14.00 mmol) was added in portions to the reaction mixture, and stirred at room temperature for 1 hour. The reaction was quenched with saturated sodium bicarbonate solution and diluted with ethyl acetate (500 mL). The mixture was filtered, and the filtrate was washed successively 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 = 5:95) and reverse-phase column chromatography (90% acetonitrile in ammonium bicarbonate aqueous solution) to give title compounds 1-4 (3400 mg, 58%) as pale yellow solids. MS (ESI): m / z = 824.2 [M+H] + .

[0280] (1r,4r)-4-(((4-((2,2'-dichloro-3'-(6-(difluoromethoxy)-4-methyl-5-(((((S)-5-carbonyl) Pyrrolidine-2-yl)methyl)amino)methyl)pyridin-2-yl)-[1,1'-biphenyl]-3-yl)amino)thiazo[4,5-c]pyridine (Pyridine-2-yl)methyl)amino)cyclohexane-1-carboxylic acid

[0281]

[0282] Compounds 1-4 (730 mg, 0.89 mmol) and 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 solution was neutralized with dilute hydrochloric acid (1 mol / L), filtered, and concentrated. The residue was purified by reversed-phase rapid separation column to give title compound 1 (400 mg, 55%) as a white solid. MS (ESI): m / z = 810.2 [M+H] + .

[0283] 1 H NMR (400MHz, DMSO-d6) δ8.90(d,J=8.4Hz,1H),8.64(s,1H),8.11(d,J=5.6Hz,1H),7.72(t,J=73Hz,1H),7.67(dd, J=7.8,1.6Hz,1H),7.65(s,1H),7.59(s,1H),7.56(dd,J=11.6,3.8Hz,2H),7.50-7.42(m,3H),7.05(dd,J=7.6,1.2 Hz,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),2.20-2.0 2(m,4H),1.96(d,J=10.6Hz,2H),1.88(d,J=11.6Hz,2H),1.74-1.62(m,1H),1.39-1.19(m,2H),1.18-1.03(m,2H).

[0284] Example 2: (1r,4r)-4-(((4-((2,2'-dichloro-3'-(6-methoxy-4-methyl-5-((((5-carbonylpyridine) (Pyrrolidone-2-yl)methyl)amino)methyl)pyridin-2-yl)-[1,1'-biphenyl]-3-yl)amino)thiazo[4,5-c]pyr (Pyridine-2-yl)methyl)amino)cyclohexane-1-carboxylic acid

[0285]

[0286] Methyl(1r,4r)-4-(((4-((2,2'-dichloro-3'-(5-formyl-6-methoxy-4-methylpyridine-2- )-[1,1'-biphenyl]-3-yl)amino)thiazo[4,5-c]pyridin-2-yl)methyl)amino)cyclohexane-1-carboxylic acid ester

[0287]

[0288] Under nitrogen protection, 1-1 (2000 mg, 3.92 mmol), 6-(2-chloro-3-(4,4,5,5-tetramethyl-1,3,2-dioxoboropentane-2-yl)phenyl)-2-methoxy-4-methylnicotine aldehyde (2279 mg, 5.88 mmol), tetraphenylphosphine palladium (231 mg, 0.20 mmol), and potassium carbonate (1355 mg, 9.80 mmol) were mixed thoroughly. 1,4-dioxane (20 mL) and water (2 mL) were added, and the mixture was stirred at 90 °C for 2 hours. After the reaction was complete, 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 dissolved in ethyl acetate (3 mL), then slowly added dropwise to stirred petroleum ether (30 mL). The mixture was filtered, the filter cake was washed with petroleum ether, and the dried filter cake yielded a crude product. This crude product was then slurried with methyl tert-butyl ether to give the title compound 2-1 (1260 mg, 46%) as a gray solid. MS (ESI): m / z = 690.2 [M+H] + .

[0289] Methyl(1r,4r)-4-(((4-((2,2'-dichloro-3'-(6-methoxy-4-methyl-5-((((5-carbonylpyrrole)) Alkyl-2-yl)methyl)amino)methyl)pyridin-2-yl)-[1,1'-biphenyl]-3-yl)amino)thiazo[4,5-c]pyridine- 2-yl)methyl)amino)cyclohexane-1-carboxylic acid ester

[0290]

[0291] A mixture of 2-1 (180 mg, 0.26 mmol) and 5-(aminomethyl)pyrrolidone-2-one (59 mg, 0.52 mmol) was added to a solution of glacial acetic acid in anhydrous dichloromethane (3 mL), and stirred at room temperature for 4 hours. Then, sodium triacetoxyborohydride (66 mg, 0.31 mmol) was added in portions to the reaction mixture, and the mixture was 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 was washed twice with water, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by normal-phase column chromatography (methanol:dichloromethane = 6:94) to give the title compound 2-2 (145 mg, 71%) as a pale yellow solid. MS (ESI): m / z = 788.1 [M+H] + .

[0292] (1r,4r)-4-(((4-((2,2'-dichloro-3'-(6-methoxy-4-methyl-5-((((5-carbonylpyrrolidine-2- (1,1'-Biphenyl]-3-yl)amino)thiazo[4,5-c]pyridin-2-yl) (Methyl)amino)cyclohexane-1-carboxylic acid

[0293]

[0294] Compound 2-2 (145 mg, 0.18 mmol) and lithium hydroxide monohydrate (30 mg, 0.72 mmol) were 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 solution was neutralized with dilute hydrochloric acid (1 mol / L) and concentrated. The residue was purified by reversed-phase column chromatography (85% methanol in 0.1% ammonium bicarbonate aqueous solution) to give title compound 2 (85 mg, 61%) as a white solid. MS (ESI): m / z = 774.1 [M+H] + .

[0295] 1 H NMR (400MHz, DMSO-d6) δ8.87(dd,J=8.4,1.4Hz,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=7.6,1.8Hz,1H),7.10(s,1H),7.02(dd,J=7.6,1 .6Hz,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,3 H),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).

