Biphenyl derivative, pharmaceutical composition and use thereof

Abstract

A biphenyl derivative having a structure represented by formula I or a pharmaceutically acceptable salt, an ester, an amide, a solvate, an active metabolite, a polymorph, an isotopically labeled substance, an isomer or a prodrug thereof, and a pharmaceutical composition containing the same. The derivative is designed on the basis of a plurality of Wnt upstream kinases with similar structures, and can regulate the pathway by means of at least one Wnt upstream pathway. The derivative increases the expression of reprogramming core genes Oct4, Lin28A and c-Myc when existing independently, and can thus be well applied to cell reprogramming.

Description

Biphenyl derivatives, pharmaceutical compositions and applications thereof Technical Field

[0001] The present application relates to the field of medical technology, and in particular to a biphenyl derivative, a pharmaceutical composition and applications thereof. Background Art

[0002] In 2006, Shinya Yamanaka's team proposed a "cocktail" method consisting of four transcription factors: Oct4, Sox2, KlF4, and c-Myc. This method can successfully reprogram terminally differentiated skin fibroblasts into stem cells with differentiation pluripotency. These stem cells are called induced pluripotent stem cells (induced pluripotent cells) (Takahashi K, et al., Cell, 2006, 126(4)pp.663-676; Takahashi K and Yamanaka S, Cell, 2007, 131(5)pp.861-872). These stem cells have differentiation potential similar to embryonic stem cells and can form the three most basic germ layers of human development: ectoderm, mesoderm, and endoderm, and ultimately form a variety of adult cells. The introduction of this method broke through the ethical restrictions on the use of human embryonic stem cells in medicine and greatly expanded the application potential of stem cell technology in clinical medicine.

[0003] The currently widely used reprogramming methods mostly use viruses or other types of vectors to overexpress reprogramming transcription factors represented by Oct4 (Takahashi K, et al., Cell, 2006, 126(4): 663-676; Takahashi K and Yamanaka S, Cell, 2007, 131(5): 861-872); Yu J, et al. Science. 2007; 318: 1917–1920). Such methods have potential clinical risks in the clinical use of induced pluripotent stem cells (iPSCs), such as the potential tumorigenicity risks brought about by the use of viral vectors; in addition, the complex GMP production process of the vectors also brings complexity to the clinical supervision of induced pluripotent stem cells. Furthermore, the use of vectors will bring high costs to such products. Therefore, if chemical substances can be used to achieve selective gene regulation, thereby changing the expression state of reprogramming transcription factors, then the ectopic expression of "reprogramming genes" will not be required.

[0004] The Wnt / β-catenin pathway is an evolutionarily conserved signaling cascade that is crucial for embryonic development, cell viability, and tissue regeneration, and its dysregulation is associated with tumorigenesis (H. Clevers and R. Nusse, Cell, 2012, 149:1192-1205). Therefore, upstream and downstream regulation of the Wnt signaling pathway has become a research hotspot in multiple fields.

[0005] Currently, a variety of core protein inhibitors in the Wnt signaling pathway have been developed and applied. GSK3 is a serine / threonine kinase and a key inhibitor in the Wnt pathway. CHIR99021 is a widely used GSK3 inhibitor that has a very significant effect on maintaining the pluripotency of mammalian embryos (Meek et al., STEM CELLS. 2007, 31, 10, p. 2104-2115). In addition, combining CHIR99021 with compounds such as valproic acid can enable fibroblasts to become pluripotent stem cells through pure chemical reprogramming (without genetic factors) (Guan et al., Nature. 2022 May; 605, 790,: 325-331). A recent study showed that the use of CHIR99021 allows β-catenin downstream of GSK3 to form a complex with Oct4 in a TCF-independent manner, thereby enhancing the pluripotency of cells (Fernando F et al., Development, 2013: 140, 1171-1183).

[0006] The CMGC kinase family is named after the abbreviations of its subfamily members, including cyclin-dependent kinases (CDKs), mitogen-activated protein kinases (MAPKs), glycogen synthase kinases (GSKs), and CDC-like kinases (CLKs). CMGC kinases are highly conserved in organisms (Chowdhury I et al., Cancers (Basel). 2023 Jul 28; 15(15): 3838). Taking CLK as an example, it can phosphorylate serine, threonine, and tyrosine residues (Manning G et al., Science. 2002; 298: 1912–1934). CLK has a highly typical structure, consisting of an N-lobe and a C-lobe connected by a "hinge" region of the protein backbone. The catalytic domain, consisting of β-strands and α-helices, lies between the N- and C-lobe regions (Bullock A et al., Structure. 2009;17:352–362; Keri G et al., Curr. Signal Transduct. Ther. 2006;1:67–95). CLK further regulates the Wnt signaling pathway by modulating alternative splicing of key Wnt-related genes (Tam BY et al., Cancer Lett. 2019:S0304383519304732). Representative CLK kinases include CLK3 and CLK4. Mitogen-activated protein kinase (MAPK), another subfamily member of CMGC, is a group of serine-threonine protein kinases that can be activated by various extracellular stimuli, such as cytokines, neurotransmitters, hormones, cell stress, and cell adhesion. Its typical representatives include JNK1 kinase (c-Jun N-terminal kinase 1).

[0007] The STE kinase family is involved in regulating the MAP kinase and CMGC kinase families, and plays a central role in the transduction of various extracellular and intracellular signals. For example, hPAK1, a member of the STE20 kinase family, regulates the JNK MAP kinase pathway through GTPases (Brown JL et al., Curr Biol. 1996 May 1; 6(5): 598-605). TNIK (Traf2 and Nck-interacting kinase, TNIK), a member of the STE kinase family, is also a regulatory component of the β-catenin-TCF4 transcriptional complex. TNIK can promote the activation of downstream target genes in the Wnt signaling pathway by directly interacting with β-catenin and TCF4, and plays an important role in cytoskeleton formation and development (Fu et al., JBC, 1999, (274): 30729-30737; Mahmoudi et al., EMBO, 2009, (28): 3329-3340). The TNIK inhibitor NCB-0846 can negatively regulate the TGF-β / SMAD signaling pathway by downregulating the expression of TGFBRI, thereby inhibiting the epithelial-mesenchymal transition process of tumors (Sugano et al., Br J Cancer, 2021, (124): 228-236).

[0008] In addition to the key proteins in the Wnt pathway, a variety of kinases upstream and downstream of the Wnt pathway have also been shown to be important regulatory factors of Wnt signals, such as tyrosine kinases. Tyrosine kinases are enzymes that catalyze the transfer of phosphate groups from ATP to tyrosine residues of proteins in cells, which regulate the “on” and “off” of signaling pathways in cells. Receptor tyrosine kinases (RTKs) are one of the tyrosine kinases that are activated by binding of ligands to their extracellular domains (Hanks SK et al., Science. 1988 Jul 1; 241 (4861): 42-52). PDGFRs are one of the core members of the receptor tyrosine kinase (RTKs) family, including two subtypes, PDGFRα and PDGFRβ (Guérit et al., Cellular and Molecular Life Sciences, 2021, 78: 3867-3881). PDGFRα can also promote the early development of multiple cell lineages by inhibiting the Wnt9a / β-catenin signaling pathway (Bartoletti et al., Developmental Biology, 2020, (1): 36-46; Sun et al., Cell Stem Cell, 2020 (26): 707-721). SU5402 is an inhibitor that can simultaneously act on the FGF, VEGF, and PDGF signaling pathways. SU5402 can regulate Wnt signaling by acting on platelet-derived growth factor receptors (PDGFRs).

[0009] It is worth noting that although the upstream and downstream pathways of the above-mentioned Wnt pathway can have a certain impact on the Wnt pathway, including the GSK3 inhibitor CHIR99021, the PDGF signaling pathway inhibitor SU5402, and the TNIK inhibitor NCB-0846 can regulate certain physiological changes, they can neither simultaneously inhibit multiple Wnt pathway upstream kinases at the same time, nor increase the expression of the reprogramming core gene OCT4 during somatic cell reprogramming, thereby further affecting other reprogramming genes and increasing the probability of somatic cell reprogramming.

[0010] Summary of the Invention

[0011] Based on this, it is necessary to provide a biphenyl derivative and a pharmaceutical composition containing the same. The derivative is designed based on multiple structurally similar Wnt upstream kinases, can regulate the pathway through at least one Wnt upstream pathway, and can increase the expression of reprogramming core genes Oct4, Lin28A, and c-Myc reprogramming core genes when it exists independently. Therefore, it can be well applied to cell reprogramming.

[0012] In a first aspect of the present application, a biphenyl derivative is provided, wherein the biphenyl derivative has a structure as shown in Formula I, or is a pharmaceutically acceptable salt, ester, amide, solvate, active metabolite, polymorph, isotope-labeled substance, isomer or prodrug of the structure as shown in Formula I;

[0013] wherein Ring A is a five-membered ring substituted or unsubstituted with methyl or amino groups, and the ring atoms of Ring A contain one or two nitrogen atoms;

[0014] R 1 It is a donor or acceptor of hydrogen bonds, and its structure contains one or more of amino, imino, hydroxyl and ether bonds.

[0015] In a second aspect of the present application, a pharmaceutical composition is provided, comprising the aforementioned biphenyl derivative and at least one pharmaceutically acceptable carrier.

[0016] The third aspect of the present application provides the use of the aforementioned biphenyl derivative or pharmaceutical composition in cell reprogramming.

[0017] The fourth aspect of the present application provides a method for cell reprogramming, comprising the following steps:

[0018] The cells are contacted with the aforementioned biphenyl derivative pharmaceutical composition.

[0019] The fifth aspect of the present application provides the use of the aforementioned biphenyl derivative or pharmaceutical composition in kinase inhibition, wherein the kinase includes one or more of the TK kinase family, the STE kinase family, and the CMGC kinase family.

[0020] In a sixth aspect of the present application, a method for inhibiting kinase is provided, comprising the following steps:

[0021] contacting the kinase with the aforementioned biphenyl derivative or pharmaceutical composition;

[0022] The kinases include one or more of the TK kinase family, the STE kinase family and the CMGC kinase family. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 shows a comparative analysis of the three-dimensional structures of PDGFR, TNIK, CLK4, and JNK1. Figure 1A is a heatmap displaying the relative structural similarity (RMSD) between pairwise comparisons of the proteins, with the numbers indicating the RMSD between the two structures. Figures 1B, 1C, 1D and 1E are cartoon models of the three-dimensional structures of PDGFR (PDB ID: 5GRN) (Liang L et al., Biochem Biophys Res Commun. 2016 Sep 2; 477(4): 667-672), TNIK (PDB ID: 5AX9) (Masuda M et al., Nat Commun. 2016 Aug 26; 7: 12586), CLK4 (PDB ID: 6FYV) (Kallen J et al., ChemMedChem. 2018 Sep 19; 13(18): 1997-2007) and JNK1 (PDB ID: 3ELJ) (Chamberlain SD et al., Bioorg Med Chem Lett. 2009 Jan 15; 19(2): 360-4), respectively; the key sites of the active center of the protein (Figure 1B) are shown as stick models.

