WO2025145323A1 - Biphenyl derivative, pharmaceutical composition and use thereof - Google Patents

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

The present application relates to the field of medical technology, and in particular to a biphenyl derivative, a pharmaceutical composition and application thereof.

Background Art

In 2006, Shinya Yamanaka's team proposed a "cocktail" method consisting of four transcription factors, Oct4, Sox2, KlF4 and c-Myc, which can successfully reprogram terminally differentiated skin fibroblasts into stem cells with differentiation pluripotency. Such stem cells are called induced pluripotent stem 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 finally form a variety of adult cells. The introduction of this method breaks through the ethical restrictions on the use of human embryonic stem cells in medicine and greatly expands the application potential of stem cell technology in clinical medicine.

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 tumorigenicity risks caused by the use of viral vectors; in addition, the complex GMP production process of the vector also brings complexity to the clinical supervision of induced pluripotent stem cells. Furthermore, such products will be expensive due to the use of vectors. Therefore, if chemical substances can be used to achieve selective gene regulation, thereby changing the state of expression of reprogramming transcription factors, then ectopic expression of "reprogramming genes" will not be required.

The Wnt/β-catenin pathway is an evolutionarily conserved signaling cascade that is essential 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 many fields.

At present, 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).

The CMGC kinase family is named after the acronyms 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). The structure of CLK is very typical, consisting of an N lobe and a C lobe, which are connected by the "hinge" region of the protein main chain. The structure distributed between the N region and the C region is a catalytic domain of β-strands and α-helices (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 regulating the alternative splicing of key Wnt-related genes (Tam BY et al., Cancer Lett. 2019: S0304383519304732). Typical representatives of 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 different extracellular stimuli such as cytokines, neurotransmitters, hormones, cell stress and cell adhesion. Its typical representatives include JNK1 kinase (c-Jun N-terminal kinase 1).

The STE kinase family is involved in regulating the MAP kinase and CMGC kinase families, and plays a core 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 GTPase (Brown JL et al., Curr Biol. 1996 May 1; 6(5): 598-605). TNIK (Traf2and Nck-interacting kinase, TNIK), a member of the STE kinase family, is also one of the regulatory components of the β-catenin-TCF4 transcription complex. TNIK can promote the activation of downstream target genes of 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 the tumor (Sugano et al., Br J Cancer, 2021, (124): 228-236).

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 a type of tyrosine kinase that is activated by the 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).

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, but they can neither 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.

Summary of the invention

Based on this, it is necessary to provide a biphenyl derivative and a pharmaceutical composition containing the same, which 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 they exist independently, and therefore, can be well applied to cell reprogramming.

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;

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;

^R1 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.

The second aspect of the present application provides a pharmaceutical composition comprising the aforementioned biphenyl derivative and at least one pharmaceutically acceptable carrier.

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

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

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

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.

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

contacting the kinase with the aforementioned biphenyl derivative or pharmaceutical composition;

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

Figure 1 is a comparative analysis of the three-dimensional structures of PDGFR, TNIK, CLK4 and JNK1. 1A is a heat map display of the structural similarity (RMSD) obtained by comparing the protein structures two by two, and the numbers in the figure show the RMSD of the two-way structural comparison. 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 model.

Figure 2 shows the binding ability and chemical bonding details of biphenyl derivative I-1 and TNIK in dynamic simulation. A is a graph showing the RMSD of the complex of TNIK and I-1 changing with simulation time during the simulation process. The results show that TNIK can bind to I-1, and the complex structure formed by the binding is in a stable binding state; B is an energy decomposition diagram of the interaction between each amino acid site of TNIK and I-1. The x-axis is the amino acid sequence of TNIK, and the y-axis is the energy value of each amino acid site contributing to ligand binding. The names of amino acid sites with a contribution of ligand binding energy less than -0.5 kJ/mol have been marked; C is the complex structure of TNIK and I-1 after molecular dynamics simulation stabilization. The protein is displayed as a cartoon model, and the color of each site indicates its energy value of contributing to ligand binding. The main chain or side chain of the small molecule and the residue interacting with it are displayed as a stick model, the hydrogen bond interaction is displayed as a long dashed line, and the hydrophobic interaction is displayed as a short dashed line. This result shows that the active catalytic center of the kinase target bound by TNIK and I-1 and the chemical basis of their interaction are clear, that is, the basis for chemical bond formation is consistent.

Figure 3 shows the binding ability and chemical bonding details of biphenyl derivative I-4 and TNIK in dynamic simulation. A is a graph showing the RMSD of the complex of TNIK and I-1 changing with simulation time during the simulation process. The results show that TNIK can bind to I-4, and the complex structure formed by the binding is in a stable binding state. B is an energy decomposition diagram of the interaction between each amino acid site of TNIK and I-4. The x-axis is the amino acid sequence of TNIK, and the y-axis is the energy value of each amino acid site contributing to ligand binding. The names of amino acid sites that contribute ligand binding energy less than -0.5 kJ/mol have been marked; C is the complex structure of TNIK and I-4 after molecular dynamics simulation stabilization. The protein is displayed as a cartoon model, and the color of each site indicates the energy value of its contribution to ligand binding. The main chain or side chain of the small molecule and the residues that interact with it are displayed as a stick model, the hydrogen bond interaction is displayed as a long dashed line, and the hydrophobic interaction is displayed as a short dashed line. This result shows that the active catalytic center of the kinase target bound by TNIK and I-4 and the chemical basis of their interaction are clear, that is, the basis for chemical bond formation is consistent.