[0296] Example 3: (S)-5-((((6-(2,2'-dichloro-3'-((2-methylthioazolo[4,5-c]pyridin-4-yl)amino (1,1'-Biphenyl)-3-yl)-2-(difluoromethoxy)-4-methylpyridin-3-yl)methyl)amino)methyl)pyrrolidine- 2-keto

[0297]

[0298] 6-(2-chloro-3-(4,4,5,5-tetramethyl-1,3,2-dioxoboropentane-2-yl)phenyl)-2-(difluoromethoxy)- 4-Methylnicotinaldehyde

[0299]

[0300] Under nitrogen protection, 6-chloro-2-(difluoromethoxy)-4-methylnicotine aldehyde (500 mg, 2.26 mmol), intermediate B (1.37 g, 3.76 mmol), tetrakis(triphenylphosphine)palladium (77 mg, 0.066 mmol), and potassium acetate (461 mg, 4.70 mmol) were mixed thoroughly, and 1,4-dioxane (10 mL) was added. The mixture was then stirred at 80 °C for 5 hours. The reaction solution was filtered and concentrated. The residue was purified by normal-phase column chromatography (petroleum ether:ethyl acetate = 100:4) to give the title compound 3-1 (900 mg, 62%, purity 66%) as a white solid. MS (ESI): m / z = 424.0 [M+H] + .

[0301] N-(3-Bromo-2-chlorophenyl)-2-methylthiozo[4,5-c]pyridine-4-amine

[0302]

[0303] A solution of intermediate A (500 mg, 1.15 mmol) in dimethyl sulfoxide (20 mL) was stirred at 0 °C and sodium borohydride (430 mg, 11.5 mmol) was added. The mixture was stirred at room temperature for 2 hours. The reaction solution was diluted with dichloromethane (100 mL), then washed with brine and water. The organic phase was concentrated. The residue was purified by normal-phase column chromatography (petroleum ether: ethyl acetate = 60:40) to give the title compound 3-3 (160 mg, 39%) as a yellow solid. MS (ESI): m / z = 356.0 [M+H] + .

[0304] 6-(2,2'-dichloro-3'-((2-methylthioazolo[4,5-c]pyridin-4-yl)amino)-[1,1'-biphenyl]-3- 2-(difluoromethoxy)-4-methylnicotine aldehyde

[0305]

[0306] Under nitrogen protection, 3-1 (248 mg, 0.59 mmol), 3-3 (160 mg, 0.45 mmol), tetrakis(triphenylphosphine)palladium (52 ​​mg, 0.045 mmol), and potassium carbonate (155 mg, 1.12 mmol) were mixed thoroughly. Then, 1,4-dioxane (5 mL) and water (1 mL) were added, and the mixture was stirred at 90 °C for 2 hours. The reaction solution was filtered and concentrated. The residue was purified by normal-phase column chromatography (petroleum ether:ethyl acetate = 84:16) to obtain the target product 3-2 (140 mg, 54%) as a yellow solid. MS (ESI): m / z = 571.1 [M+H] + .

[0307] (S)-5-((((6-(2,2'-dichloro-3'-((2-methylthiozo[4,5-c]pyridin-4-yl)amino)-[1,1'- [Biphenyl]-3-yl)-2-(difluoromethoxy)-4-methylpyridin-3-yl)methyl)amino)methyl)pyrrolidone-2-one

[0308]

[0309] A mixture of 3-2 (140 mg, 0.25 mmol) and (S)-5-(aminomethyl)pyrrolidone-2-one (56 mg, 0.49 mmol), acetic acid (0.1 mL), and anhydrous dichloromethane (2 mL) was stirred at room temperature for 16 hours. Then, sodium triacetoxyborohydride (104 mg, 0.49 mmol) was added in portions, and the reaction was continued with stirring at room temperature for 2 hours. The reaction mixture was dissolved in dichloromethane, washed with brine, washed with water, and concentrated. The residue was purified by preparative high-performance liquid chromatography to give title compound 3 (11.4 mg, 7%) as a white solid. MS (ESI): m / z = 669.2 [M+H] + .

[0310] 1 H NMR (400MHz, MeOD) δ8.87(dd,J=8.4,1.4Hz,1H),8.09(d,J=5.8Hz,1H),7.65(s,1H),7.63(dd,J=7.8,1.6Hz,1H),7.52(t,J=7.6Hz,1H),7.48-7.38(m, 4H),7.02(dd,J=7.6,1.4Hz,1H),3.96(s,1H),3.87(t,J=12.2,1H),2.89(s ,3H),2.77-2.74(m,2H),2.55(s,3H),2.39-2.26(m,3H),1.90-1.80(m,1H).

[0311] Example 4: (1r,4r)-4-(((4-((2,2'-dichloro-3'-((E)-5-(((((S)-5-carbonylpyrrolidine-2- (1,1'-Biphenyl)-3-yl)amino)thiazo[4,5-yl) c]pyridin-2-yl)methyl)amino)cyclohexane-1-carboxylic acid

[0312]

[0313] (S,E)-5-(((3-(3-bromo-2-chlorostyryl)-4-(trifluoromethyl)benzyl)amino)methyl)pyrrolidine- 2-keto

[0314]

[0315] A mixture of (E)-3-(3-bromo-2-chlorostyryl)-4-(trifluoromethyl)benzaldehyde (1000 mg, 1.75 mmol, 68%) and (S)-5-(aminomethyl)pyrrolidine-2-one hydrochloride (527 mg, 3.50 mmol) was added to a solution of N-ethyl-N-isopropylpropane-2-amine (226 mg, 1.75 mmol) in anhydrous dichloromethane (3 mL) and methanol (9 mL), and stirred at room temperature for 16 hours. Then, sodium triacetoxyborohydride (1133 mg, 5.25 mmol) was added in portions, and stirring continued for 4 hours at room temperature. The reaction mixture was quenched with saturated sodium bicarbonate solution and stirred at room temperature for half an hour. The mixture was extracted three times with dichloromethane, washed twice with saturated salt water, dried, and concentrated. The residue was purified by normal-phase column chromatography (methanol:dichloromethane = 3:97) to give the title compound 4-1 (750 mg, 78%) as a pale yellow solid. MS (ESI): m / z = 487.0 [M+H] + .