[0024] Figure 2 shows the binding ability and chemical bonding details of the biphenyl derivative I-1 and TNIK during dynamics simulations. A plot shows the RMSD of the TNIK-I-1 complex as a function of simulation time. This result demonstrates that TNIK binds to I-1 and that the resulting complex is stable. B plots the energy decomposition of the interaction between each amino acid site of TNIK and I-1. The x-axis represents the amino acid sequence of TNIK, and the y-axis represents the energy contribution to ligand binding. Amino acid sites with a ligand binding energy contribution of less than -0.5 kJ / mol are labeled. C shows the stabilized structure of the TNIK-I-1 complex after molecular dynamics simulations. The protein is shown as a cartoon model, with the color of each site indicating its ligand binding energy contribution. The small molecule and its interacting residues are shown as stick figures. Hydrogen bonds are shown as long dashed lines, and hydrophobic interactions are shown as short dashed lines. This result demonstrates that the active catalytic site of the kinase target bound by TNIK and I-1 and the chemical basis of their interaction are clear, indicating a consistent chemical bond formation.

[0025] Figure 3 shows the binding ability and chemical bonding details of the biphenyl derivative I-4 and TNIK from dynamics simulations. A shows the RMSD of the TNIK / I-1 complex as a function of simulation time during the simulation. The results demonstrate that TNIK binds to I-4 and that the resulting complex is stable. B shows the energy decomposition of the interaction between each amino acid site of TNIK and I-4. The x-axis represents the amino acid sequence of TNIK, and the y-axis represents the energy contribution to ligand binding for each amino acid site. Amino acid sites with a ligand binding energy contribution of less than -0.5 kJ / mol are labeled. C shows the stabilized structure of the TNIK / I-4 complex after molecular dynamics simulations. The protein is shown as a cartoon model, and the color of each site indicates its ligand binding energy contribution. The small molecule and its interacting residues are shown as stick figures. Hydrogen bonds are shown as long dashed lines, and hydrophobic interactions are shown as short dashed lines. This result demonstrates that the active catalytic site of the kinase target bound by TNIK and I-4 and the chemical basis of their interaction are clear, indicating a consistent chemical bond formation.

[0026] Figure 4 shows the binding ability and chemical bonding details of the biphenyl derivative I-12 and TNIK during dynamics simulations. A shows the RMSD of the TNIK-I-12 complex as a function of simulation time. The results indicate that TNIK can bind to I-12 and the resulting complex is stable. B shows the energy decomposition of the interaction between each amino acid site of TNIK and I-12. The x-axis represents the amino acid sequence of TNIK, and the y-axis represents the energy contribution to ligand binding for each amino acid site. Amino acid sites with a ligand binding energy contribution of less than -0.5 kJ / mol are labeled. C shows the structure of the TNIK-I-12 complex after molecular dynamics simulations. The protein is shown as a cartoon model, and the color of each site indicates its ligand binding energy contribution. The small molecule and its interacting residue main chain or side chain are shown as stick models. Hydrogen bonding interactions are shown as long dashed lines, and hydrophobic interactions are shown as short dashed lines. This result indicates that the active catalytic center of the kinase target bound by TNIK and I-12 and the chemical basis of their interaction are clear, that is, the basis of chemical bond formation is consistent.

[0027] Figure 5 shows the binding ability and chemical bonding details of the biphenyl derivative I-1 and PDGFRα during dynamics simulations. A shows the RMSD of the PDGFRα-I-1 complex as a function of simulation time. The results indicate that PDGFRα can bind to I-1 and the resulting complex is stable. B shows the energy decomposition of the interaction between PDGFRα and I-1 at each amino acid site. The x-axis represents the amino acid sequence of PDGFRα, and the y-axis represents the energy contribution to ligand binding for each amino acid site. Amino acid sites with a ligand binding energy contribution of less than -0.5 kJ / mol are labeled. C shows the stabilized structure of the PDGFRα-I-1 complex after molecular dynamics simulations. The protein is shown as a cartoon model, and the color of each site indicates its ligand binding energy contribution. The small molecule and its interacting residues are shown as stick figures. Hydrogen bonds are shown as long dashed lines, and hydrophobic interactions are shown as short dashed lines. This result indicates that the active catalytic center of the kinase target bound by PDGFRα and I-1 and the chemical basis of their interaction are clear, that is, the basis of chemical bond formation is consistent.

[0028] Figure 6 shows the binding ability and chemical bonding details of the biphenyl derivative I-4 to PDGFRα during dynamics simulations. A shows the RMSD of the PDGFRα-I-1 complex as a function of simulation time. The results indicate that PDGFRα can bind to I-1 and the resulting complex is stable. B shows the energy decomposition of the interaction between PDGFRα and I-4 at each amino acid site. The x-axis represents the amino acid sequence of PDGFRα, and the y-axis represents the energy contribution to ligand binding for each amino acid site. Amino acid sites with a ligand binding energy contribution of less than -0.5 kJ / mol are labeled. C shows the stabilized structure of the PDGFRα-I-4 complex after molecular dynamics simulations. The protein is shown as a cartoon model, and the color of each site indicates its ligand binding energy contribution. The small molecule and its interacting residues are shown as stick figures. Hydrogen bonds are shown as long dashed lines, and hydrophobic interactions are shown as short dashed lines. This result indicates that the active catalytic center of the kinase target bound by PDGFRα and I-4 and the chemical basis of their interaction are clear, that is, the basis of chemical bond formation is consistent.

[0029] Figure 7 shows the binding ability and chemical bonding details of the biphenyl derivative I-12 to PDGFRα during dynamics simulations. A shows the RMSD of the PDGFRα-I-12 complex as a function of simulation time. The results indicate that PDGFRα can bind to I-12 and the resulting complex is stable. B shows the energy decomposition of the interaction between PDGFRα and I-12 at each amino acid site. The x-axis represents the amino acid sequence of PDGFRα, and the y-axis represents the energy contribution to ligand binding for each amino acid site. Amino acid sites with a ligand binding energy contribution of less than -0.5 kJ / mol are labeled. C shows the structure of the PDGFRα-I-12 complex after molecular dynamics simulations. The protein is shown as a cartoon model, and the color of each site indicates its energy contribution to ligand binding. The small molecule and its interacting residues are shown as stick figures. Hydrogen bonds are shown as long dashed lines, and hydrophobic interactions are shown as short dashed lines. This result indicates that the active catalytic center of the kinase target bound by PDGFRα and I-12 and the chemical basis of their interaction are clear, that is, the basis of chemical bond formation is consistent.

[0030] Figure 8 shows the binding ability and chemical bonding details of the biphenyl derivative I-1 and CLK4 during dynamics simulations. A plot shows the RMSD of the CLK4-I-1 complex as a function of simulation time during the simulation. The results demonstrate that CLK4 binds to I-1 and that the resulting complex is stable. B plots the energy decomposition of the interaction between each amino acid site of CLK4 and I-1. The x-axis represents the amino acid sequence of CLK4, and the y-axis represents the energy contribution to ligand binding for each amino acid site. Amino acid sites with a ligand binding energy contribution of less than -0.5 kJ / mol are labeled. C shows the stabilized structure of the CLK4-I-1 complex after molecular dynamics simulations. The protein is shown as a cartoon model, with the color of each site indicating its ligand binding energy contribution. The small molecule and its interacting residues are shown as stick figures. Hydrogen bonds are shown as long dashed lines, and hydrophobic interactions are shown as short dashed lines. This result demonstrates that the active catalytic site of the kinase target bound by CLK4 and I-1 and the chemical basis of their interaction are clear, i.e., the chemical bond formation is consistent.

[0031] Figure 9 shows the binding ability and chemical bonding details of the biphenyl derivative I-4 and CLK4 during dynamics simulations. A shows the RMSD of the CLK4-I-1 complex as a function of simulation time during the simulation. The results demonstrate that CLK4 can bind to I-4, and the resulting complex is stable. B shows the energy decomposition of the interaction between each amino acid site of CLK4 and I-4. The x-axis represents the amino acid sequence of CLK4, and the y-axis represents the energy contribution to ligand binding for each amino acid site. Amino acid sites with a ligand binding energy contribution of less than -0.5 kJ / mol are labeled. C shows the structure of the CLK4-I-4 complex stabilized after molecular dynamics simulations. The protein is shown as a cartoon model, with the color of each site indicating its ligand binding energy contribution. The small molecule and its interacting residue main chain or side chain are shown as stick models. Hydrogen bonds are shown as long dashed lines, and hydrophobic interactions are shown as short dashed lines. This result demonstrates that the active catalytic center of the kinase target bound by CLK4 and I-4 and the chemical basis of their interaction are clear, i.e., the basis for chemical bond formation is consistent.

[0032] Figure 10 shows the binding ability and chemical bonding details of the biphenyl derivative I-12 and CLK4 during dynamics simulations. A shows the RMSD of the CLK4-I-1 complex as a function of simulation time during the simulation. The results indicate that CLK4 can bind to I-12, and the resulting complex is in a stable binding state. B shows the energy decomposition of the interaction between each amino acid site of CLK4 and I-12. The x-axis shows the amino acid sequence of CLK4, and the y-axis shows the energy contribution of each amino acid site to ligand binding. The names of amino acid sites with a ligand binding energy contribution of less than -0.5 kJ / mol are marked. C shows the structure of the CLK4-I-12 complex after molecular dynamics simulation stabilization. The protein is shown as a cartoon model, and the color of each site indicates its energy contribution to ligand binding. The small molecule and its interacting residue main chain or side chain are shown as stick models. Hydrogen bond interactions are shown as long dashed lines, and hydrophobic interactions are shown as short dashed lines. This result indicates that the active catalytic center of the kinase target bound by CLK4 and I-12 and the chemical basis of their interaction are clear, that is, the basis of chemical bond formation is consistent.

[0033] Figure 11 shows the binding ability and chemical bonding details of the biphenyl derivative I-1 and JNK1 during dynamics simulations. A shows the RMSD of the JNK1-I-1 complex as a function of simulation time during the simulation. The results demonstrate that JNK1 binds to I-1 and the resulting complex is stable. B shows the energy decomposition of the interaction between each amino acid site of JNK1 and I-1. The x-axis represents the amino acid sequence of JNK1, and the y-axis represents the energy contribution to ligand binding for each amino acid site. Amino acid sites with a ligand binding energy contribution of less than -0.5 kJ / mol are labeled. C shows the stabilized structure of the JNK1-I-1 complex after molecular dynamics simulations. The protein is shown as a cartoon model, with the color of each site indicating its ligand binding energy contribution. The small molecule and its interacting residues are shown as stick figures. Hydrogen bonds are shown as long dashed lines, and hydrophobic interactions are shown as short dashed lines. This result demonstrates that the active catalytic sites of the kinase targets bound by JNK1 and I-1 and the chemical basis of their interaction are clear, indicating a consistent chemical bond formation.