Figure 4 shows the binding ability and chemical bonding details of biphenyl derivative I-12 and TNIK in dynamic simulation. A is a graph showing the change of RMSD of the complex of TNIK and I-12 with simulation time during the simulation process. The results show that TNIK can bind to I-12, and the complex structure formed by the binding is in a stable binding state; B is an energy decomposition diagram of the interaction between each amino acid site of TNIK and I-12. The x-axis is the amino acid sequence of TNIK, and the y-axis is the energy value of each amino acid site contributing to ligand binding. The names of amino acid sites that contribute ligand binding energy less than -0.5 kJ/mol are marked; C is the complex structure of TNIK and I-12 after molecular dynamics simulation stabilization. The protein is displayed as a cartoon model, and the color of each site indicates its energy value contributing to ligand binding. The main chain or side chain of the small molecule and the residue interacting with it is displayed as a stick model, the hydrogen bond interaction is displayed as a long dashed line, and the hydrophobic interaction is displayed as a short dashed line. 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 for chemical bond formation is consistent.

Figure 5 shows the binding ability and chemical bonding details of the biphenyl derivative I-1 and PDGFRα during the dynamics simulation. A is a graph showing the changes in the RMSD of the complex of PDGFRα and I-1 over the simulation time during the simulation process. The results show that PDGFRα can bind to I-1, and the complex structure formed by the binding is in a stable binding state. B is an energy decomposition diagram of the interaction between each amino acid site of PDGFRα and I-1. The x-axis is the amino acid sequence of PDGFRα, and the y-axis is the energy value of each amino acid site contributing to ligand binding. The names of amino acid sites that contribute ligand binding energy less than -0.5 kJ/mol are marked; C is the complex structure of PDGFRα and I-1 after molecular dynamics simulation stabilization. The protein is displayed as a cartoon model, and the color of each site indicates its energy value contributing to ligand binding. Small molecules and the main chains or side chains of residues that interact with them are displayed as stick models, and hydrogen bond interactions are displayed as long dashed lines. The 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.

Figure 6 shows the binding ability and chemical bonding details of biphenyl derivative I-4 and PDGFRα in dynamic simulation. A is a graph showing the change of RMSD of the complex of PDGFRα and I-1 with simulation time during the simulation process. The results show that PDGFRα can bind to I-1, and the complex structure formed by the binding is in a stable binding state. B is an energy decomposition diagram of the interaction between each amino acid site of PDGFRα and I-4. The x-axis is the amino acid sequence of PDGFRα, and the y-axis is the energy value of each amino acid site contributing to ligand binding. The names of amino acid sites that contribute less than -0.5 kJ/mol to ligand binding are marked; C is the complex structure of PDGFRα and I-4 after molecular dynamics simulation stabilization. The protein is displayed as a cartoon model, and the color of each site indicates its energy value contributing to ligand binding. The main chain or side chain of the small molecule and the residue interacting with it is displayed as a stick model, the hydrogen bond interaction is displayed as a long dashed line, and the hydrophobic interaction is displayed as a short dashed line. 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 for chemical bond formation is consistent.

Figure 7 shows the binding ability and chemical bonding details of biphenyl derivative I-12 and PDGFRα in dynamic simulation. A is a graph showing the change of RMSD of the complex of PDGFRα and I-12 with simulation time during the simulation process. The results show that PDGFRα can bind to I-12, and the complex structure formed by the binding is in a stable binding state. B is an energy decomposition diagram of the interaction between each amino acid site of PDGFRα and I-12. The x-axis is the amino acid sequence of PDGFRα, and the y-axis is the energy value of each amino acid site contributing to ligand binding. The names of amino acid sites that contribute ligand binding energy less than -0.5 kJ/mol are marked; C is the complex structure of PDGFRα and I-12 after molecular dynamics simulation stabilization. The protein is displayed as a cartoon model, and the color of each site indicates the energy value of its contribution to ligand binding. The main chain or side chain of the small molecule and the residue interacting with it is displayed as a stick model, the hydrogen bond interaction is displayed as a long dashed line, and the hydrophobic interaction is displayed as a short dashed line. 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 for chemical bond formation is consistent.

Figure 8 shows the binding ability and chemical bonding details of biphenyl derivative I-1 and CLK4 in dynamic simulation. A is a graph showing the RMSD of the complex of CLK4 and I-1 changing with simulation time during the simulation process. The results show that CLK4 can bind to I-1, and the complex structure formed by the binding is in a stable binding state. B is an energy decomposition diagram of the interaction between each amino acid site of CLK4 and I-1. The x-axis is the amino acid sequence of CLK4, and the y-axis is the energy value of each amino acid site contributing to ligand binding. The names of amino acid sites that contribute ligand binding energy less than -0.5 kJ/mol have been marked; C is the complex structure of CLK4 and I-1 after molecular dynamics simulation stabilization. The protein is displayed as a cartoon model, and the color of each site indicates the energy value of its contribution to ligand binding. The main chain or side chain of the small molecule and the residues that interact with it are displayed as a stick model, the hydrogen bond interaction is displayed as a long dashed line, and the hydrophobic interaction is displayed as a short dashed line. This result shows that the active catalytic center of the kinase target bound by CLK4 and I-1 and the chemical basis of their interaction are clear, that is, the basis for chemical bond formation is consistent.