[0316] (S,E)-(2-chloro-3-(5-((((5-carbonylpyrrolidin-2-yl)methyl)amino)methyl)-2-(trifluoromethyl)benzene vinyl(phenyl)boronic acid

[0317]

[0318] Under nitrogen protection, 4-1 (488 mg, 1.00 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxoboronylcyclopentane) (381 mg, 1.50 mmol), [1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride (41 mg, 0.05 mmol), and anhydrous potassium acetate (245 mg, 2.50 mmol) were thoroughly mixed, and then 1,4-dioxane (5 mL) was added. The mixture was stirred at 100 °C for 10 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 = 3:97) to give the title compound 4-2 (113 mg, 25%) as a white solid. MS(ESI): m / z = 535.1 [M+H] + .

[0319] (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)thiazo[4,5-c]pyridine-2- 1-(methyl)amino)cyclohexane-1-carboxylic acid

[0320]

[0321] Under nitrogen protection, 6-4 (65 mg, 0.13 mmol), 4-2 (77 mg, 0.17 mmol), tetraphenylphosphine palladium (8 mg, 0.0065 mmol), and anhydrous potassium carbonate (46 mg, 0.33 mmol) were thoroughly mixed, followed by the addition of 1,4-dioxane (3 mL) and water (0.5 mL), and then stirred at 90 °C for 2 hours. The reaction mixture 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 title compound 4 (35 mg, 33%) as a white solid. MS (ESI): m / z = 823.3 [M+H] + .

[0322] 1 H NMR (400MHz, DMSO-d6) δ8.85(d,J=7.2Hz,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=6.4Hz,1H),6.98(d,J= 6.4Hz,1H),4.15(s,2H),3.81-3.79(m,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).

[0323] Example 5: (S)-1-(5-chloro-4-(((S)-4-(2-chloro-3-((E)-5-(((((S)-5-carbonylpyrrolidine-2- (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

[0324]

[0325] (S)-1-(4-(((S)-4-bromo-2,3-dihydro-1H-inden-1-yl)oxo)-5-chloro-2-((5-cyanopyridine-3- 3-methyl-3-piperidine-3-carboxylic acid

[0326]

[0327] 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 catalytic amounts of glacial acetic acid and anhydrous dichloromethane (3 mL) was stirred at room temperature for 16 hours. Then, sodium triacetoxyborohydride (262 mg, 1.24 mmol) was added in portions, and the reaction was continued with stirring at room temperature for 2 hours. The reaction mixture was quenched with saturated sodium bicarbonate solution, 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 (ethyl acetate: petroleum ether = 60:40) to give the title compound 5-1 (110 mg, 30%) as a white solid. MS(ESI): m / z = 596.1 [M+H] + .

[0328] (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-cyanopyridine) (Pyridine-3-yl)methoxy)benzyl)piperidine-3-carboxylic acid

[0329]

[0330] Under nitrogen protection, 5-1 (110 mg, 0.18 mmol), 4-2 (109 mg, 0.24 mmol), tetraphenylphosphine palladium (46 mg, 0.04 mmol), and anhydrous potassium carbonate (62 mg, 0.45 mmol) were thoroughly mixed, followed by the addition of 1,4-dioxane (3 mL) and water (0.5 mL), and then stirred at 90 °C for 2 hours. The reaction mixture 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 = 13:87) and Prep-HPLC to give title compound 5 (45 mg, 27%) as a white solid. MS (ESI): m / z = 924.3 [M+H] + .

[0331] 1H NMR (400MHz, DMSO-d6) δ9.01-8.95(m,2H),8.39(s,1H),7.92(s,1H),7.79(d,J=6.6Hz,1H),7.69(d,J=8.2Hz,1H),7.62( s,1H),7.56(d,J=15.8Hz,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),3.81-3.79(m,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).

[0332] Example 6: (1r,4r)-4-(((4-((2-chloro-3-(2-((((((S)-5-carbonylpyrrolidine-2-yl)methyl)amino) ) methyl) benzo[d]oxazol-5-yl) phenyl) amino) thiazo[4,5-c]pyridin-2-yl) methyl) amino) cyclohexane-1- carboxylic acid

[0333]

[0334] 5-Bromo-2-(chloromethyl)benzo[d]oxazole

[0335]

[0336] A solution of 2-amino-4-bromophenol (2 g, 10.64 mmol) and 2-chloro-1,1,1-trimethoxyethane (1.64 g, 10.64 mmol) in ethanol (10 mL) was heated to 60 °C and stirred for 3 hours. The reaction solution was concentrated, and the residue was purified by normal-phase column chromatography (petroleum ether:ethanol = 100:5) to give the title compound 6-1 (2.3 g, 88%) as a pale yellow solid. MS (ESI): m / z = 247.9 [M+H] + .

[0337] 5-((((5-bromobenzo[d]oxazol-2-yl)methyl)amino)methyl)pyrrolidone-2-one

[0338]

[0339] A mixture of 6-1 (1 g, 4.00 mmol), 5-(aminomethyl)pyrrolidone-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 °C and stirred for 3.5 h. The reaction mixture 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 6-2 (780 mg, 59%) as a pale yellow solid. MS (ESI): m / z = 324.0 [M+H] + .

[0340] 5-((((5-(4,4,5,5-tetramethyl-1,3,2-dioxoboropentane-2-yl)benzo[d]oxazol-2-yl)methyl) (amino)methyl)pyrrolidone-2-one

[0341]

[0342] Under nitrogen protection, 6-2 (700 mg, 2.16 mmol), pinacol diboronate (1.10 g, 4.32 mmol), a dichloromethane complex of [1,1'-bis(di-phenylphosphino)ferrocene]palladium(II) chloride (1:1) (106 mg, 0.13 mmol), and potassium acetate (424 mg, 3.07 mmol) were mixed thoroughly, and 1,4-dioxane (6 mL) was added. The mixture was then stirred at 80 °C for 5 hours. The reaction solution was concentrated, and the residue was purified by normal-phase column chromatography (petroleum ether:ethyl acetate = 82:18) to give the title compound 6-3 (350 mg, 43%) as a pale yellow solid. MS (ESI): m / z = 372.2 [M+H] + .