[0034] Figure 12 shows the binding ability and chemical bonding details of the biphenyl derivative I-4 to JNK1 as determined by dynamics simulations. A shows the RMSD of the JNK1-I-4 complex as a function of simulation time during the simulation. The results demonstrate that JNK1 can bind to I-4, and the resulting complex is stable. B shows the energy decomposition of the interaction between each amino acid site of JNK1 and I-4. The x-axis represents the amino acid sequence of JNK1, and the y-axis represents the energy contribution to ligand binding for each amino acid site. Amino acid sites with a ligand binding energy contribution of less than -0.5 kJ / mol are labeled. C shows the stabilized structure of the JNK1-I-4 complex after molecular dynamics simulations. The protein is shown as a cartoon model, with the color of each site indicating its ligand binding energy contribution. The small molecule and its interacting residues are shown as stick figures. Hydrogen bonds are shown as long dashed lines, and hydrophobic interactions are shown as short dashed lines. This result demonstrates that the active catalytic sites of the kinase targets bound by JNK1 and I-4 and the chemical basis of their interaction are clear, i.e., the chemical bond formation is consistent.

[0035] Figure 13 shows a visualization of the kinome-wide activity analysis of the biphenyl derivative I-1. Each node represents a kinase, and the size and color of the node indicate the inhibitory effect of I-1 on that kinase. The results demonstrate that I-1 has substantial binding activity against each target kinase. The kinome evolutionary tree is available from Cell Signaling Technology: www.cellsignal.com.

[0036] Figure 14 shows the kinase targets of biphenyl derivatives based on kinase activity screening, showing the effect of biphenyl derivative I-1 on the enzymatic activity of 330 kinases (the x-axis shows the name of a kinase every 10 kinases).

[0037] Figure 15 shows the IC values ​​of biphenyl derivatives for target kinases 50 As a result, the data showed that biphenyl derivatives exhibited the ability to bind to the target kinase and had significant antagonistic ability against the target kinase.

[0038] Figure 16 shows that biphenyl derivatives have a significant enhancing effect on the expression of reprogramming core genes OCT4, c-Myc and Lin28A. Compared with the blank control (CK) without added compounds, biphenyl derivatives can simultaneously enhance the expression of OCT4, c-Myc and Lin28A, a group of reprogramming core genes, when present independently. DETAILED DESCRIPTION

[0039] To facilitate understanding of the present application, a more comprehensive description of the present application will be provided below with reference to the accompanying drawings. The accompanying drawings illustrate preferred embodiments of the present application. However, the present application may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the disclosure of the present application.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0041] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0042] In this application, when referring to numerical ranges, unless otherwise specified, the numerical ranges are considered continuous and include the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges subsumed therein.

[0043] Unless otherwise specified, the percentage contents mentioned in this application refer to mass percentage for solid-liquid mixture and solid-solid mixture, and refer to volume percentage for liquid-liquid mixture.

[0044] The percentage concentrations mentioned in this application, unless otherwise specified, refer to the final concentration, which refers to the percentage of the added component in the system after the addition of the component.

[0045] The temperature parameters in this application, unless otherwise specified, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows for temperature fluctuations within the precision range of instrument control.

[0046] Explanation of terms

[0047] As used herein, "ring atoms" refer to atoms that form the backbone of ring A and do not include substituents on ring A. For example, if ring A has the structure shown in A-1-1, its ring atoms are the carbon atoms at the two attachment sites, the two nitrogen atoms, and the carbon atom forming the carbon-nitrogen double bond. The carbon atom in the methyl group substituted on the nitrogen atom is not considered a ring atom.

[0048] "Hydrogen bond donor" refers to a group containing hydrogen atoms connected to atoms with strong electronegativity and small radius, such as oxygen, nitrogen, fluorine, etc., for example, groups containing amino groups, hydroxyl groups, etc. "Hydrogen bond acceptor" refers to a group containing atoms with strong electronegativity and small radius, such as oxygen, nitrogen, fluorine, etc. 1 It can be a hydrogen bond donor or acceptor, inhibiting kinases by forming hydrogen bonds with protein structures.

[0049] "Prodrug" refers to any compound that, when administered to an organism, produces a drug, i.e., an active ingredient, as a result of a spontaneous chemical reaction, an enzyme-catalyzed chemical reaction, photolysis, and / or metabolic chemical reaction. A prodrug is thus a covalently modified analog or latent form of a therapeutically active compound. Suitable examples include, but are not limited to, carboxylate, carbonate, phosphate, nitrate, sulfate, sulfone, sulfoxide, amide, carbamate, azo compound, phosphoramide, glucoside, ether, acetal, and the like forms of the compound.

[0050] "Pharmaceutically acceptable" refers to those ligands, materials, compositions, and / or dosage forms that are suitable for administration to a patient within the scope of sound medical judgment and commensurate with a reasonable benefit / risk ratio.

[0051] A "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. As used herein, the language "pharmaceutically acceptable carrier" includes buffers, sterile water for injection, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with drug administration. Each carrier must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients in the formulation and not harmful to the patient. Suitable examples include, but are not limited to: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch, potato starch, and substituted or unsubstituted β-cyclodextrins; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn starch, and maltose. Rice oil and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer; and (21) other nontoxic compatible substances used in pharmaceutical formulations.

[0052] "Pharmaceutically acceptable salt" refers to a salt suitable for use as a drug formed by any compound of the structure shown with an acid or base. Pharmaceutically acceptable salts include inorganic salts and organic salts. Among them, one type of salt is a salt formed by the compound of the present application with an acid. Acids suitable for forming salts include but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, and naphthalenesulfonic acid; and amino acids such as proline, phenylalanine, aspartic acid, and glutamic acid. Another type of salt is a salt formed by a compound of the present invention and a base. Suitable bases for forming salts include, but are not limited to, alkali metal salts (e.g., sodium salts or potassium salts), alkaline earth metal salts (e.g., magnesium salts or calcium salts), ammonium salts (e.g., lower alkanolammonium salts and other pharmaceutically acceptable amine salts), for example, methylamine salts, ethylamine salts, propylamine salts, dimethylamine salts, trimethylamine salts, diethylamine salts, triethylamine salts, tert-butylamine salts, ethylenediamine salts, hydroxyethylamine salts, dihydroxyethylamine salts, trihydroxyethylamine salts, and amine salts formed from morpholine, piperazine, and lysine, respectively.

[0053] "Pharmaceutically acceptable esters and amides" refer to esters or amides formed by any compound in the structure shown and other compounds that are suitable for use as drugs. Pharmaceutically acceptable esters include organic esters and inorganic esters.

[0054] When the compound represented by Formula I has a hydroxyl group, the compound represented by Formula I can be subjected to a condensation reaction with a carboxylic acid, an acyl chloride, an acid anhydride, etc. according to conventional methods to obtain a pharmaceutically acceptable ester.

[0055] The aforementioned esters may be, for example, C1-C6 alkyl esters such as methyl ester, ethyl ester, propyl ester, isopropyl ester, butyl ester, sec-butyl ester, tert-butyl ester, pentyl ester, and hexyl ester; C3-C6 cycloalkyl esters such as cyclopentyl ester and cyclohexyl ester; C6-C6 cycloalkyl esters such as phenyl ester and naphthyl ester; 10 Aryl esters; benzyl ester, phenylethyl ester, α-methylbenzyl ester, 3-phenylpropyl ester, 4-phenylbutyl ester, 6-phenylhexyl ester, diphenylmethyl ester, triphenylmethyl ester, etc. C6-C 10 Aryl C1-C6 alkyl esters; or esters that can be hydrolyzed in vivo, such as (5-methyl-2-oxo-1,3-dioxol-4-yl)methyl ester, (pivaloyloxy)methyl ester, benzofuranonyl ester, [(isopropoxycarbonyl)oxy]methyl ester, [(cyclohexyloxycarbonyl)oxy]methyl ester, and 1-[(cyclohexyloxycarbonyl)oxy]ethyl ester.

[0056] The aforementioned amides may be, for example, amide (-CONH2); mono-C1-C6 alkylamides or mono-C3-C6 cycloalkylamides such as N-formamide, N-acetamide, N-propionamide, N-isopropylamide, N-butyramide, N-sec-butyramide, N-tert-butyramide, N-pentanamide, N-hexanamide, N-cyclopropanamide, N-cyclopentanamide, and N-cyclohexanamide; or N,N-diformamide, N,N-diethylamide, N,N-dipropylamide, N-isopropylamide, N-butyramide, N-sec-butyramide, N-tert-butyramide, N-pentanamide, N-hexanamide, N-cyclopropanamide, N-cyclopentanamide, and N-cyclohexanamide; N-C1-C6 alkylamides, N-C1-C6 alkyl-N-C3-C6 cycloalkylamides or di-C3-C6 cycloalkylamides such as amide, N,N-diisopropylamide, N-methyl-N-acetamide, N-methyl-N-propionamide, N-methyl-N-butyramide, N-ethyl-N-propionamide, N-ethyl-N-butyramide, N-butyl-N-cyclopentylamide, N-ethyl-N-cyclopropionamide, N,N-dicyclohexylamide, etc.

[0057] "Solvate" refers to a complex formed by the compound represented by general formula (I) and solvent molecules in a specific ratio. "Hydrate" refers to a complex formed by the compound of the present application and water.

[0058] "Active metabolite" refers to an active derivative of a compound that is formed when the compound is metabolized.

[0059] "Polymorphs" refer to compounds of the present invention that exist in different crystal lattice forms.

[0060] "Isotope-labeled" refers to a compound of the present invention that is isotopically labeled. For example, the isotopes in the compound of the present invention may include various isotopes of elements such as H, C, N, O, P, F, and S, such as 2 H. 3 H. 13 C. 14 C. 15 N. 18 O. 17 O. 31 P. 32 P. 35 S. 18 F and 36 S.

[0061] "Isomers" refer to isomers produced by the different spatial arrangement of atoms in a molecule. The compounds of the present application contain structures such as asymmetric or chiral centers, double bonds, etc. Therefore, the compounds of the present application may include multiple isomer forms such as optical isomers, geometric isomers, tautomers, atropisomers, and these isomers and their single isomers, racemates, etc. are all included in the scope of the present application. For example, for optical isomers, optically active (R)- and (S)-isomers and D and L isomers can be prepared by chiral resolution, chiral synthesis or chiral reagents or other conventional techniques. For example, diastereomers can be converted into diastereomers by reacting with appropriate optically active substances (such as chiral alcohols or Mosher's acyl chlorides), which are separated and converted (such as hydrolyzed) into corresponding single isomers. For another example, separation can also be carried out by chromatographic column.

[0062] "Pharmaceutical compositions" may be prepared in a manner well known in the pharmaceutical art and may be administered or applied by a variety of routes depending upon whether local or systemic treatment is desired and upon the area to be treated.

[0063] Administration

[0064] The dosage form and administration method of the compound of the present application or its pharmaceutical composition are not particularly limited.

[0065] Representative routes of administration include, but are not limited to, oral, intratumoral, rectal, parenteral (intravenous, intramuscular, or subcutaneous) injection, and topical administration.

[0066] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets, and pills, the dosage form may also include a buffer. Solid dosage forms such as tablets, dragees, capsules, pills, and granules may be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may include opacifying agents, and the release of the active compound or compounds in such compositions may be delayed in a certain portion of the digestive tract. Examples of embedding components that may be used are polymeric substances and waxes. If desired, the active compound may also be microencapsulated with one or more of the above-mentioned excipients.