Figure 9 shows the binding ability and chemical bonding details of the biphenyl derivative I-4 and CLK4 in dynamic simulation. A is a graph showing the change in RMSD of the CLK4 and I-1 complex with simulation time during the simulation process. The results show that CLK4 can bind to I-4, and the complex structure formed by the combination is in a stable binding state. B is an energy decomposition diagram of the interaction between each amino acid site of CLK4 and I-4. The x-axis is the amino acid sequence of CLK4, and the y-axis is the energy value of each amino acid site contributing to ligand binding. The names of amino acid sites that contribute ligand binding energy less than -0.5 kJ/mol are marked; C is the complex structure of CLK4 and I-4 after molecular dynamics simulation stabilization. The protein is displayed as a cartoon model, and the color of each site indicates its contribution to ligand binding. The energy values of the small molecule and the main chain or side chain of the residues interacting with it 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 shows that the active catalytic center of the kinase target bound by CLK4 and I-4 and the chemical basis of their interaction are clear, that is, the basis of chemical bond formation is consistent.

Figure 10 shows the binding ability and chemical bonding details of biphenyl derivative I-12 and CLK4 in dynamic simulation. A is a graph showing the change of RMSD of the complex of CLK4 and I-1 with simulation time during the simulation process. The results show that CLK4 can bind to I-12, and the complex structure formed by the binding is in a stable binding state. B is an energy decomposition diagram of the interaction between each amino acid site of CLK4 and I-12. The x-axis is the amino acid sequence of CLK4, and the y-axis is the energy value of each amino acid site contributing to ligand binding. The names of amino acid sites that contribute ligand binding energy less than -0.5 kJ/mol are marked; C is the complex structure of CLK4 and I-12 after molecular dynamics simulation stabilization. The protein is displayed as a cartoon model, and the color of each site indicates the energy value of its contribution to ligand binding. The main chain or side chain of the small molecule and the residue interacting with it is displayed as a stick model, the hydrogen bond interaction is displayed as a long dashed line, and the hydrophobic interaction is displayed as a short dashed line. 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.

Figure 11 shows the binding ability and chemical bonding details of biphenyl derivative I-1 and JNK1 in dynamic simulation. A is a graph showing the change of RMSD of the complex of JNK1 and I-1 with simulation time during the simulation process. The results show that JNK1 can bind to I-1, and the complex structure formed by the binding is in a stable binding state. B is an energy decomposition diagram of the interaction between each amino acid site of JNK1 and I-1. The x-axis is the amino acid sequence of JNK1, and the y-axis is the energy value of each amino acid site contributing to ligand binding. The names of amino acid sites that contribute ligand binding energy less than -0.5 kJ/mol have been marked; C is the complex structure of JNK1 and I-1 after molecular dynamics simulation stabilization. The protein is displayed as a cartoon model, and the color of each site indicates the energy value of its contribution to ligand binding. The main chain or side chain of the small molecule and the residue interacting with it is displayed as a stick model, the hydrogen bond interaction is displayed as a long dashed line, and the hydrophobic interaction is displayed as a short dashed line. This result shows that the active catalytic center of the kinase target bound by JNK1 and I-1 and the chemical basis of their interaction are clear, that is, the basis for chemical bond formation is consistent.

Figure 12 shows the binding ability and chemical bonding details of biphenyl derivative I-4 and JNK1 in dynamic simulation. A is a graph showing the change of RMSD of the complex of JNK1 and I-4 with simulation time during the simulation process. The results show that JNK1 can bind to I-4, and the complex structure formed by the binding is in a stable binding state. B is an energy decomposition diagram of the interaction between each amino acid site of JNK1 and I-4. The x-axis is the amino acid sequence of JNK1, and the y-axis is the energy value of ligand binding contributed by each amino acid site. The names of amino acid sites with a contribution of ligand binding energy less than -0.5 kJ/mol have been marked; C is the complex structure of JNK1 and I-4 after molecular dynamics simulation stabilization. The protein is displayed as a cartoon model, and the color of each site indicates the energy value of its contribution to ligand binding. The main chain or side chain of the small molecule and the residue interacting with it is displayed as a stick model, the hydrogen bond interaction is displayed as a long dashed line, and the hydrophobic interaction is displayed as a short dashed line. This result shows that the active catalytic center of the kinase target bound by JNK1 and I-4 and the chemical basis of their interaction are clear, that is, the basis for chemical bond formation is consistent.

Figure 13 is a visualization analysis of the results of the kinase group activity test 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 each kinase. The results show that I-1 has an actual binding effect on each target kinase. The kinase group evolution tree is referenced from Cell Signaling Technology: www.cellsignal.com.

FIG14 is a kinase target screening analysis based on kinase activity of biphenyl derivatives. It shows the effects of biphenyl derivative I-1 on 330 kinases. Analysis of the effect of enzyme activity (the x-axis shows the name of a kinase every 10 kinases).

FIG. 15 shows the IC ₅₀ results of biphenyl derivatives against target kinases. The data show that biphenyl derivatives exhibit binding ability to target kinases and have significant antagonistic ability against target kinases.

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 they exist independently.

DETAILED DESCRIPTION

In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present application are given in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

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

In the present 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.

In this application, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are deemed to be continuous and include the minimum and maximum values of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.