[0343] (1r,4r)-4-(((4-((3-bromo-2-chlorophenyl)amino)thiazo[4,5-c]pyridin-2-yl)methyl)amino) Cyclohexane-1-carboxylic acid

[0344]

[0345] Compound 1-1 (400 mg, 0.78 mmol) and lithium hydroxide hydrate (128 mg, 3.2 mmol) were added to a mixture of tetrahydrofuran (0.4 mL), methanol (2 mL), and water (0.4 mL), and stirred at room temperature for 2 hours. The reaction mixture was neutralized with 2 mol / L hydrochloric acid, the solvent was evaporated to dryness, and the residue was purified by normal-phase column chromatography (dichloromethane:methanol = 100:6) to give the title compound 6-4 (300 mg, 77%) as a white solid. MS (ESI): m / z = 496.9 [M+H] + .

[0346] (1r,4r)-4-(((4-((2-chloro-3-(2-((((((S)5-carbonylpyrrolidine-2-yl)methyl)amino)methyl)benzene) [d]oxazol-5-yl)phenyl)amino)thiazo[4,5-c]pyridin-2-yl)methyl)amino)cyclohexane-1-carboxylic acid

[0347]

[0348] Under nitrogen protection, 6-3 (350 mg, 0.94 mmol), 6-4 (437 mg, 0.88 mmol), tetrakis(triphenylphosphine)palladium (100 mg, 0.086 mmol), and potassium carbonate (237 mg, 1.72 mmol) were mixed thoroughly. 1,4-dioxane (5 mL) and water (1 mL) were added, and the mixture was stirred at 80 °C for 2 hours. The reaction mixture 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 target product 6 (24.0 mg, 4%) as a white solid. MS (ESI): m / z = 660.2 [M+H] + .

[0349] 1 H NMR (400MHz, DMSO-d6) δ8.80(dd,J=8.4,1.4Hz,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=7.6,1.6Hz,1 H),4.15(s,2H),4.03-4.01(m,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),1.95-1.93(m,2H),1.87-1.86(m,2H),1.70-1.62(m,1H),1.27-1.25(m,2H),1.08-1.06(m,2H).

[0350] Example 7: (S)-1-((5-chloro-2-methoxy-6-(((S)-4-(2-(1-(((S)-5-carbonylpyrrolidine-2- (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

[0351]

[0352] 5-Bromo-2-(piperidin-4-yl)benzo[d]oxazole

[0353]

[0354] 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 to a sealed tube, and the mixture was stirred at 190 °C for 3.5 h. The reaction solution was cooled to room temperature, diluted with water in an ice bath, and the pH was adjusted to 9 with sodium hydroxide. The solution was then extracted three times with ethyl acetate, the organic phase was dried, and the concentrate was used to give the title compound 7-1 (1.10 g, 74%) as a pale yellow solid. MS (ESI): m / z = 283.0 [M+H] + .

[0355] (S)-5-((4-(5-bromobenzo[d]oxazol-2-yl)piperidin-1-yl)methyl)pyrrolidine-2-one

[0356]

[0357] A solution of 7-1 (500 mg, 1.78 mmol), (S)-(5-carbonylpyrrolidone-2-yl)methyl 4-methylbenzenesulfonate (719 mg, 2.67 mmol), and anhydrous potassium carbonate (495 mg, 3.58 mmol) in acetonitrile (5 mL) was sequentially added to a sealed tube and stirred at 100 °C for 20 h. The reaction solution was diluted with ethyl acetate, washed with water and brine, dried over anhydrous sodium sulfate, concentrated, and the residue was purified by normal-phase column chromatography (methanol:dichloromethane = 3:97) to give the title compound 7-2 (490 mg, 73%) as a pale yellow solid. MS (ESI): m / z = 378.0 [M+H] + .

[0358] (S)-5-((4-(5-(4,4,5,5-tetramethyl-1,3,2-dioxoboropentane-2-yl)benzo[d]oxazol-2-yl) Piperidin-1-yl)methyl)pyrrolidone-2-one.

[0359]

[0360] Under nitrogen protection, 7-2 (490 mg, 1.30 mmol), 4,4,4',4',5,5,5',5'-octamethyl-2,2'-bi(1,3,2-dioxoboronylcyclohexane) (495 mg, 1.95 mmol), tetraphenylphosphine palladium (75 mg, 0.065 mmol), and anhydrous potassium acetate (319 mg, 3.25 mmol) were mixed thoroughly, and 1,4-dioxane (4 mL) and water (0.5 mL) were added. The mixture was then stirred at 80 °C for 6 hours. The reaction solution was filtered, concentrated, and the residue was purified by normal-phase column chromatography (ethyl acetate:petroleum ether = 60:40) to give the title compound 7-3 (445 mg, 81%) as a yellow solid. MS (ESI): m / z = 426.2 [M+H] + .

[0361] 5-Chloro-2-methoxy-6-(((S)-4-(2-(1-((((S)-5-carbonylpyrrolidine-2-yl)methyl)piperidin-4-yl) Benz[d]oxazol-5-yl)-2,3-dihydro-1H-inden-1-yl)oxo)nicotinaldehyde

[0362]

[0363] Under nitrogen protection, 7-3 (98 mg, 0.23 mmol), intermediate D (70 mg, 0.15 mmol), tetraphenylphosphine palladium (8 mg, 0.0075 mmol), and anhydrous potassium carbonate (62 mg, 0.45 mmol) were mixed thoroughly. 1,4-dioxane (4 mL) and water (0.5 mL) were added, and the mixture was stirred at 90 °C 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 7-4 (85 mg, 83%) as a pale yellow solid. MS (ESI): m / z = 690.2 [M+H] + .

[0364] (S)-1-((5-chloro-2-methoxy-6-(((S)-4-(2-(1-((((S)-5-carbonylpyrrolidine-2-yl)methyl)piperyl) (Pinidin-4-yl)benzo[d]oxazol-5-yl)-2,3-dihydro-1H-inden-1-yl)oxo)pyridin-3-yl)methyl)piperidin-3-carboxylic acid

[0365]

[0366] A mixture of 7-4 (40 mg, 0.067 mmol) and (S)-piperidine-3-carboxylic acid (43 mg, 0.335 mmol) was added to a catalytic amount of anhydrous dichloromethane (1 mL) and methanol (0.5 mL) in glacial acetic acid, and stirred at room temperature for 16 hours. Then, sodium triacetoxyborohydride (28 mg, 0.13 mmol) was added to the reaction mixture, and stirring continued for 2 hours at room temperature. The reaction mixture was quenched with saturated sodium bicarbonate solution, concentrated, and the residue was purified by Prep-HPLC to give title compound 7 (15 mg, 31%) as a white solid. MS (ESI): m / z = 714.1 [M+H] + .