[0067] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers, specifically ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures thereof. In addition to these inert diluents, the composition may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances. For example, a suspension may contain a suspending agent, specifically ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide, and agar, or mixtures thereof.

[0068] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous or nonaqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0069] Dosage forms for topical administration include ointments, powders, patches, sprays and inhalants, which are prepared by mixing the active ingredient with a pharmaceutically acceptable carrier and any preservatives, buffers, or propellants as necessary under sterile conditions.

[0070] As used herein, "drug" includes any agent, compound, composition, or mixture that provides a physiological and / or pharmacological effect in vivo or in vitro, often with a beneficial effect. The scope of the physiological and / or pharmacological effect produced by the "drug" in vivo is not particularly limited and may be systemic or localized. The activity of the "drug" is not particularly limited and may be an active substance that interacts with other substances or an inert substance that does not interact.

[0071] In a first aspect of the present application, a biphenyl derivative is provided, wherein the biphenyl derivative has a structure as shown in Formula I, or is a pharmaceutically acceptable salt, ester, amide, solvate, active metabolite, polymorph, isotope-labeled substance, isomer or prodrug of the structure as shown in Formula I;

[0072] wherein Ring A is a five-membered ring substituted or unsubstituted with methyl or amino groups, and the ring atoms of Ring A contain one or two nitrogen atoms;

[0073] R 1 It is a donor or acceptor of hydrogen bonds, and its structure contains one or more of amino, imino, hydroxyl and ether bonds.

[0074] The biphenyl derivatives of the present application are structurally designed. A five-membered ring containing one or two nitrogen atoms is fused to the 2nd and 3rd positions of a benzene ring, and a substituent that can serve as a hydrogen bond donor or acceptor is connected to the 3' position of the other benzene ring. It can bind well to protein targets of kinases such as PDGFRα (belonging to the TK kinase family, more specifically the RTK kinase family), TNIK (belonging to the STE kinase family), CLK4 (belonging to the CMGC kinase family, more specifically the CLK kinase family) and JNK1 (belonging to the CMGC kinase family, more specifically the MAPK kinase family), thereby simultaneously inhibiting the activity of these kinases and regulating the Wnt pathway from the upstream. In addition, it can also significantly increase the expression of reprogramming core genes such as OCT4, Lin28A and c-Myc, which can effectively promote cell reprogramming. This proves that the simultaneous inhibition of multiple kinases upstream of the Wnt pathway can produce a reprogramming effect.

[0075] In some embodiments, R 1 Select one of the following structures:

[0076] Wherein, “*” indicates the connection site.

[0077] In some embodiments, R 1 Select one of the following structures:

[0078] Wherein, “*” indicates the connection site.

[0079] Preferably, R 1 Selected from R-1-1, R-1-2, R-2 or R-3-1.

[0080] In some embodiments, Ring A is selected from one of the following structures:

[0081] Where X is CR 3 R 4 or NR 5 , R 2 ~R 5 Each independently selected from -H, -CH3 or -NH2;

[0082] “*” indicates the attachment site.

[0083] In some embodiments, Ring A is selected from one of the following structures:

[0084] Wherein, “*” indicates the connection site.

[0085] Preferably, the A ring is selected from one of the following structures:

[0086] Wherein, “*” indicates the connection site.

[0087] In some embodiments, the biphenyl derivative has a structure shown in any one of Formulas I-1 to I-18, or is a pharmaceutically acceptable salt, ester, amide, solvate, active metabolite, polymorph, isotope-labeled substance, isomer or prodrug of the structure shown in any one of Formulas I-1 to I-18;

[0088] The inventors of this application designed over 1,900 compounds and, based on comprehensive considerations including target protein calculations, kinase inhibition efficacy, and ease of synthesis, screened 18 compounds represented by Formulas I-1 to I-18. Preferably, the compounds represented by Formulas I-1 to I-4 and I-12 are more effective and easier to synthesize.

[0089] In a second aspect of the present application, a pharmaceutical composition is provided, comprising one or more biphenyl derivatives according to the aforementioned embodiments, and at least one pharmaceutically acceptable carrier.

[0090] The third aspect of the present application provides the use of the biphenyl derivative of one or more of the aforementioned embodiments or the aforementioned pharmaceutical composition in cell reprogramming.

[0091] In some embodiments, when the biphenyl derivative is used for cell reprogramming, the concentration of the biphenyl derivative is 1 μM to 50 μM. Alternatively, the concentration of the biphenyl derivative can be, for example, 2 μM, 4 μM, 6 μM, 8 μM, 10 μM, 12 μM, 14 μM, 16 μM, 18 μM, 20 μM, 22 μM, 24 μM, 26 μM, 28 μM, 30 μM, 32 μM, 34 μM, 36 μM, 38 μM, 40 μM, 42 μM, 44 μM, 46 μM, or 48 μM.

[0092] The fourth aspect of the present application provides a method for cell reprogramming, comprising the following steps:

[0093] The cells are contacted with the biphenyl derivative according to one or more embodiments or the pharmaceutical composition.

[0094] In some embodiments, the concentration of the biphenyl derivative during the contact treatment is 1 μM to 50 μM. Alternatively, the concentration of the biphenyl derivative during the contact treatment can be, for example, 2 μM, 4 μM, 6 μM, 8 μM, 10 μM, 12 μM, 14 μM, 16 μM, 18 μM, 20 μM, 22 μM, 24 μM, 26 μM, 28 μM, 30 μM, 32 μM, 34 μM, 36 μM, 38 μM, 40 μM, 42 μM, 44 μM, 46 μM, or 48 μM.

[0095] The fifth aspect of the present application provides the use of the aforementioned biphenyl derivative or pharmaceutical composition in kinase inhibition, wherein the kinase includes one or more of the TK kinase family, the STE kinase family, and the CMGC kinase family.

[0096] In some embodiments, the kinase comprises one or more of PDGFRα, TNIK, CLK4, and JNK1.

[0097] In a sixth aspect of the present application, a method for inhibiting kinase is provided, comprising the following steps:

[0098] contacting the kinase with the biphenyl derivative of one or more embodiments described above or the pharmaceutical composition described above;

[0099] The kinases include one or more of the TK kinase family, the STE kinase family and the CMGC kinase family.

[0100] In some embodiments, the kinase comprises one or more of PDGFRα, TNIK, CLK4, and JNK1.

[0101] Synthesis routes of compounds I-1 to I-18:

[0102] (1) Synthesis of I-1

[0103] (S)-4-(3-(1-methyl-1H-indazol-4-yl)phenyl)pyrrolidin-2-one

[0104] To a solution of Pd(PPh3)4 (1.05 g, 0.91 mmol) in ethylene glycol dimethyl ether was added a solution of (S)-4-(3-bromophenyl)pyrrolidin-2-one (4.51 g, 18.77 mmol) in ethylene glycol dimethyl ether (60 mL), and the mixture was stirred under nitrogen for 15 minutes. To the mixture from the previous step was added a solution of 1-methyl-1H-indazole-4-boronic acid (5.00 g, 28.40 mmol) in ethanol (15 mL), and the mixture was stirred for 10 minutes. To the resulting mixture was added a 2M Na2CO3 solution (80 mL), and the mixture was refluxed and dried for 20 hours. After cooling the mixture, the organic layer was separated, rinsed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:benzene=5:1) to give (S)-4-(3-(1-methyl-1H-indazol-4-yl)phenyl)pyrrolidin-2-one (5.25 g, 96%). LC-MS m / z=292.1 [M+H] + .

[0105] (2) Synthesis of I-2

[0106] (R)-4-(3-(1-methyl-1H-indazol-4-yl)phenyl)pyrrolidin-2-one

[0107] To a solution of Pd(PPh3)4 (1.05 g, 0.91 mmol) in ethylene glycol dimethyl ether was added a solution of (R)-4-(3-bromophenyl)pyrrolidin-2-one (4.51 g, 18.77 mmol) in ethylene glycol dimethyl ether (60 mL), and the mixture was stirred under nitrogen for 15 minutes. To the mixture from the previous step was added a solution of 1-methyl-1H-indazole-4-boronic acid (5.00 g, 28.40 mmol) in ethanol (15 mL), and the mixture was stirred for 10 minutes. To the resulting mixture was added a 2M Na2CO3 solution (80 mL), and the mixture was dried under reflux for 20 hours. After cooling the mixture, the organic layer was separated, rinsed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:benzene=5:1) to give (R)-4-(3-(1-methyl-1H-indazol-4-yl)phenyl)pyrrolidin-2-one (5.25 g, 96%). LC-MS m / z=292.1 [M+H] + .

[0108] (3) Synthesis of I-3

[0109] (3-Amino-1H-isoindol-7-yl)boronic acid

[0110] Under nitrogen, 1.2 mL of a 1:1 solution of alkyllithium-tetramethylethylenediamine complex (2.8 M in hexane, 3.36 mmol) in anhydrous tetrahydrofuran was added to 20 mL of a solution of 1H-isoindol-3-amine (445.5 mg, 3.37 mmol) in anhydrous tetrahydrofuran over 2.5 minutes. The mixture was stirred at -78°C for 30 minutes. To the mixture was added 403.1 mg of trimethyl borate (3.88 mmol), and the mixture was stirred at -78°C for 5 minutes before the cryostat reaction bath was removed. After the mixture returned to room temperature, the tetrahydrofuran was removed in vacuo, 5% aqueous HCl was added, and the mixture was washed with diethyl ether. The organic layer was separated, dried over magnesium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate: hexane = 3:7) to give (3-amino-1H-isoindol-7-yl)boronic acid (474.5 mg, 80%). LC-MS m / z = 177.1 [M+H] + .

[0111] (S)-4-(3-(3-amino-1H-isoindol-7-yl)phenyl)pyrrolidin-2-one

[0112] To a solution of Pd(PPh3)4 (1.05 g, 0.91 mmol) in ethylene glycol dimethyl ether was added a solution of (R)-4-(3-bromophenyl)pyrrolidin-2-one (4.51 g, 18.77 mmol) in ethylene glycol dimethyl ether (60 mL). The mixture was stirred under nitrogen for 15 minutes. To the mixture from the previous step was added a solution of (3-amino-1H-isoindol-7-yl)boronic acid (5.00 g, 28.40 mmol) in ethanol (15 mL). The mixture was stirred for 10 minutes. To the resulting mixture was added a 2M Na2CO3 solution (80 mL). The mixture was dried under reflux for 20 hours. After cooling, the organic layer was separated, rinsed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:benzene=5:1) to give (R)-4-(3-(3-amino-1H-isoindol-7-yl)phenyl)pyrrolidin-2-one (5.25 g, 96%). LC-MS m / z=292.1 [M+H] + .

[0113] (4) Synthesis of I-4

[0114] 3'-Bromo-[1,1'-biphenyl]-3-amine

[0115] To a solution of Pd(PPh3)4 (1.05 g, 0.91 mmol) in ethylene glycol dimethyl ether was added a solution of 3-iodoaniline (4.10 g, 18.77 mmol) in ethylene glycol dimethyl ether (60 mL), and the mixture was stirred under nitrogen for 15 minutes. A solution of 3-bromophenylboronic acid (5.70 g, 28.40 mmol) in ethanol (15 mL) was added to the mixture from the previous step, and the mixture was stirred for 10 minutes. A 2M Na2CO3 solution (80 mL) was added to the resulting mixture, and the mixture was dried under reflux for 20 hours. After cooling the mixture, the organic layer was separated, rinsed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:benzene = 5:1) to yield 3'-bromo-[1,1'-biphenyl]-3-amine (3.12 g, 67%). LC-MS: m / z = 248.0 [M+H] + .