The percentage contents involved in this application, unless otherwise specified, refer to mass percentage for solid-liquid mixing and solid-solid mixing, and refer to volume percentage for liquid-liquid mixing.

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.

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 the temperature to fluctuate within the accuracy range of instrument control.

Explanation of terms

The "ring atoms" described herein refer to atoms that participate in forming the skeleton of ring A, excluding substituents on ring A. For example, if ring A has the structure shown in A-1-1, its ring atoms are the carbon atoms of the two connection sites, the two nitrogen atoms, and the carbon atoms that form the carbon-nitrogen double bond, and the carbon atom in the substituted methyl group on the nitrogen atom does not belong to the ring atoms.

"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., such as 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. In this application, ^R1 can be a hydrogen bond donor or acceptor, which inhibits kinases by forming hydrogen bonds with protein structures.

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

"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.

"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, etc. 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 buffered saline; and (21) other non-toxic compatible substances used in pharmaceutical formulations.

"Pharmaceutically acceptable salt" refers to a salt suitable for use as a drug formed by any compound in the structure shown and 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 and 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 application and a base. Suitable bases for forming salts include but are not limited to alkali metal salts (such as sodium salts or potassium salts), alkaline earth metal salts (such as magnesium salts or calcium salts), ammonium salts (such as 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.

"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.

In the case where 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 a conventional method to obtain a pharmaceutically acceptable ester.

The aforementioned esters may be, for example, C ₁ -C ₆ alkyl esters such as methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl, and hexyl; C ₃ -C ₆ cycloalkyl esters such as cyclopentyl and cyclohexyl; C 6 -C ₁₀ aryl esters such as phenyl and naphthyl; C _{6 -C 10} aryl C ₁ -C ₆ alkyl esters such as benzyl, phenethyl, α-methylbenzyl, 3-phenylpropyl, 4-phenylbutyl, 6-phenylhexyl, diphenylmethyl, and triphenylmethyl; or esters hydrolyzable in vivo such as (5-methyl-2-oxo-1,3-dioxol-4-yl)methyl, (pivaloyloxy)methyl, benzofuranonyl, [(isopropoxycarbonyl)oxy]methyl, [(cyclohexyloxycarbonyl)oxy]methyl, and 1-[(cyclohexyloxycarbonyl)oxy]ethyl.

The aforementioned amides may be, for example, amide (—CONH ₂ ); mono-C 1 -C 6 alkylamides or mono-C ₃ -C ₆ cycloalkylamides such as N-formamide, N-acetamide, N-propionamide, N-isopropionamide, N-butyramide, N-sec-butyramide, N-tert-butyramide, N-pentylamide, N-hexylamide, N-cyclopropionamide, N-cyclopentylamide, and N-cyclohexylamide; or di-C ₁ -C ₆ alkylamides such as N,N-diformamide, N,N-diethylamide, N,N-dipropionamide, N,N-diisopropionamide, 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, and N,N-dicyclohexylamide; or N-C ₁ -C ₆ alkylamides such as N-C _{3 -C 6} alkylamides. C 3 -C ₆ cycloalkylamide or di- _{C 3} -C ₆ cycloalkylamide.

"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.

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

"Polymorph" refers to a compound of the present invention that exists in different crystal lattice forms.

"Isotope-labeled substance" refers to a compound of the present application that is labeled with an isotope. For example, the isotopes in the compound of the present application may include various isotopes of elements such as H, C, N, O, P, F, 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.

"Isomers" refer to isomers produced by different spatial arrangements 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 isomeric forms such as optical isomers, geometric isomers, tautomers, atropisomers, etc., 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, it 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 a chromatographic column.

"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.

Mode of administration

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

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

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound 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, such as starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, such as hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and gum arabic; (c) humectants, such as glycerol; (d) disintegrators, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) dispersants, such as paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may also contain a buffer. Solid dosage forms such as tablets, pills, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifiers, and the release of the active compound or compounds in such compositions can be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active compound can also be formed into microencapsulated form with one or more of the above-mentioned excipients.

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

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

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 that may be required under sterile conditions.

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

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;

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;

^R1 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.

The biphenyl derivatives of the present application are structurally designed to condense a five-membered ring containing one or two nitrogen atoms at the 2nd and 3rd positions of a benzene ring, and to connect a substituent that can serve as a donor or acceptor of a hydrogen bond at the 3' position of another benzene ring, and 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, and can effectively promote cell reprogramming. This proves that the simultaneous inhibition of multiple kinases upstream of the Wnt pathway can produce a reprogramming effect.

In some embodiments, R ¹ is selected from one of the following structures:

Wherein, “*” indicates the connection site.

In some embodiments, R ¹ is selected from one of the following structures:

Wherein, “*” indicates the connection site.

Preferably, ^R1 is selected from R-1-1, R-1-2, R-2 or R-3-1.

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

Wherein, X is CR ³ R ⁴ or NR ⁵ , and R ² to R ⁵ are independently selected from -H, -CH ₃ or -NH ₂ ;

“*” indicates the attachment site.

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

Wherein, “*” indicates the connection site.

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

Wherein, “*” indicates the connection site.

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;

The inventors of the present application have comprehensively considered and selected 18 compounds shown in formulas I-1 to I-18 from more than 1,900 compounds designed, based on the calculation results of target proteins, kinase inhibition effects, and the difficulty of synthesis. Preferably, the compounds shown in formulas I-1 to I-4 and I-12 have better effects and are easy to synthesize.

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

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.