[0367] 1H NMR (400MHz, DMSO-d6) δ7.76(s,1H),7.71(d,J=8.4Hz,1H),7.68(s,1H),7.53(s,1H),7.44(d,J=8.6H z,1H),7.41(d,J=7.0Hz,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),2.75-2.73(m,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).

[0368] Examples 8-15 were prepared using a method similar to that used in Examples 1-7:

[0369]

[0370]

[0371]

[0372] Example 16: 2-(1-((4-((2,2'-dichloro-3'-(6-(difluoromethoxy)-5-(((((S)-5-carbonylpyrrole)) Alkyl-2-yl)methyl)amino)methyl)pyrazin-2-yl)-[1,1'-biphenyl]-3-yl)amino)thiazo[4,5-c]pyridine- 2-yl)methyl)pyrrolidine-3-yl)acetic acid

[0373]

[0374] Ethyl 2-(1-((4-((2,2'-dichloro-3'-(6-(difluoromethoxy)-5-(((((S)-5-carbonylpyrrolidine-2-yl)methyl)amino)methyl)pyrazin-2-yl)-[1,1'-biphenyl]-3-yl)amino)thiazo[4,5-c]pyridin-2-yl)methyl)pyrrolidine-3-yl)acetate

[0375]

[0376] Compound 16-1 was synthesized by substituting the corresponding starting materials according to the synthesis method of Example 1. MS (ESI): m / z = 811.3 [M+H] + .

[0377] Ethyl 2-(1-((4-((2,2'-dichloro-3'-(6-(difluoromethoxy)-5-(((((S)-5-carbonylpyrrolidine-2-yl)methyl)amino)methyl)pyrazin-2-yl)-[1,1'-biphenyl]-3-yl)amino)thiazo[4,5-c]pyridin-2-yl)methyl)pyrrolidine-3-yl)acetate

[0378]

[0379] A mixture of 16-1 (50 mg, 0.06 mmol) and hydrochloric acid (1 mol / L, 2 mL) was heated to 40 °C and stirred for 18 hours. The reaction solution was neutralized with saturated sodium bicarbonate, evaporated to dryness, and the residue was subjected to preparative high-performance liquid chromatography (HPLC) to obtain the target product 2-(1-((4-((2,2'-dichloro-3'-(6-(difluoromethoxy)-5-(((((S)-5-carbonylpyrrolidine-2-yl)methyl)amino)methyl)pyrazin-2-yl)-[1,1'-biphenyl]-3-yl)amino)thiazo[4,5-c]pyridin-2-yl)methyl)pyrrolidine-3-yl)acetic acid (19.5 mg, 40%) as a white solid. MS (ESI): m / z = 783.1 [M+H] + .

[0380] 1 H NMR (400MHz, DMSO-d6) δ8.87(d,J=7.3Hz,1H),8.81(s,1H),8.67(s,1H),8.13(d,J=5.6H z,1H),7.80(s,1H),7.78(dd,J=7.8,1.6Hz,1H),7.70-7.58(m,3H),7.55(dd,J=7.6,1.6H z,1H),7.50(dd,J=8.0,8.0Hz,1H),7.07(dd,J=7.6,1.4Hz,1H),4.14-4.10(m,2H),3.96( s,2H),3.67-2.60(m,1H),2.88-2.85(m,1H),2.73-2.70(m,2H),2.69-2.62(m,2H),2.48- 2.43(m,1H),2.43-2.37(m,1H),2.27-2.25(m,2H),2.15-1.95(m,4H),1.76-1.67(m,1H),1.48-1.38(m,1H).

[0381] Examples 11, 42, and 47 were prepared using a method similar to that of Example 16:

[0382]

[0383] Example 17: 8-((4-((2,2'-dichloro-3'-(6-(difluoromethoxy)-4-methyl-5-(((((S)-5-carbonyl) Pyrrolidine-2-yl)methyl)amino)methyl)pyridin-2-yl)-[1,1'-biphenyl]-3-yl)amino)thiazo[4,5-c]pyridine (Pyridine-2-yl)methyl)-8-azabicyclo[3.2.1]octane-3-carboxylic acid

[0384]

[0385] N-(2,2'-dichloro-3'-(4,4,5,5-tetramethyl-1,3,2-dioxoboropentane-2-yl)-[1,1'-biphenyl]- 3-yl)-2-(methoxymethyl)thiazo[4,5-c]pyridine-4-amine

[0386]

[0387] Compound A-2 (1 g, 2.6 mmol), compound B (1.66 g, 4.5 mmol), tetratetraphenylphosphine 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 protection, mixed thoroughly, and then stirred at 80 °C for 4 hours. After the reaction was complete, the reaction solution was filtered, the filtrate was concentrated, and the residue was purified by normal-phase silica gel column chromatography (petroleum ether: ethyl acetate = 0–30%) to give the target compound 17-1 (470 mg, 31%) as a yellow solid. MS (ESI): m / z = 542.0 [M+H] + .

[0388] 6-(2,2'-dichloro-3'-((2-(methoxymethyl)thiazo[4,5-c]pyridin-4-yl)amino)-[1,1'-bi [Phenyl]-3-yl)-2-(difluoromethoxy)-4-methylnicotine aldehyde

[0389]

[0390] Compound 17-1 (11.0 g, 20.3 mmol), compound C (4.08 g, 18.4 mmol), tetraphenylphosphine 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 protection and mixed thoroughly. The mixture was then stirred at 90 °C for 2 hours. After the reaction was complete, the reaction solution was filtered, the filtrate was concentrated, and the residue was purified by normal-phase silica gel column chromatography (ethyl acetate: petroleum ether = 1:2) to give the title compound 17-2 (8.00 g, 72%) as a pale yellow solid. MS (ESI): m / z = 601.2 [M+H] + .