[0116] 3'-(1-methyl-1H-indazol-4-yl)-[1,1'-biphenyl]-3-amine

[0117] To a solution of Pd(PPh3)4 (1.05 g, 0.91 mmol) in ethylene glycol dimethyl ether was added a solution of 3'-bromo-[1,1'-biphenyl]-3-amine (4.66 g, 18.77 mmol) in ethylene glycol dimethyl ether (60 mL). The mixture was stirred under nitrogen for 15 minutes. To the previous mixture was added a solution of (1-methyl-1H-indazol-4-yl)boronic acid (5.00 g, 28.40 mmol) in ethanol (15 mL). The mixture was stirred for 10 minutes. To the resulting mixture was added a 2M Na2CO3 solution (80 mL). The mixture was dried under reflux for 20 hours. After cooling, the organic layer was separated, rinsed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:benzene=5:1) to give 3'-(1-methyl-1H-indazol-4-yl)-[1,1'-biphenyl]-3-amine (5.39 g, 96%). LC-MS m / z=300.1 [M+H] + .

[0118] (5) Synthesis of I-5

[0119] 3-(1-methyl-1H-indazol-4-yl)aniline

[0120] To a solution of Pd(PPh3)4 (1.05 g, 0.91 mmol) in ethylene glycol dimethyl ether was added a solution of 4-bromo-1-methyl-1H-indazole (3.96 g, 18.77 mmol) in ethylene glycol dimethyl ether (60 mL), and the mixture was stirred under nitrogen for 15 minutes. A solution of 3-aminophenylboronic acid (3.89 g, 28.40 mmol) in ethanol (15 mL) was added to the previous mixture, and the mixture was stirred for 10 minutes. A 2M Na2CO3 solution (80 mL) was added to the resulting mixture, and the mixture was dried under reflux for 20 hours. After cooling the mixture, the organic layer was separated, rinsed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:benzene = 5:1) to yield 3-(1-methyl-1H-indazol-4-yl)aniline (4.02 g, 96%). LC-MS: m / z = 224.1 [M+H] + .

[0121] (S)-2-Methyl-1-((3-(1-methyl-1H-indazol-4-yl)phenyl)amino)-2-butanol

[0122] To a 20 mL pressure tube, (R)-2-ethyl-2-methyloxirane (86.1 mg, 1.0 mmol), 3-(1-methyl-1H-indazol-4-yl)aniline (335.0 mg, 1.5 mmol), and 6.7 mL of reagent-grade DMF were added. The mixture was stirred and sealed at 60°C for 12 hours. After cooling to room temperature, 6.7 mL of deionized water was added, and the mixture was stirred and sealed at 60°C for 12 hours. The solvent was removed by rotary evaporation (22.5 mbar, 35°C), and the residue was purified by silica gel column chromatography (ethyl acetate:hexane = 1:2) to yield (S)-2-methyl-1-((3-(1-methyl-1H-indazol-4-yl)phenyl)amino)-2-butanol (281.6 mg, 91%). LC-MS / MS: m / z = 310.2 [M+H]. + .

[0123] (6) Synthesis of I-6

[0124] N-(3-(1-methyl-1H-indazol-4-yl)phenyl)isobutyramide

[0125] To a solution of Pd(PPh3)4 (1.05 g, 0.91 mmol) in ethylene glycol dimethyl ether was added a solution of 4-bromo-1-methyl-1H-indazole (3.96 g, 18.77 mmol) in ethylene glycol dimethyl ether (60 mL), and the mixture was stirred under nitrogen for 15 minutes. To the mixture from the previous step was added a solution of 3-isobutyramidophenylboronic acid (5.88 g, 28.40 mmol) in ethanol (15 mL), and the mixture was stirred for 10 minutes. To the resulting mixture was added a 2M Na2CO3 solution (80 mL), and the mixture was refluxed and dried for 20 hours. After cooling the mixture, the organic layer was separated, rinsed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:benzene=5:1) to give N-(3-(1-methyl-1H-indazol-4-yl)phenyl)isobutyramide (5.29 g, 96%). LC-MS m / z=294.2 [M+H] + .

[0126] (7) Synthesis of I-7

[0127] (R)-2-Methyl-4-(3-(1-methyl-1H-indazol-4-yl)phenyl)morpholine

[0128] To La[N(SiMe3)2]3 (124 mg, 0.20 mmol, stored and weighed in a fume hood) were added CH2Cl2 (1.25 mL), 4-(3-fluorophenyl)-1-methyl-1H-indazole (200 μL, 0.20 mmol, 1.0 M CH2Cl2 solution), and (R)-2-methylmorpholine (37 μL, 0.22 mmol). The mixture was stirred at room temperature for 1 minute. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH=95:5) to give (R)-2-methyl-4-(3-(1-methyl-1H-indazol-4-yl)phenyl)morpholine (57.2 mg, 93%). LC-MS / MS: m / z=308.2 [M+H]. + .

[0129] (8) Synthesis of I-8

[0130] Ethyl 2-(3-bromophenyl)oxazole-5-carboxylate

[0131] To a solution of Pd(PPh3)4 (1.05 g, 0.91 mmol) in ethylene glycol dimethyl ether was added a solution of ethyl 2-chlorooxazole-5-carboxylate (3.30 g, 18.77 mmol) in ethylene glycol dimethyl ether (60 mL), and the mixture was stirred under nitrogen for 15 minutes. A solution of 3-bromophenylboronic acid (5.70 g, 28.40 mmol) in ethanol (15 mL) was added to the mixture from the previous step, and the mixture was stirred for 10 minutes. A 2M Na2CO3 solution (80 mL) was added to the resulting mixture, and the mixture was refluxed for 20 hours. After cooling the mixture, the organic layer was separated, rinsed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:benzene = 5:1) to afford ethyl 2-(3-bromophenyl)oxazole-5-carboxylate (2.72 g, 49%). The LC-MS / MS spectra were m / z = 296.0 [M+H]. + .

[0132] (2-(3-Bromophenyl)oxazol-5-yl)methanol

[0133] To a methanol solution (100 mL, 0° C.) of ethyl 2-(3-bromophenyl)oxazole-5-carboxylate (11.4 g, 38.6 mmol) was added NaBH4 (4.4 g, 115.8 mmol, 3 eq) in portions. The mixture was stirred at room temperature for 3 hours. Water (100 mL) was added to terminate the reaction, and the product was extracted with ethyl acetate (3×40 mL). The organic layer was separated, rinsed with brine, dried over sodium sulfate, and concentrated under reduced pressure to give (2-(3-bromophenyl)oxazol-5-yl)methanol (9.3 g, 95%). Liquid chromatography mass spectrum: m / z=254.0 [M+H] + .

[0134] (2-(3-(1-methyl-1H-indazol-4-yl)phenyl)oxazol-5-yl)methanol

[0135] To a solution of Pd(PPh3)4 (1.05 g, 0.91 mmol) in ethylene glycol dimethyl ether was added a solution of (2-(3-bromophenyl)oxazol-5-yl)methanol (4.77 g, 18.77 mmol) in ethylene glycol dimethyl ether (60 mL). The mixture was stirred under nitrogen for 15 minutes. To the mixture from the previous step was added a solution of (1-methyl-1H-indazol-4-yl)boronic acid (5.00 g, 28.40 mmol) in ethanol (15 mL), and the mixture was stirred for 10 minutes. To the resulting mixture was added a 2M Na2CO3 solution (80 mL), and the mixture was dried under reflux for 20 hours. After cooling the mixture, the organic layer was separated, rinsed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:benzene=5:1) to give (2-(3-(1-methyl-1H-indazol-4-yl)phenyl)oxazol-5-yl)methanol (5.50 g, 96%). LC-MS m / z=306.1 [M+H] + .

[0136] (9) Synthesis of I-9

[0137] (3-(1-Methyl-1H-indazol-4-yl)phenyl)methanol

[0138] To a solution of Pd(PPh3)4 (1.05 g, 0.91 mmol) in ethylene glycol dimethyl ether was added a solution of 4-bromo-1-methyl-1H-indazole (3.96 g, 18.77 mmol) in ethylene glycol dimethyl ether (60 mL), and the mixture was stirred under nitrogen for 15 minutes. A solution of 3-hydroxymethylphenylboronic acid (4.32 g, 28.40 mmol) in ethanol (15 mL) was added to the previous mixture, and the mixture was stirred for 10 minutes. A 2M Na2CO3 solution (80 mL) was added to the resulting mixture, and the mixture was dried under reflux for 20 hours. After cooling the mixture, the organic layer was separated, rinsed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:benzene = 5:1) to yield (3-(1-methyl-1H-indazol-4-yl)phenyl)methanol (4.29 g, 96%), with a liquid chromatography mass spectrum of m / z = 239.1 [M+H]. + .

[0139] 2-Methyl-1-((3-(1-methyl-1H-indazol-4-yl)phenyl)oxy)isopropanol

[0140] To a solution of 2,2-butylene oxide (15.0 mL, 168 mmol) and (3-(1-methyl-1H-indazol-4-yl)phenyl)methanol (11.0 g, 46 mmol) in toluene (40 mL) was added 50% aq NaOH (12 mL), and the mixture was stirred at 100°C for 30 hours. Water and ethyl acetate were added to the mixture, and the organic layer was separated, washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1) to give 2-methyl-1-((3-(1-methyl-1H-indazol-4-yl)phenyl)oxy)isopropanol (14.2 g, 99%). LC-MS m / z = 311.2 [M+H] + .

[0141] (10) Synthesis of I-10

[0142] 4-(3-((1H-pyrazol-4-yl)oxy)phenyl)-1-methyl-1H-indazole

[0143] Sodium hydride (60%, 256.8 mg, 10.7 mmol) was added to 4-hydroxypyrazole (849.4 mg, 10.1 mmol). After 15 minutes, 4-(3-fluorophenyl)-1-methyl-1H-indazole (2.3 g, 10.2 mmol) was added and stirred at room temperature for 2 hours. The product was extracted with ethyl acetate, and the organic layer was separated and dried. The residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 1:1) to give 4-(3-((1H-pyrazol-4-yl)oxy)phenyl)-1-methyl-1H-indazole (1.47 g, 50%). Liquid chromatography mass spectrum m / z = 291.1 [M+H] + .