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.

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

The cells are contacted with the biphenyl derivative according to one or more embodiments described above or the pharmaceutical composition described above.

In some embodiments, when the contact treatment is performed, the concentration of the biphenyl derivative is 1 μM to 50 μM. Optionally, when the contact treatment is performed, 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.

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.

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

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

contacting the kinase with the biphenyl derivative of one or more of the aforementioned embodiments or the aforementioned pharmaceutical composition;

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

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

Synthesis route of compounds I-1 to I-18:

(1) Synthesis of I-1

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

To a solution of Pd(PPh ₃ ) ₄ (1.05 g, 0.91 mmol) in ethylene glycol dimethyl ether, add a solution of (S)-4-(3-bromophenyl)pyrrolidin-2-one (4.51 g, 18.77 mmol) in ethylene glycol dimethyl ether (60 mL), and stir the mixture for 15 minutes under nitrogen protection. To the mixture from the previous step, add a solution of 1-methyl-1H-indazole-4-boronic acid (5.00 g, 28.40 mmol) in ethanol (15 mL), and stir the mixture for 10 minutes. To the resulting mixture, add a 2M Na ₂ CO ₃ solution (80 mL), reflux and dry for 20 hours, cool the mixture, separate the organic layer, rinse with brine, dry over sodium sulfate, and concentrate 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%), liquid chromatography-mass spectrum: m/z=292.1 [M+H] ⁺ .

(2) Synthesis of I-2

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

To a solution of Pd(PPh ₃ ) 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 for 15 minutes under nitrogen protection. A solution of 1-methyl-1H-indazole-4-boronic acid (5.00 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 Na ₂ CO ₃ solution (80 mL) was added to the resulting mixture, and the mixture was refluxed and dried for 20 hours. After the mixture was cooled, 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:benzene=5:1) to give (R)-4-(3-(1-methyl-1H-indazol-4-yl)phenyl)pyrrolidin-2-one (5.25 g, 96%), liquid chromatography mass spectrum m/z=292.1 [M+H] ⁺ .

(3) Synthesis of I-3

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

Under nitrogen protection, 1.2 mL of anhydrous tetrahydrofuran solution of alkyl lithium-tetramethylethylenediamine 1:1 complex (2.8 M hexane solution, 3.36 mmol) was added to 20 mL of anhydrous tetrahydrofuran solution of 1H-isoindole-3-amine (445.5 mg, 3.37 mmol) within 2.5 minutes, and the mixture was stirred at -78°C for 30 minutes. 403.1 mg of trimethyl borate (3.88 mmol) was added to the mixture, and the mixture was stirred at -78°C for 5 minutes before the low temperature constant temperature reaction bath was stopped. After the mixture returned to room temperature, tetrahydrofuran was removed in vacuo, 5% HCl aqueous solution was added, 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%). HPLC mass spectrum: m/z=177.1 [M+H] ⁺ .

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

To a solution of Pd(PPh ₃ ) 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 for 15 minutes under nitrogen protection. A solution of (3-amino-1H-isoindol-7-yl)boric acid (5.00 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 Na ₂ CO ₃ solution (80 mL) was added to the resulting mixture, and the mixture was dried under reflux for 20 hours. After the mixture was cooled, 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:benzene=5:1) to give (R)-4-(3-(3-amino-1H-isoindol-7-yl)phenyl)pyrrolidin-2-one (5.25 g, 96%), liquid chromatography-mass spectrum: m/z=292.1 [M+H] ⁺ .

(4) Synthesis of I-4

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

To a solution of Pd(PPh ₃ ) 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 for 15 minutes under nitrogen protection. 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 Na ₂ CO ₃ solution (80 mL) was added to the resulting mixture, and the mixture was refluxed and dried for 20 hours. After the mixture was cooled, 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: benzene = 5: 1) to give 3'-bromo-[1,1'-biphenyl]-3-amine (3.12 g, 67%), liquid phase mass spectrum m/z = 248.0 [M+H] ⁺ .

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

To a solution of Pd(PPh ₃ ) 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), and the mixture was stirred for 15 minutes under nitrogen protection. The mixture was stirred for 10 minutes with a solution of 1H-indazol-4-yl)boronic acid (5.00 g, 28.40 mmol) in ethanol (15 mL). A 2M Na ₂ CO ₃ solution (80 mL) was added to the resulting mixture and dried under reflux for 20 hours. After the mixture was cooled, 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: benzene = 5: 1) to give 3'-(1-methyl-1H-indazol-4-yl)-[1,1'-biphenyl]-3-amine (5.39 g, 96%), liquid phase mass spectrum m/z = 300.1 [M+H] ⁺ .

(5) Synthesis of I-5

3-(1-Methyl-1H-indazol-4-yl)aniline

To a solution of Pd(PPh ₃ ) 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 for 15 minutes under nitrogen protection. A solution of 3-aminophenylboronic acid (3.89 g, 28.40 mmol) in ethanol (15 mL) was added to the mixture in the previous step, and the mixture was stirred for 10 minutes. A 2M Na ₂ CO ₃ solution (80 mL) was added to the resulting mixture, and the mixture was refluxed and dried for 20 hours. After the mixture was cooled, 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:benzene=5:1) to give 3-(1-methyl-1H-indazol-4-yl)aniline (4.02 g, 96%), liquid mass spectrum m/z=224.1 [M+H] ⁺ .