[0391] (S)-5-((((6-(2,2'-dichloro-3'-((2-(methoxymethyl)thiazo[4,5-c]pyridin-4-yl)amino (1,1'-Biphenyl)-3-yl)-2-(difluoromethoxy)-4-methylpyridin-3-yl)methyl)amino)methyl)pyrrolidine- 2-keto

[0392]

[0393] A solution of compound 17-2 (8.0 g, 13.3 mmol), (S)-5-(aminomethyl)pyrrolidine-2-one hydrochloride (6.0 g, 39.9 mmol), and N,N-diisopropylethylamine (2.58 g, 19.95 mmol) in anhydrous dichloromethane (40 mL) was stirred at room temperature for 12 hours. Then, sodium triacetoxyborohydride (8.46 g, 39.90 mmol) was added in portions, and the reaction was continued at room temperature with stirring for 3 hours. After the reaction was complete, the mixture was quenched with a saturated ammonium bicarbonate solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, concentrated, and the residue purified by normal-phase silica gel column chromatography (methanol:dichloromethane = 6:100) to give compound 17-3 (8.76 g, 94%) as a pale yellow solid. MS (ESI): m / z = 699.3 [M+H] + .

[0394] (S)-5-((((6-(3'-(((2-(bromomethyl)thiazo[4,5-c]pyridin-4-yl)amino)-2,2'-dichloro- [1,1'-Biphenyl]-3-yl)-2-(difluoromethoxy)-4-methylpyridin-3-yl)methyl)amino)methyl)pyrrolidone-2-one

[0395]

[0396] Compound 17-3 (2.00 g, 2.86 mmol) was added to a hydrobromic acid (20 mL) acetic acid solution. The mixture was heated to 60 °C and stirred for 10 hours. After the reaction was complete, the reaction solution was slowly added dropwise to a saturated aqueous solution of sodium bicarbonate under ice bath conditions. The mixture was extracted twice with dichloromethane (50 mL), and the combined organic phases were washed with brine, dried over anhydrous sodium sulfate, and concentrated to give the target compound 17-4 (1.75 g, 57%) as a yellow solid. MS (ESI): m / z = 747.1 [M+H] + .

[0397] Methyl(1R,5S)-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)thiazo[4,5- c]pyridin-2-yl)methyl)-8-azabicyclo[3.2.1]octane-3-carboxylic acid ester

[0398]

[0399] Cesium carbonate (391 mg, 1.20 mmol) and methyl (1R,5S)-8-azabicyclo[3.2.1]octane-3-carboxylic acid hydrochloride (165 mg, 0.80 mmol) were 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, and stirring continued at room temperature until the starting material was completely dissolved. The reaction solution was diluted with dichloromethane (50 mL), washed successively with water and saturated brine, dried, concentrated, and the residue purified using a reversed-phase rapid separation column (85% acetonitrile in an aqueous solution of ammonium bicarbonate (0.1%)) to give the target compound 17-5 (122 mg, 38%) as a pale yellow solid. MS (ESI): m / z = 836.4 [M+H] + .

[0400] 8-((4-((2,2'-dichloro-3'-(6-(difluoromethoxy)-4-methyl-5-(((((S)-5-carbonylpyrrolidine-2- (1,1'-Biphenyl]-3-yl)amino)thiazo[4,5-c]pyridin-2-yl) Methyl)-8-azabicyclo[3.2.1]octane-3-carboxylic acid

[0401]

[0402] 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). The reaction mixture was stirred overnight at room temperature. After the reaction was complete, the solution was neutralized with dilute hydrochloric acid, concentrated, and the residue was purified by preparative high-performance liquid chromatography to obtain the target product 17 (95 mg, 80%) as a white solid. MS (ESI): m / z = 822.3 [M+H] + .

[0403] 1 H NMR (400MHz, DMSO-d6) 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=7.6Hz,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,1 H),2.49(s,3H),2.18-2.05(m,3H),2.04-1.92(m,2H),1.82-1.80(m,2H),1.74-1.58(m,5H).

[0404] The following compounds can be synthesized by replacing the corresponding starting materials according to the synthesis method of Example 17.

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411] Test Example 1: Detecting the inhibitory effect of compounds on the binding of PD-1 / PD-L1 proteins.

[0412] Homogenous time-resolved fluorescence (HTRF) was used to detect the in vitro inhibitory activity of the compound on the binding of PD-1 and PD-L1.

[0413] The PD-1 / PD-L1 binding assay kit (Cisbio, Cat#63ADK000CPDECP) was used. This kit contains two proteins, Tag 1-PD-L1 and Tag 2-PD-1, and Anti-Tag 1-Eu. 3+ Two antibodies, Anti-Tag2-XL 665 and Anti-Tag1-Eu. Detection principle: Anti-tag1-Eu 3+ Anti-Tag2-XL 665, acting as an HTRF donor and acceptor, showed that when Tag1-PD-L1 and Tag2-PD-1 interacted, the added HTRF donor and acceptor approached each other. Upon receiving excitation energy, the donor transferred some energy to the acceptor, producing 665nm emission. When the compound blocked the PD-1 / PD-L1 interaction, only 620nm emission was produced. The inhibitory effect of the compound was determined by comparing the 665nm / 620nm ratio. Tag1-PD-L1 was diluted to a working concentration of 10nM with Diluent buffer (cat#62DLBDDF), and Tag2-PD-1 was diluted to a working concentration of 500nM with Diluent buffer. Anti-Tag1-Eu... 3+Dilute Anti-Tag2-XL 665 1:100 with detection buffer (CAT#62DB1FDG), and dilute it 1:20 with detection buffer. Serially dilute the target compound to a 10X final concentration using diluent buffer. Add 2 μL of the compound to each well of a 384-well plate, followed by 4 μL of Tag 1-PD-L1 and 4 μL of Tag 2-PD-1, and incubate at room temperature for 15 minutes. Add 5 μL of Anti-Tag1-Eu 3+ Incubate overnight at room temperature with 5 μL Anti-Tag2-XL 665, then use BioTek Synergy TM The Neo2 multi-functional microplate reader was used to detect the 665nm / 620nm ratio. The IC50 curve was fitted using Prism.