[0144] (11) Synthesis of I-11

[0145] (5-(3-(1-methyl-1H-indazol-4-yl)phenyl)oxazol-4-yl)methanol

[0146] To a solution of Pd(PPh3)4 (1.05 g, 0.91 mmol) in ethylene glycol dimethyl ether was added a solution of (5-(3-chlorophenyl)oxazol-4-yl)methanol (3.93 g, 18.77 mmol) in ethylene glycol dimethyl ether (60 mL), and the mixture was stirred under nitrogen for 15 minutes. To the mixture from the previous step was added a solution of (1-methyl-1H-indazol-4-yl)boronic acid (5.00 g, 28.40 mmol) in ethanol (15 mL), and the mixture was stirred for 10 minutes. To the resulting mixture was added a 2M Na2CO3 solution (80 mL), and the mixture was refluxed and dried for 20 hours. After cooling the mixture, the organic layer was separated, rinsed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:benzene=5:1) to give (5-(3-(1-methyl-1H-indazol-4-yl)phenyl)oxazol-4-yl)methanol (5.50 g, 96%). LC-MS m / z=306.1 [M+H] + .

[0147] (12) Synthesis of I-12

[0148] 2-(3-chlorophenyl)-4-hydroxypyridine

[0149] Under stirring, Aliquat-336 (10% of the substrate weight) was added in one portion to a mixture of 2-(3-chlorophenyl)-4-methoxypyridine (20 mmol) and 47% HBr (4.5 mmol). The mixture was heated to 105±5°C and the reaction progress was continuously monitored by TLC. After the reaction was completed, the mixture was cooled to room temperature and water (25 mL) was added to terminate the reaction. The product was extracted with ethyl acetate (3×30 mL), the organic layer was separated, washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane: ethyl acetate = 2:1) to give 2-(3-chlorophenyl)-4-hydroxypyridine (3.94 g, 96%), liquid phase mass spectrum m / z = 206.0 [M+H] + .

[0150] 2-(3-(1-methyl-1H-indazol-4-yl)phenyl)-4-hydroxypyridine

[0151] To a solution of Pd(PPh3)4 (1.05 g, 0.91 mmol) in ethylene glycol dimethyl ether was added a solution of 2-(3-chlorophenyl)-4-hydroxypyridine (3.86 g, 18.77 mmol) in ethylene glycol dimethyl ether (60 mL), and the mixture was stirred under nitrogen for 15 minutes. To the mixture from the previous step was added a solution of (1-methyl-1H-indazol-4-yl)boronic acid (5.00 g, 28.40 mmol) in ethanol (15 mL), and the mixture was stirred for 10 minutes. To the resulting mixture was added a 2M Na2CO3 solution (80 mL), and the mixture was refluxed and dried for 20 hours. After cooling the mixture, the organic layer was separated, rinsed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:benzene=5:1) to give 2-(3-(1-methyl-1H-indazol-4-yl)phenyl)-4-hydroxypyridine (5.43 g, 96%). LC-MS m / z=302.1 [M+H] + .

[0152] (13) Synthesis of I-13

[0153] (3-(1-Methyl-1H-indazol-4-yl)phenyl)methanol

[0154] To a solution of Pd(PPh3)4 (1.05 g, 0.91 mmol) in ethylene glycol dimethyl ether was added a solution of 4-bromo-1-methyl-1H-indazole (3.96 g, 18.77 mmol) in ethylene glycol dimethyl ether (60 mL), and the mixture was stirred under nitrogen for 15 minutes. A solution of 3-hydroxymethylphenylboronic acid (4.32 g, 28.40 mmol) in ethanol (15 mL) was added to the previous mixture, and the mixture was stirred for 10 minutes. A 2M Na2CO3 solution (80 mL) was added to the resulting mixture, and the mixture was dried under reflux for 20 hours. After cooling the mixture, the organic layer was separated, rinsed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:benzene = 5:1) to yield (3-(1-methyl-1H-indazol-4-yl)phenyl)methanol (4.29 g, 96%), with a liquid chromatography mass spectrum of m / z = 239.1 [M+H]. + .

[0155] 1-Methyl-4-(3-((4-methyl-1H-pyrazol-1-yl)methyl)phenyl)-1H-indazole

[0156] To a 100 mL round-bottom flask were added 2.38 g (10 mmol) of (3-(1-methyl-1H-indazol-4-yl)phenyl)methanol, 1.64 g (20 mmol) of 4-methyl-1H-pyrazole, and 0.26 g (1 mmol) of Ni(ClO4)2. Finally, 20 mL of anhydrous 1,2-dichloroethane was added, and the mixture was stirred at 85°C for 6 hours. After cooling the mixture to room temperature, it was poured into an ice-water mixture, and the product was extracted with dichloromethane (3 × 50 mL). The organic layer was separated, rinsed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:benzene = 5:1) to give 1-methyl-4-(3-((4-methyl-1H-pyrazol-1-yl)methyl)phenyl)-1H-indazole (2.78 g, 92%). LC-MS: m / z = 303.1 [M+H]. + .

[0157] (14) Synthesis of I-14

[0158] 2-Methyl-N-(3-(1-methyl-1H-indazol-4-yl)phenyl)isopropylamine

[0159] To a solution of Pd(PPh3)4 (1.05 g, 0.91 mmol) in ethylene glycol dimethyl ether was added a solution of N-(3-bromobenzyl)-2-methylisopropylamine (4.55 g, 18.77 mmol) in ethylene glycol dimethyl ether (60 mL), and the mixture was stirred under nitrogen for 15 minutes. To the mixture from the previous step was added a solution of (1-methyl-1H-indazol-4-yl)boronic acid (5.00 g, 28.40 mmol) in ethanol (15 mL), and the mixture was stirred for 10 minutes. To the resulting mixture was added a 2M Na2CO3 solution (80 mL), and the mixture was refluxed and dried for 20 hours. After cooling the mixture, the organic layer was separated, rinsed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:benzene=5:1) to give 2-methyl-N-(3-(1-methyl-1H-indazol-4-yl)phenyl)isopropylamine (5.29 g, 96%). LC-MS m / z=294.2 [M+H] + .

[0160] (15) Synthesis of I-15

[0161] 1-(Chloromethyl)-3-iodobenzene

[0162] Under nitrogen, (3-iodophenyl)methanol (41.2 g), toluene (300 mL), and pyridine (0.5 mL) were mixed and stirred at 45°C for 1 hour. Sulfur dichloride (14.0 mL) was added to the mixture at a temperature between 45 and 55°C, and the mixture was heated under reflux for 2 hours. The mixture was cooled to 25°C, and water (300 mL) and toluene (300 mL) were added and mixed. The organic layer was separated, rinsed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (n-heptane:toluene = 1:1) to give 1-(chloromethyl)-3-iodobenzene (42.79 g, 94%). LC-MS m / z = 252.9 [M+H] + .

[0163] 3-(3-iodophenyl)-1-methylazetidin-3-ol

[0164] To a solution of magnesium powder (3.2 g, 131.66 mmol) in diethyl ether (190 mL) was added a solution of 1-(chloromethyl)-3-iodobenzene (31.97 g, 126.62 mmol) in diethyl ether (32 mL), and the mixture was stirred at 45 ° C for 4 hours. After cooling the reactants to room temperature, a solution of 1-methylazetidin-3-one (16.81 g, 197.49 mmol) in diethyl ether (32 mL) at 0 ° C was added and stirred at room temperature overnight. The mixture was placed on ice, and dilute hydrochloric acid (60 mL) and NaHSO4 solution (1.0 M, 50 mL) were added. The product was extracted with ethyl acetate (3×100 mL), the organic layer was separated, rinsed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 1:1) to give 3-(3-iodophenyl)-1-methylazetidin-3-ol (36.46 g, 95%). LC-MS m / z = 304.0 [M+H] + .

[0165] 1-Methyl-3-(3-(1-methyl-1H-indazol-4-yl)phenyl)azetidin-3-ol

[0166] To a solution of Pd(PPh3)4 (1.05 g, 0.91 mmol) in ethylene glycol dimethyl ether was added a solution of 3-(3-iodophenyl)-1-methylazetidine-3-ol (5.69 g, 18.77 mmol) in ethylene glycol dimethyl ether (60 mL), and the mixture was stirred under nitrogen for 15 minutes. To the mixture from the previous step was added a solution of (1-methyl-1H-indazol-4-yl)boronic acid (5.00 g, 28.40 mmol) in ethanol (15 mL), and the mixture was stirred for 10 minutes. To the resulting mixture was added a 2M Na2CO3 solution (80 mL), and the mixture was refluxed and dried for 20 hours. After cooling the mixture, the organic layer was separated, rinsed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:benzene=5:1) to give 1-methyl-3-(3-(1-methyl-1H-indazol-4-yl)phenyl)azetidin-3-ol (5.54 g, 96%). LC-MS m / z=308.2 [M+H] + .

[0167] (16) Synthesis of I-16

[0168] 3-(3-Chlorophenyl)-N-ethylpropionamide

[0169] Under nitrogen, a solution of methyllithium in diethyl ether (1.6 M, 0.8 mL, 0.5 mmol) was mixed with a mixture of copper iodide and anhydrous tetrahydrofuran (12 mL) and stirred on ice for 15 minutes. The ice was then brought to -78°C, and anhydrous hexamethylphosphoramide (1.7 mL, 10 mmol) was added while stirring, followed by a solution of diisobutylaluminum hydride in cyclohexane (1 M, 8.0 mL, 8 mmol). The temperature was maintained at this temperature and stirred for 70 minutes. (E)-3-(3-chlorophenyl)-N-ethylacrylamide (1.0 mmol) was added, and the temperature was maintained at this temperature and stirred for 70 minutes. Methanol (5 mL) was added, and after the mixture returned to room temperature, saturated potassium sodium citrate (20 mL) and diethyl ether (30 mL) were added, and the mixture was stirred at room temperature overnight. The organic layer was separated, rinsed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate: petroleum ether = 1:1) to give 3-(3-chlorophenyl)-N-ethylpropionamide (203.2 mg, 96%), HPLC mass spectrum m / z = 212.1 [M+H] + .

[0170] N-ethyl-3-(3-(1-methyl-1H-indazol-4-yl)phenyl)propanamide

[0171] To a solution of Pd(PPh3)4 (1.05 g, 0.91 mmol) in ethylene glycol dimethyl ether was added a solution of 3-(3-chlorophenyl)-N-ethylpropionamide (3.97 g, 18.77 mmol) in ethylene glycol dimethyl ether (60 mL), and the mixture was stirred under nitrogen for 15 minutes. To the mixture from the previous step was added a solution of (1-methyl-1H-indazol-4-yl)boronic acid (5.00 g, 28.40 mmol) in ethanol (15 mL), and the mixture was stirred for 10 minutes. To the resulting mixture was added a 2M Na2CO3 solution (80 mL), and the mixture was refluxed and dried for 20 hours. After cooling the mixture, the organic layer was separated, rinsed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:benzene=5:1) to give N-ethyl-3-(3-(1-methyl-1H-indazol-4-yl)phenyl)propanamide (5.54 g, 96%). LC-MS m / z=308.2 [M+H] + .