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

Add (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 to a 20 mL pressure tube. The mixture was sealed and stirred at 60°C for 12 hours. After cooling the reaction to room temperature, 6.7 mL of deionized water was added, and the mixture was sealed and stirred at 60°C for 12 hours. The solvent was removed by rotary evaporator (22.5 mbar, 35°C), and the residue was purified by silica gel column chromatography (ethyl acetate: hexane = 1:2) to obtain (S)-2-methyl-1-((3-(1-methyl-1H-indazol-4-yl)phenyl)amino)-2-butanol (281.6 mg, 91%), liquid phase mass spectrum m/z = 310.2 [M+H] ⁺ .

(6) Synthesis of I-6

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

To a solution of Pd(PPh ₃ ) 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 for 15 minutes under nitrogen protection. A solution of 3-isobutyramidophenylboronic acid (5.88 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 Na ₂ CO ₃ solution (80 mL) was added to the resulting mixture, and the mixture was refluxed and dried for 20 hours. After the mixture was cooled, 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:benzene=5:1) to give N-(3-(1-methyl-1H-indazol-4-yl)phenyl)isobutyramide (5.29 g, 96%), liquid mass spectrum m/z=294.2 [M+H] ⁺ .

(7) Synthesis of I-7

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

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

(8) Synthesis of I-8

Ethyl 2-(3-bromophenyl)oxazole-5-carboxylate

To a solution of Pd(PPh ₃ ) ₄ (1.05 g, 0.91 mmol) in ethylene glycol dimethyl ether was added a solution of 2-chlorooxazole-5-carboxylic acid ethyl ester (3.30 g, 18.77 mmol) in ethylene glycol dimethyl ether (60 mL), and the mixture was stirred for 15 minutes under nitrogen protection. 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 Na ₂ CO ₃ solution (80 mL) was added to the resulting mixture, and the mixture was refluxed and dried for 20 hours. After the mixture was cooled, 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: benzene = 5: 1) to give 2-(3-bromophenyl)oxazole-5-carboxylic acid ethyl ester (2.72 g, 49%), with a liquid phase mass spectrum of m/z = 296.0 [M+H] ⁺ .

(2-(3-Bromophenyl)oxazol-5-yl)methanol

To a methanol solution (100 mL, 0°C) of ethyl 2-(3-bromophenyl)oxazole-5-carboxylate (11.4 g, 38.6 mmol) was added NaBH ₄ (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, washed 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 mass spectrum: m/z=254.0 [M+H] ⁺ .

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

To a solution of Pd(PPh ₃ ) ₄ (1.05 g, 0.91 mmol) in ethylene glycol dimethyl ether was added (2-(3-bromophenyl)oxazol-5-yl)methanol (4.77 g, 18.77 mmol) in ethylene glycol dimethyl ether solution (60 mL), and the mixture was stirred for 15 minutes under nitrogen protection. A solution of (1-methyl-1H-indazol-4-yl)boric acid (5.00 g, 28.40 mmol) in ethanol (15 mL) was added to the mixture in the previous step, and the mixture was stirred for 10 minutes. A 2M Na ₂ CO ₃ solution (80 mL) was added to the resulting mixture, and refluxed for 20 hours. After the mixture was cooled, 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: benzene = 5: 1) to give (2-(3-(1-methyl-1H-indazol-4-yl)phenyl)oxazol-5-yl)methanol (5.50 g, 96%), liquid phase mass spectrum m/z = 306.1 [M+H] ⁺ .

(9) Synthesis of I-9

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

To a solution of Pd(PPh ₃ ) 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 for 15 minutes under nitrogen protection. A solution of 3-hydroxymethylphenylboronic acid (4.32 g, 28.40 mmol) in ethanol (15 mL) was added to the mixture in the previous step, and the mixture was stirred for 10 minutes. A 2M Na ₂ CO ₃ solution (80 mL) was added to the resulting mixture, and the mixture was refluxed and dried for 20 hours. After the mixture was cooled, 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:benzene=5:1) to give (3-(1-methyl-1H-indazol-4-yl)phenyl)methanol (4.29 g, 96%), liquid mass spectrum m/z=239.1 [M+H] ⁺ .

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

50% aqNaOH (12 mL) was added to a toluene (40 mL) 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), 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%), liquid phase mass spectrum m/z = 311.2 [M+H] ⁺ .

(10) Synthesis of I-10

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

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 obtain 4-(3-((1H-pyrazol-4-yl)oxy)phenyl)-1-methyl-1H-indazole (1.47 g, 50%), liquid phase mass spectrum m/z = 291.1 [M+H] ⁺ .

(11) Synthesis of I-11

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

To a solution of Pd(PPh ₃ ) ₄ (1.05 g, 0.91 mmol) in ethylene glycol dimethyl ether, add a solution of (5-(3-chlorophenyl)oxazol-4-yl)methanol (3.93 g, 18.77 mmol) in ethylene glycol dimethyl ether (60 mL), and stir the mixture for 15 minutes under nitrogen protection. To the mixture in the previous step, add a solution of (1-methyl-1H-indazol-4-yl)boric acid (5.00 g, 28.40 mmol) in ethanol (15 mL), and stir the mixture for 10 minutes. To the resulting mixture, add a 2M Na ₂ CO ₃ solution (80 mL), reflux and dry for 20 hours, cool the mixture, separate the organic layer, rinse with brine, dry over sodium sulfate, and concentrate 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%). HPLC mass spectrum m/z=306.1 [M+H] ⁺ .