[0414] Table 1 shows the IC50 values ​​of some compounds of the present invention in Test Example 1. 50 value

[0415] 1 A 23 A 2 A 25 A 3 A 30 A 4 A 31 A 5 A 32 A 6 A 33 A 7 A 34 A 8 B 36 A

[0416] 10 A 38 A 11 A 39 A 12 A 41 A 13 A 42 B 14 A 43 A 15 A 44 A 16 A 45 A 18 A 46 A 19 A 47 A 20 A 48 A 22 A

[0417] The letter A represents IC 50 Less than 5 nM;

[0418] The letter B represents IC 50 The range is from 5 nM to 1 μM;

[0419] The letter C stands for IC 50 It is greater than 1 μM;

[0420] The results showed that the compounds in this invention can effectively inhibit the binding of PD-1 / PD-L1 at different concentrations. Therefore, they can be used in the treatment of diseases related to the interaction of PD-1 / PD-L1.

[0421] Test Example 2: Cellular NFAT Reporter Gene Assay

[0422] Cellular experiments involving PD-1 / PD-L1 require two cell types: PD-1 effector cells and PD-L1 aAPC / CHO-K1 cells. PD-1 effector cells express human PD-1 protein and an NFAT-driven luciferase reporter gene, while PD-L1 aAPC / CHO-K1 cells express PD-L1 protein and an anti-CD3 antibody. When these two cell types are co-cultured, the PD-1 / PD-L1 interaction inhibits TCR-NFAT-RE signaling, interrupting NFAT-RE-mediated fluorescence. Adding an inhibitor of PD-1 or PD-L1 blocks the PD-1 / PD-L1 interaction, relieving the inhibition of the TCR-NFAT-RE pathway and enhancing the fluorescence signal. The intensity of the fluorescence signal indicates the inhibitor's blocking effect.

[0423] On the first day of the experiment, the resuscitated PD-L1 aAPC / CHO-K1 cells were digested, centrifuged, and then the concentration was adjusted to 2.0 × 10⁻⁶ cells using culture medium (90% Ham's F-12 / 10% FBS). 5 / mL, at 40μL per well, 8×10 3 Cells were seeded in 384-well plates and incubated overnight. The next day, the test compound was serially diluted to twice the desired concentration using a detection buffer (99% RPMI 1640 / 1% FBS). PD-1 cells were centrifuged and then diluted with the detection buffer to a concentration of 6.25 × 10⁻⁶. 5 / mL. After removing the culture medium from the overnight cultured 384-well plate, add 20 μL of compound dilution and 20 μL of diluted PD-1 effector cells to each well. Incubate in a cell culture incubator for 6 hours, then add 20 μL of Bio-Glo reagent (Promega, cat#G7940) to each well. After about 10 minutes, read the plate using a multi-plate reader. Each plate should have a negative control (cells only, no compound) and a blank control (detection buffer only).

[0424] Prism was used to analyze the inhibitory activity of the compound based on the fluorescence value.

[0425] Table 2 EC values ​​of some compounds of the present invention in Test Example 2 50 value

[0426] 1 A 27 B 2 A 28 B 3 B 29 B

[0427] 5 A 31 B 6 B 32 B 7 B 33 A 9 A 34 B 10 A 35 B 11 A 36 B 12 A 37 B 13 A 38 B 14 A 39 B 15 B 40 B 16 A 41 B 17 B 42 B 18 B 43 A 19 B 44 B 20 B 45 B 21 B 46 B 22 B 47 B 23 A 48 A 24 B 49 B 25 B 50 B 26 B

[0428] The letter A represents IC 50 Less than 100 nM;

[0429] The letter B represents IC50 The range is from 100 nM to 1 μM;

[0430] The letter C stands for IC 50 It is greater than 1 μM;

[0431] The results showed that the compounds of the present invention can effectively block the interaction between PD-1 and PD-L1, with half-maximal inhibitory concentrations comparable to or better than those of PD-1 / PD-L1 inhibitors in clinical trials.

[0432] Test Example 3: Human PBMC Functional Experiment

[0433] SEB (Toxin Technology, cat#BT202) induces T cell activation and IL-2 secretion. Adding anti-PD-1 / PD-L1 antibodies or small molecule inhibitors to the reaction system promotes T cell activation and increases IL-2 secretion. The activity of the compound is assessed by detecting the IL-2 content in the cell supernatant using an IL-2 ELISA kit.

[0434] On the first day of the experiment, prepare the test compound, antibody solution, and control solution. Add 50 μL of the compound, antibody, or control solution to each well. Resuscitate human PBMC cells according to the manufacturer's instructions and adjust the cell concentration to 3 × 10⁶ cells / well using culture medium (90% RPMI 1640 + 10% FBS). 6 / mL, at 100μL per well, 3×10 5 Cells were seeded in 96-well cell culture plates (Corning-3599) and cultured at 37°C for 4 days. The cell supernatant was collected by centrifugation at 300g for 5 minutes. Changes in the cytokine IL-2 were then detected using an IL-2 ELISA kit (R&D-DY202-05).

[0435] Table 3. Effective concentrations of some compounds of the present invention in Test Example 3

[0436] 2 B 10 B

[0437] The letter A indicates that the effective concentration is less than 1 nM;

[0438] The letter B indicates an effective concentration of 1-10 nM;

[0439] The letter C indicates an effective concentration of 10-100 nM;

[0440] The results showed that the compound of the present invention can increase the secretion of IL-2, and the secretion of IL-2 is dose-dependent with the concentration of the compound. Under high concentration conditions, the effect of the compound is comparable to that of the control antibody.