[0172] (17) Synthesis of I-17

[0173] 4-(3-chlorophenyl)-1-methylindazole

[0174] To a solution of Pd(PPh3)4 (1.05 g, 0.91 mmol) in ethylene glycol dimethyl ether was added a solution of 4-bromo-1-methyl-1H-indazole (3.96 g, 18.77 mmol) in ethylene glycol dimethyl ether (60 mL), and the mixture was stirred under nitrogen for 15 minutes. A solution of (3-chlorophenyl)boronic acid (4.44 g, 28.40 mmol) in ethanol (15 mL) was added to the previous mixture, and the mixture was stirred for 10 minutes. A 2M Na2CO3 solution (80 mL) was added to the resulting mixture, and the mixture was dried under reflux for 20 hours. After cooling the mixture, the organic layer was separated, rinsed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:benzene = 5:1) to yield 4-(3-chlorophenyl)-1-methylindazole (4.37 g, 96%). LCMS: m / z = 243.1 [M+H] + .

[0175] 4-(3-(1-methylindazol-4-yl)phenyl)pyridin-3-amine

[0176] To a solution of Pd(PPh3)4 (1.05 g, 0.91 mmol) in ethylene glycol dimethyl ether was added a solution of 4-(3-chlorophenyl)-1-methylindazole (4.37 g, 18.02 mmol) in ethylene glycol dimethyl ether (60 mL). The mixture was stirred under nitrogen for 15 minutes. To the mixture from the previous step was added a solution of (3-aminopyridin-4-yl)boronic acid (3.92 g, 28.40 mmol) in ethanol (15 mL). The mixture was stirred for 10 minutes. To the resulting mixture was added a 2M Na2CO3 solution (80 mL). The mixture was dried under reflux for 20 hours. After cooling, the organic layer was separated, rinsed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:benzene=5:1) to give 4-(3-(1-methylindazol-4-yl)phenyl)pyridin-3-amine (5.19 g, 96%). LC-MS m / z=301.1 [M+H] + .

[0177] (18) Synthesis of I-18

[0178] 3-(3-chlorophenyl)pyrazin-2-ol

[0179] Under stirring, Aliquat-336 (10% of the substrate weight) was added in one portion to a mixture of 2-(3-chlorophenyl)-3-methoxypyrazine (20 mmol) and 47% HBr (4.5 mmol). The mixture was heated to 105±5°C and the reaction progress was continuously monitored by TLC. After the reaction was completed, the mixture was cooled to room temperature and water (25 mL) was added to terminate the reaction. The product was extracted with ethyl acetate (3×30 mL), and the organic layer was separated, washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane: ethyl acetate = 2:1) to give 3-(3-chlorophenyl)pyrazin-2-ol (3.96 g, 96%), with a liquid chromatography mass spectrum of m / z = 207.0 [M+H]. + .

[0180] 3-(3-(1-methylindazol-4-yl)phenyl)pyrazin-2-ol

[0181] To a solution of Pd(PPh3)4 (1.05 g, 0.91 mmol) in ethylene glycol dimethyl ether was added a solution of 3-(3-chlorophenyl)pyrazin-2-ol (3.87 g, 18.77 mmol) in ethylene glycol dimethyl ether (60 mL), and the mixture was stirred under nitrogen for 15 minutes. To the mixture from the previous step was added a solution of (1-methyl-1H-indazol-4-yl)boronic acid (5.00 g, 28.40 mmol) in ethanol (15 mL), and the mixture was stirred for 10 minutes. To the resulting mixture was added a 2M Na2CO3 solution (80 mL), and the mixture was dried under reflux for 20 hours. After cooling the mixture, the organic layer was separated, rinsed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane:benzene=5:1) to give 3-(3-(1-methylindazol-4-yl)phenyl)pyrazin-2-ol (5.44 g, 96%). LC-MS m / z=303.1 [M+H] + .

[0182] Example 1 Virtual screening of target kinases and biphenyl derivative molecules

[0183] Virtual screening is a computational technique used in the drug discovery process to search through small molecule compound libraries to identify small chemical molecules that may bind to drug targets. Compared to traditional high-throughput drug screening techniques, virtual screening has advantages such as high efficiency and cost-effectiveness. Therefore, this application uses virtual screening to predict the effects of compounds. The specific theory and methods are based on "Docking Screens for Novel Ligands Conferring New Biology" (Irwin JJ and Shoichet BK, J Med Chem. 2016 May 12; 59(9): 4103-20).

[0184] Comparative analysis of protein structures showed that PDGFR, TNIK, CLK4, and CLK3 have similar three-dimensional structures (RMSD ≤ 8.4), with the structural similarity between PDGFR and CLK3 (RMSD = 8.4) lower than the structural similarity between any two other proteins (RMSD ≤ 2.9) (Figure 1A). Within the protein structure, the positions of key sites in the active centers of each kinase are highly conserved (Figure 1B). Based on the above analysis, the biphenyl derivatives of the present application have a structural basis for simultaneously inhibiting PDGFR, TNIK, and CLK.

[0185] Using AutoDock Vina software, molecular docking of biphenyl derivative molecules I-1 to I-18 with multiple target proteins was performed. The binding energy and ligand efficiency of each biphenyl derivative molecule docking with the target protein were calculated. The specific docking results are shown in Table 1: Columns 2-5 are the binding free energies (binding energies) of the compounds of the present application with different target proteins calculated by the docking software. The larger the absolute value, the stronger the binding ability of the small molecule ligand to the target protein. The binding energy levels of the compounds involved in this application were predicted. The results showed that the binding energies of the compounds in this application were all far greater than the threshold value of 3 set according to the target characteristics of this application. This result shows that the biphenyl derivative molecules in this application have the ability to bind to the target.

[0186] Table 1. Energy analysis of the binding process between the compounds of the present application and the target protein

[0187] Further, the biphenyl derivative molecules in this application are subjected to dynamic simulation, and molecular dynamics simulation is a computational method, based on Newtonian mechanics and intermolecular interaction force field, to simulate and predict the behavior of molecular systems by numerical integration. Molecular dynamics simulation can provide detailed information on protein structure, dynamics and function, and help understand the conformational changes of proteins, the characteristics of binding sites and the interactions with small molecules. By simulating the interaction of molecules, potential drug molecules can be screened, their interaction modes with proteins can be predicted, and their binding abilities can be evaluated. Therefore, the application tests the chemical basis of biphenyl derivative binding by molecular dynamics simulation, and its principles and methods are shown in "Molecular Dynamics Simulations of Protein-Drug Complexes:A Computational Protocol for Investigating the Interactions of Small-Molecule Therapeutics with Biological Targets and Biosensors" (Hadden JA and Perilla JR, Methods Mol Biol.2018;1762:245-270).

[0188] The stability of each complex system was studied by Amber molecular dynamics simulation software. In the molecular dynamics simulation experiment, ff14SB and Gaff force fields were used for the receptor protein and ligand small molecule respectively. The input files were prepared using Antechamber and Leap programs of Amber tools and the simulation calculation was carried out for 100ns. By introducing 0.15M Na+ and Cl- ions and a side length of A TIP3P water box was used to simulate physiological salt concentrations and neutralize the system. A shaking algorithm was used to constrain the bond lengths of all hydrogen bonds, and the particle-mesh Ewald method was used to calculate long-range electrostatic interactions. The system was minimized for 10,000 steps, followed by another 10,000 steps of water equilibration. The system temperature was gradually equilibrated from 200 K to 300 K over 5,000 steps. After equilibration, the prepared complex system was subjected to molecular dynamics simulations at a constant temperature of 300 K and 1 atmosphere of pressure.

[0189] Molecular dynamics simulations showed that the complex structures of each small molecule and protein target gradually reached a stable state during the 100-ns simulation (Figures 2-10, Series A). The binding energies of each small molecule and protein target were calculated using the MMGPSA program of the Amber tool, as shown in Table 1. Analysis of the energetic contributions of the amino acid sites of each protein target to the binding of the ligand small molecule revealed that I-1, I-4, and I-12 have similar binding sites within the same target (Figures 2-10, Series B). However, the energetic contributions of the same amino acid site to the binding of different small molecules vary. For example, the energetic contributions of LYS43 of TNIK to the binding of I-1, I-4, and I-12 are -0.39 kJ / mol, -2.36 kJ / mol, and -0.58 kJ / mol, respectively. This may be one of the reasons for the different sensitivities of TNIK to the three ligands. Analysis of the complex structure stabilized by molecular dynamics simulation revealed that each small molecule was stably bound to the active catalytic center of the kinase target, and the amino acid sites involved in small molecule binding (binding energy contribution less than -0.5 kJ / mol) mainly interacted directly with the small molecules through hydrogen bonds and hydrophobic interactions (Figures 2-10, series C).

[0190] As shown in Figures 2-4, Figures A show the RMSD of the complexes of TNIK with I-1, I-4, and I-12 as a function of simulation time. These results indicate that TNIK binds to all three, and that the resulting complexes are stable. Figures B show the energy decomposition of the interactions between each amino acid site of TNIK and I-1, I-4, and I-12. The x-axis represents the amino acid sequence of TNIK, and the y-axis represents the energy contribution to ligand binding. Amino acid sites with a contribution of less than -0.5 kJ / mol are labeled. This indicates that the binding sites of TNIK with I-1, I-4, and I-12 are similar. Furthermore, Figures C show the structures of the complexes of TNIK with I-1, I-4, and I-12 after stabilization by molecular dynamics simulation. The protein is displayed as a cartoon model, with the color of each site indicating its contribution to the energy of ligand binding. The small molecule and its interacting residue main chain or side chain are shown as stick models, hydrogen bonding interactions are shown as long dashed lines, and hydrophobic interactions are shown as short dashed lines. This result indicates that the active catalytic sites of the kinase targets bound by TNIK and I-1, I-4, and I-12 and the chemical basis of their interactions are clear, indicating that the chemical bond formation is consistent.

[0191] As shown in Figures 5-7, Figures A show the RMSD of the complexes of PDGFRα with I-1, I-4, and I-12 over simulation time. These results indicate that PDGFRα binds to all three, and that the resulting complexes are stable. Figures B show the energy decomposition of the interactions between each amino acid site of PDGFRα and I-1, I-4, and I-12. The x-axis represents the amino acid sequence of PDGFRα, and the y-axis represents the energy contribution to ligand binding. Amino acid sites with a contribution of less than -0.5 kJ / mol are labeled. This indicates that the binding sites of PDGFRα with I-1, I-4, and I-12 are similar. Furthermore, Figures C show the structures of the complexes of PDGFRα with I-1, I-4, and I-12 after molecular dynamics simulations. The protein is displayed as a cartoon model, with the color of each site indicating its contribution to the energy of ligand binding. The small molecule and its interacting residue main chain or side chain are shown as stick models, hydrogen bonds are shown as long dashed lines, and hydrophobic interactions are shown as short dashed lines. This result indicates that the active catalytic center of PDGFRα binding to kinase targets I-1, I-4, and I-12 and the chemical basis of their interactions are clear, indicating that the chemical bond formation is consistent.