(12) Synthesis of I-12

2-(3-Chlorophenyl)-4-hydroxypyridine

Aliquat-336 (10% of the weight of the substrate) was added to a mixture of 2-(3-chlorophenyl)-4-methoxypyridine (20 mmol) and 47% HBr (4.5 mmol) under stirring, and the mixture was heated to 105±5°C, and the progress of the reaction was continuously monitored by TLC experiment. 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 2-(3-chlorophenyl)-4-hydroxypyridine (3.94 g, 96%), liquid phase mass spectrum m/z = 206.0 [M+H] ⁺ .

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

To a solution of Pd(PPh ₃ ) 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 for 15 minutes under nitrogen protection. A solution of (1-methyl-1H-indazol-4-yl)boric acid (5.00 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 Na ₂ CO ₃ solution (80 mL) was added to the resulting mixture, and the mixture was refluxed and dried for 20 hours. After the mixture was cooled, 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:benzene=5:1) to give 2-(3-(1-methyl-1H-indazol-4-yl)phenyl)-4-hydroxypyridine (5.43 g, 96%), liquid mass spectrum m/z=302.1 [M+H] ⁺ .

(13) Synthesis of I-13

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

To a solution of Pd(PPh ₃ ) 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 for 15 minutes under nitrogen protection. A solution of 3-hydroxymethylphenylboronic acid (4.32 g, 28.40 mmol) in ethanol (15 mL) was added to the mixture in the previous step, and the mixture was stirred for 10 minutes. A 2M Na ₂ CO ₃ solution (80 mL) was added to the resulting mixture, and the mixture was refluxed and dried for 20 hours. After the mixture was cooled, 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:benzene=5:1) to give (3-(1-methyl-1H-indazol-4-yl)phenyl)methanol (4.29 g, 96%), liquid mass spectrum m/z=239.1 [M+H] ⁺ .

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

2.38 g (10 mmol) (3-(1-methyl-1H-indazol-4-yl)phenyl)methanol, 1.64 g (20 mmol) 4-methyl-1H-pyrazole and 0.26 g (1 mmol) Ni(ClO ₄ ) ₂ were added to a 100 mL round-bottom flask, and finally 20 mL of anhydrous 1,2-dichloroethane was added, and the mixture was stirred at 85°C for 6 hours. After the mixture was cooled 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, washed 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%), liquid phase mass spectrum m/z = 303.1 [M+H] ⁺ .

(14) Synthesis of I-14

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

To a solution of Pd(PPh ₃ ) 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 for 15 minutes under nitrogen protection. A solution of (1-methyl-1H-indazol-4-yl)boric acid (5.00 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 Na ₂ CO ₃ solution (80 mL) was added to the resulting mixture, and the mixture was refluxed and dried for 20 hours. After the mixture was cooled, 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:benzene=5:1) to give 2-methyl-N-(3-(1-methyl-1H-indazol-4-yl)phenyl)isopropylamine (5.29 g, 96%), liquid mass spectrum m/z=294.2 [M+H] ⁺ .

(15) Synthesis of I-15

1-(Chloromethyl)-3-iodobenzene

Under nitrogen protection, (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 range of 45-55°C and heated under reflux for 2 hours. The mixture was cooled to 25°C, water (300 mL) and toluene (300 mL) were added and mixed, 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 (n-heptane: toluene = 1: 1) to obtain 1-(chloromethyl)-3-iodobenzene (42.79 g, 94%), liquid phase mass spectrum m/z = 252.9 [M+H] ⁺ .

3-(3-Iodophenyl)-1-methylazetidin-3-ol

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 the reactants were cooled to room temperature, a solution of 1-methylazetidine-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 NaHSO ₄ solution (1.0 M, 50 mL) were added. The product was extracted with ethyl acetate (3×100 mL), and 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%), liquid mass spectrum m/z = 304.0 [M+H] ⁺ .

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

To a solution of Pd(PPh ₃ ) 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 for 15 minutes under nitrogen protection. A solution of (1-methyl-1H-indazol-4-yl)boronic acid (5.00 g, 28.40 mmol) in ethanol (15 mL) was added to the mixture in the previous step, and the mixture was stirred for 10 minutes. A 2M Na ₂ CO ₃ solution (80 mL) was added to the resulting mixture, and the mixture was refluxed and dried for 20 hours. After the mixture was cooled, 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:benzene=5:1) to give 1-methyl-3-(3-(1-methyl-1H-indazol-4-yl)phenyl)azetidin-3-ol (5.54 g, 96%), liquid mass spectrum m/z=308.2 [M+H] ⁺ .

(16) Synthesis of I-16

3-(3-Chlorophenyl)-N-ethylpropionamide

Under nitrogen protection, a diethyl ether solution of methyl lithium (1.6M, 0.8mL, 0.5mmol) was mixed with a mixture of copper iodide and anhydrous tetrahydrofuran (12mL), and stirred on ice for 15 minutes. The ice was changed to -78°C, and anhydrous hexamethylphosphoric triamide (1.7mL, 10mmol) was added while stirring, followed by a cyclohexane solution of diisobutylaluminum hydride (1M, 8.0mL, 8mmol), and the temperature was maintained and stirred for 70 minutes. (E)-3-(3-chlorophenyl)-N-ethylacrylamide (1.0mmol) was added, and the temperature was maintained and stirred for 70 minutes. Methanol (5mL) was added, and after the mixture naturally returned to room temperature, saturated potassium sodium citrate (20mL) and diethyl ether (30mL) 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%), liquid mass spectrum m/z = 212.1 [M+H] ⁺ .