[0441] Test Example 4: In vivo efficacy experiment of the small molecule inhibitor in the treatment of tumors according to the present invention

[0442] A mouse model of subcutaneous tumor transplantation was established to examine the in vivo inhibitory effect of these compounds on tumor growth. The method was as follows: Cultured specific tumor cells were digested, centrifuged to collect cells, washed twice with sterile saline, and counted. The cell concentration was adjusted to the desired level with saline, and 0.2 mL of the cell suspension was subcutaneously inoculated into hPD-L1C57BL / 6 or Balb / c immunocompetent mice. After inoculation, tumor growth was observed until a specific size was reached. Animals were randomly divided into groups of 6-15 mice, weighed, and administered the test compound once or twice daily. The groups included: a vector group, a control anti-PD-1 / PD-L1 antibody group, and a test compound group. Tumor growth was monitored weekly for approximately 6 weeks. After the tumor volume reached the endpoint, the mice were weighed and euthanized. Tumor tissue, spleen tissue, and blood samples were collected. The tumor inhibition rate was calculated, and the inhibition rate of hPD-L1 and the composition of immune cells in the tumor, spleen, and blood samples were detected. The immunomodulatory activity of the test compound was calculated.

[0443] The results showed that the compounds of the present invention could effectively inhibit tumor growth in tumor-bearing mice, and their inhibitory effect was comparable to or better than that of PD-1 / PD-L1 inhibitors in clinical trials.

[0444] Test Example 5: Pharmacokinetic experiments of the small molecule inhibitor in mice, rats, and monkeys in this invention.

[0445] ICR mice, rats, and monkeys were administered the test compound via single intravenous (IV) and oral (PO) administration. Blood samples were collected at different time points, and the concentration of the test substance in the animal plasma was determined by LC-MS / MS, and relevant parameters were calculated. Specifically, the required amount of the test sample was prepared into a solution of the desired concentration in 20% PEG400 + 80% (20% SBE-β-CD in 100mM citrate buffer pH 3.0) for intravenous or oral administration. Bioanalytical methods and sample detection methods were established. Pharmacokinetic parameters, such as AUC, were calculated using Phoenix WinNonlin 7.0 software based on blood drug concentration data at different time points. (0-t) AUC (0-∞) T 1 / 2 C max T max、 F% and MRT, etc.

[0446] Table 4. Pharmacokinetic parameters of some compounds in this invention in mice.

[0447]

[0448] Table 5. Pharmacokinetic parameters of some compounds of this invention in rats.

[0449]

[0450] Table 6. Pharmacokinetic parameters of some compounds of this invention in cynomolgus monkeys.

[0451]

[0452] The results showed that the compounds of this invention have excellent pharmacokinetic properties.

[0453] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A compound represented by Formula II, or a pharmaceutically acceptable salt thereof: II in, X1 is N; X2 is CH or N, and when X2 is CH, R5 or R5' can be located on CH; The R5 is selected from the group consisting of: -OCHF2, -OCH2F, and ethyl groups; R5' is a group selected from the group consisting of: H, unsubstituted or halogenated C1-C6 alkyl, and unsubstituted or halogenated C1-C6 alkoxy. R2 is n1 is 1; Ra is selected from the following group: H, C1-C8 alkyl; Rb is selected from the following group: C3-C8 cycloalkyl groups, 5-10 membered heterocyclic groups having 1-3 heteroatoms selected from N, S and O; Alternatively, Ra and Rb together with the attached N atom can form a 5-10 member saturated heterocycle; The ring formed by Rb, or Ra and Rb together, can be further substituted by 1-3 groups selected from the following group: COOH, C(R6R7). m1 -COOH; where m1 is 1, 2 or 3, and R6 and R7 are each independently selected from H, C1-C3 alkyl; R3 is independently selected from the following group: n3 is 1; Ra' is H; Rb' is CH2-Re; wherein, the Re is a 5-7 member nitrogen-containing saturated ring substituted with a carbonyl group; The additional condition is that the compound of formula II has a chemically stable structure.

2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The R5 mentioned is -OCHF2.

3. The compound of claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, Ra is H; Rb is selected from the following group: C3-C8 cycloalkyl groups, 5-10 membered heterocyclic groups having 1-3 heteroatoms selected from N, S and O; Alternatively, Ra and Rb together with the attached N atom can form a 5-10 member saturated heterocycle; In this process, Rb, or the saturated heterocycle formed by Ra and Rb, can be further substituted by 1-3 groups selected from the following group: COOH, C(R6R7). m1 -COOH; where m1 is 1, 2 or 3, and R6 and R7 are each independently H.

4. The compound of claim 3, or a pharmaceutically acceptable salt thereof, characterized in that, Ra is H; The Rb is a C3-C8 cycloalkyl group; Alternatively, Ra and Rb together with the attached N atom can form a 5-10 member saturated heterocycle; The saturated heterocycle formed by Rb, or Ra and Rb together, can be further substituted by 1-3 groups selected from the following group: COOH, C(R6R7). m1 -COOH; where m1 is 1, 2 or 3, and R6 and R7 are each independently chosen as H.

5. The compound of claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, Ra is H; The Rb mentioned is selected from the following group: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 ； Alternatively, Ra and Rb, together with the attached N atom, can form a saturated heterocycle selected from the following group: , , , , , , , , , , , , ; Furthermore, the Rb, or the group formed by Ra and Rb together with the attached N atom, can be further substituted by 1-3 groups selected from the following group: COOH, C(R6R7). m1 -COOH; where m1 is 1, 2 or 3, and R6 and R7 are each independently H.

6. The compound as claimed in claim 1, characterized in that, X 1 For N; X 2 For CH; R 5 -OCHF2; R 5 ' is a group selected from the following group: unsubstituted or halogenated C1-C6 alkyl groups.

7. The compound of claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The compounds are selected from the following group: 。 8. The compound of claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The compounds are selected from the following group:

9. The compound of claim 1, or a pharmaceutically acceptable salt thereof, characterized in that, The Rb, or the nitrogen-containing heterocycle formed by Ra and Rb, is selected from the group consisting of: 。 10. The compound of claim 9, or a pharmaceutically acceptable salt thereof, characterized in that, The compounds are selected from the following group: 。 11. A pharmaceutical composition, characterized in that, It comprises (1) the compound as claimed in claim 1, or a pharmaceutically acceptable salt thereof; and (2) a pharmaceutically acceptable carrier.

12. The use of the compound of claim 1, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 11, characterized in that, This is used to prepare pharmaceutical compositions for the prevention and / or treatment of diseases related to the activity or expression level of PD-1 / PD-L1.

13. The use as described in claim 12, wherein the disease is selected from the group consisting of: tumors, pathogen infections, and diseases related to autoimmune responses.

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