[0192] As shown in Figures 8-10, Figures A show the RMSD of the complexes of CLK4 with I-1, I-4, and I-12 as a function of simulation time. These results demonstrate that CLK4 binds to each of these complexes, and the resulting complexes are stable. Figures B show the energy decomposition of the interactions between each amino acid site of CLK4 and I-1, I-4, and I-12. The x-axis represents the amino acid sequence of CLK4, and the y-axis represents the energy contribution to ligand binding for each amino acid site. Amino acid sites with a contribution of less than -0.5 kJ / mol are labeled. This indicates that the binding sites of CLK4 with I-1, I-4, and I-12 are similar. Furthermore, Figures C show the structures of the complexes of CLK4 with I-1, I-4, and I-12 after molecular dynamics simulations stabilize them. The protein is displayed as a cartoon model, with the color of each site indicating its contribution to the energy of ligand binding. The small molecule and its interacting residue main chain or side chain are shown as stick models, hydrogen bonding interactions are shown as long dashed lines, and hydrophobic interactions are shown as short dashed lines. This result indicates that the active catalytic center of CLK4 binding to the kinase targets I-1, I-4, and I-12 and the chemical basis of their interactions are clear, that is, the chemical bond formation is consistent.

[0193] As shown in Figures 11-12, Figures A show the RMSD of the complexes of JNK1 with I-1 and I-4 over simulation time. These results demonstrate that JNK1 binds to both I-1 and I-4, and the resulting complexes are stable. Figures B show the energy decomposition of the interactions between each amino acid site of JNK1 and I-1 and I-4. The x-axis represents the amino acid sequence of JNK1, and the y-axis represents the energy contribution to ligand binding. Amino acid sites with a ligand binding energy contribution of less than -0.5 kJ / mol are labeled. This indicates that the binding sites of JNK1 with I-1 and I-4 are similar. Furthermore, Figures C show the structures of the complexes of JNK1 with I-1 and I-4 after stabilization in molecular dynamics simulations. The proteins are shown as cartoon models, with the color of each site indicating its energy contribution to ligand binding. The small molecule and its interacting residues are shown as stick figures. Hydrogen bonds are shown as long dashed lines, and hydrophobic interactions are shown as short dashed lines. This result indicates that the active catalytic center of the kinase targets bound by JNK1 and I-1 and I-4 and the chemical basis of their interaction are clear, that is, the basis of chemical bond formation is consistent.

[0194] In summary, the biphenyl derivatives in the present application have a structure that binds to a series of kinase targets and has a chemical basis for stable binding.

[0195] Example 2 Biochemical Screening of Biphenyl Derivatives and Target Kinases

[0196] Based on the results in Example 1, the biphenyl derivatives in the present application were screened for in vitro kinase activity, thereby further confirming the actual function of the biphenyl derivatives in the present application with the target kinase. By screening the inhibitory effect of activity on 330 kinases, during the kinase activity test, the kinase (15-50nM, 2.5μL, prepared in test buffer) was first mixed with the biphenyl derivative I-1 (10mM, 25nL, prepared in DMSO) and incubated at 25°C for 10 minutes. Then, a mixture of kinase peptide substrate (0.2mg / ml; supplier: GenScript) and ATP (10-60μM; supplier: Promega; product number: V915B) was added to the mixed system (a total of 2.5μL, prepared in test buffer), and the mixture was reacted at 25°C for 60-120 minutes. Finally, the reaction product was quantitatively detected using HTRF or ADP-Glo ​​method. The assay buffer consisted of 50 mM HEPES (supplier: Thermo Fisher; product number: 15630080), 10 mM MgCl2 (supplier: Sigma; product number: M1028), 0.01% Brij35 (supplier: Millipore; product number: 1018940100), 1 mM EGTA (supplier: Sigma; product number: E3889), and 2 mM DTT (supplier: MCE; product number: HY-15917). During the kinase activity assay, the consumption of each substrate was less than 10%. Two technical replicates were performed for each kinase activity assay. The results demonstrate that biphenyl derivative I-1 (Figures 13-14) can significantly inhibit multiple target kinases. This result further confirms the calculations and predictions in Example 1.

[0197] Example 3 IC of biphenyl derivatives and target kinases 50 filter

[0198] In order to further evaluate the binding ability of biphenyl derivatives to target kinases, IC50 was used to detect the inhibitory effect of biphenyl derivatives on target kinases. 50 Indicates the concentration of a drug or inhibitor required to inhibit the activity of a specified kinase by half. In pharmacy, it is used to characterize the antagonist's antagonistic ability in in vitro experiments. 50The specific operation steps are as follows: "Assessing the Inhibitory Potential of Kinase Inhibitors In Vitro: Major Pitfalls and Suggestions for Improving Comparability of Data Using CK1Inhibitors as an Example" (Roth A, et al., Molecules. 2021 Aug 12; 26(16): 4898).

[0199] First, a 2× ATP and substrate mixture and a 2× TNIK and MgCl2 mixture were prepared using assay buffer. Then, 40 nL of the small molecule and 2 μL of the 2× TNIK and MgCl2 mixture were transferred to a 384-well plate, mixed at room temperature, and incubated for 10 minutes. Then, 2 μL of the 2× ATP and substrate mixture was transferred to the 384-well plate and reacted at room temperature for 60 minutes. 4 μL of ADP-Glo ​​reagent (Promega; Product No. V9103) was added to the reaction system and incubated at room temperature for 40 minutes. Finally, 8 μL of kinase detection reagent (Promega; Product No. V9103) was added to the reaction system. After incubation at room temperature for 40 minutes, the fluorescence signal was read using a microplate reader. The concentration gradient of biphenyl derivatives I-1, I-4, and I-12 was set to 50000 μM, 12500 μM, 3125 μM, 781.3 μM, 195.3 μM, 48.83 μM, 12.21 μM, 3.05 μM, 0.76 μM, and 0.19 μM.

[0200] The formula for calculating the % inhibition rate is: % inhibition rate = 100% - (small molecule reading - positive control reading) / (negative control reading - positive control reading) * 100%. The IC of the small molecule is calculated by substituting the % inhibition rate and the Log value of the inhibitor concentration into the nonlinear regression equation. 50 :Y=Bottom+(Top-Bottom) / (1+10^((LogIC 50 =X)*hillslope)), where X is the Log value of the inhibitor concentration; Y is the inhibition rate%. As shown in FIG15 , the biphenyl derivatives I-1, I-4, and I-12 all exhibited binding ability to the target kinase and exhibited significant antagonistic ability to the target kinase.

[0201] Based on Example 2 and Example 3, the above results indicate that the biphenyl derivatives can bind to the target kinase.

[0202] Example 4 Biphenyl derivatives can induce the simultaneous expression of Oct4, Lin28A, and c-Myc reprogramming core genes

[0203] CHIR99021 is a widely used WNT pathway regulatory compound that has a very significant effect on maintaining the pluripotency of mammalian embryos (Meek et al., STEM CELLS. 2007, 31, 10, p. 2104-2115) and is considered to be a potential reprogramming inducer. To further verify whether biphenyl derivatives can initiate cell reprogramming, this application also used CHIR99021 for control experiments.

[0204] Human mesenchymal cells were cultured in T25 medium at a rate of 4 x 10 5 The cells were inoculated and cultured in serum-free Dulbecco's modified Eagle's medium (DMEM-F12 medium), to which 20uM of the above-mentioned biphenyl derivatives were added, at 37°C and 5% carbon dioxide. On the third day, total RNA was extracted using RNeasy Mini or Micro Kit (QIAGEN), and 1mg of RNA was synthesized into cDNA using SuperScript III First-Strand Synthesis System (Invitrogen). Quantitative PCR was labeled and reacted using SYBR Premix Ex Taq (TaKaRa) and Thermal Cycler Dice Real Time System (TaKaRa), and beta-Actin was used as an internal control. All data were analyzed using the delta-Ct method. Each group of experiments was repeated in three groups, and variance statistics were performed. The primer sequences used to identify the coding genes of different cell markers are shown in Table 2. The results, as shown in Figure 16, show that compared to the blank control group (CK) without the biphenyl derivative small molecule and the control group with CHIR99021 alone, only the biphenyl derivative simultaneously induced the expression of Oct4, Lin28A, and c-Myc. Therefore, the use of biphenyl derivatives alone can significantly increase the expression of multiple core reprogramming genes. The simultaneous expression of Oct4, Lin28A, and c-Myc, a core reprogramming gene group, is a necessary condition for the reprogramming of multiple somatic cells.

[0205] Table 2. QPCR primer sequences for compound-responsive genes

[0206] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0207] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art could make several modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the patent in this application shall be based on the appended claims, and the specification and drawings may be used to interpret the claims.

Claims

1. A biphenyl derivative, the biphenyl derivative having the structure shown in Formula I, or being a pharmaceutically acceptable salt, ester, amide, solvate, active metabolite, polymorph, isotope-labeled compound, isomer or prodrug of the structure shown in Formula I; Among them, Ring A is a five-membered ring substituted or unsubstituted with a methyl group or an amino group, and among the ring atoms of Ring A, one or two nitrogen atoms are included; R 1 is a hydrogen bond donor or acceptor, and its structure contains one or more of an amino group, an imino group, a hydroxyl group, and an ether bond.

2. The biphenyl derivative according to claim 1, wherein The R 1 is selected from one of the following structures: wherein, "*" represents the connection site.

3. The biphenyl derivative according to claim 2, characterized in that, Said R 1 is selected from one of the following structures: wherein, "*" represents the connection site.

4. The biphenyl derivative according to claim 1, wherein The A ring is selected from one of the following structures: wherein X is CR 3 R 4 or NR 5 , R 2 ~R 5 are each independently selected from -H, -CH3 or -NH2; "*" represents the connection site.

5. The biphenyl derivative according to claim 3, characterized in that, The A ring is selected from one of the following structures: wherein, "*" represents the connection site.

6. The biphenyl derivative according to claim 3, wherein The A ring is selected from one of the following structures: wherein, "*" represents the connection site.

7. The biphenyl derivative according to claim 1, characterized in that, The biphenyl derivative has a structure represented by any one of Formula I-1 to I-18, or is a pharmaceutically acceptable salt, ester, amide, solvate, active metabolite, polymorph, isotope-labeled compound, isomer or prodrug of the structure represented by any one of Formula I-1 to I-18; 8. A pharmaceutical composition, comprising the biphenyl derivative according to any one of claims 1 to 7, and at least one pharmaceutically acceptable carrier.

9. Use of the biphenyl derivative according to any one of claims 1 to 7 or the pharmaceutical composition according to claim 8 in cell reprogramming.

10. A method for cell reprogramming, comprising the following steps: Contacting the cell with the biphenyl derivative according to any one of claims 1 to 7 or the pharmaceutical composition according to claim 8.

11. The method according to claim 10, characterized in that, When performing the contacting treatment, the concentration of the biphenyl derivative is 1 μM to 50 μM.

12. Use of the biphenyl derivative according to any one of claims 1 to 7 or the pharmaceutical composition according to claim 8 in kinase inhibition, wherein, The kinase includes one or more of the TK kinase family, the STE kinase family, and the CMGC kinase family.

13. The application according to claim 12, wherein The kinase includes one or more of PDGFRα, TNIK, CLK4, and JNK1.

14. A method for inhibiting a kinase, comprising the following steps: Contacting the kinase with the biphenyl derivative according to any one of claims 1 to 7 or the pharmaceutical composition according to claim 8; wherein, the kinase includes one or more of the TK kinase family, the STE kinase family, and the CMGC kinase family.

15. The method according to claim 14, characterized in that, The kinase includes one or more of PDGFRα, TNIK, CLK4, and JNK1.

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