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

To a solution of Pd(PPh ₃ ) 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 for 15 minutes under nitrogen protection. To the mixture from the previous step was added a solution of (1-methyl-1H-indazol-4-yl)boric acid (5.00 g, 28.40 mmol) in ethanol (15 mL), and the mixture was stirred for 10 minutes. 2M Na ₂ CO ₃ solution (80 mL) was added to the mixture, and the mixture was dried under reflux for 20 hours. After the mixture was cooled, 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: benzene = 5: 1) to give N-ethyl-3-(3-(1-methyl-1H-indazol-4-yl)phenyl)propanamide (5.54 g, 96%), liquid phase mass spectrum m/z = 308.2 [M+H] ⁺ .

(17) Synthesis of I-17

4-(3-Chlorophenyl)-1-methylindazole

To a solution of Pd(PPh ₃ ) 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 for 15 minutes under nitrogen protection. A solution of (3-chlorophenyl)boronic acid (4.44 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 Na ₂ CO ₃ solution (80 mL) was added to the resulting mixture, and the mixture was refluxed and dried for 20 hours. After the mixture was cooled, 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: benzene = 5: 1) to give 4-(3-chlorophenyl)-1-methylindazole (4.37 g, 96%), with a liquid phase mass spectrum of m/z = 243.1 [M+H] ⁺ .

4-(3-(1-methylindazol-4-yl)phenyl)pyridin-3-amine

To a solution of Pd(PPh ₃ ) 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), and the mixture was stirred for 15 minutes under nitrogen protection. A solution of (3-aminopyridin-4-yl)boric acid (3.92 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 Na ₂ CO ₃ solution (80 mL) was added to the resulting mixture, and the mixture was refluxed and dried for 20 hours. After the mixture was cooled, the organic layer was separated, washed with brine, and dried over sodium sulfate. 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%), HPLC mass spectrum: m/z=301.1 [M+H] ⁺ .

(18) Synthesis of I-18

3-(3-Chlorophenyl)pyrazin-2-ol

Aliquat-336 (10% of the weight of the substrate) was added to a mixture of 2-(3-chlorophenyl)-3-methoxypyrazine (20 mmol) and 47% HBr (4.5 mmol) under stirring, and the mixture was heated to 105±5°C, and the progress of the reaction was continuously monitored by TLC experiment. 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)pyrazine-2-ol (3.96 g, 96%), liquid phase mass spectrum m/z = 207.0 [M+H] ⁺ .

3-(3-(1-methylindazol-4-yl)phenyl)pyrazine-2-ol

To a solution of Pd(PPh ₃ ) 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 for 15 minutes under nitrogen protection. To the mixture in 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 Na ₂ CO ₃ solution (80 mL), and the mixture was refluxed and dried for 20 hours. After the mixture was cooled, 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:benzene=5:1) to give 3-(3-(1-methylindazol-4-yl)phenyl)pyrazine-2-ol (5.44 g, 96%), liquid mass spectrum: m/z=303.1 [M+H] ⁺ .

Example 1 Virtual screening of target kinases and biphenyl derivative molecules

Virtual screening is a computational technology that searches small molecule compound libraries through computational methods to obtain chemical small molecules that may bind to drug targets during the drug discovery process. Compared with traditional high-throughput drug screening technology, virtual screening has advantages such as high efficiency and economy. Therefore, this application predicts the effects of compounds through virtual screening. Its specific theories and methods refer to "Docking Screens for Novel Ligands Conferring New Biology" (Irwin JJ and Shoichet BK, J Med Chem. 2016 May 12; 59(9): 4103-20).

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

The AutoDock Vina software was used to perform molecular docking of biphenyl derivative molecules I-1 to I-18 with multiple target proteins, and the binding energy and ligand efficiency of the docking results of each biphenyl derivative molecule with the target protein were calculated. The specific docking results are shown in Table 1: The 2nd to 5th columns were calculated by the docking software to obtain the binding free energy (binding energy) of the compounds of the present application with different target proteins. The larger the absolute value, the stronger the binding ability of the small molecule ligand with the target protein. The binding energy level of the compounds involved in this application was predicted, and the results showed that the binding energy of the compounds in this application was much greater than the threshold value 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.

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

Furthermore, the biphenyl derivative molecules in the present application were subjected to dynamic simulation. Molecular dynamics simulation is a computational method that simulates and predicts the behavior of molecular systems through numerical integration based on the principles of Newtonian mechanics and the force field of intermolecular interactions. Molecular dynamics simulation can provide Provide detailed information about protein structure, dynamics and function, 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 present application tests the chemical basis of the binding of biphenyl derivatives by molecular dynamics simulation, and its principles and methods refer to "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).

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 performed for 100ns. The TIP3P water box was used to simulate physiological salt concentration and neutralize the whole system. The shaking algorithm was used to constrain the bond lengths of all hydrogen bonds, and the particle mesh Ewald method was used to calculate the long-range electrostatic interactions. The system was minimized for 10,000 steps and then equilibrated with water for 10,000 steps. The temperature of the system was gradually equilibrated from 200K to 300K in 5000 steps. After equilibration, the prepared complex system was subjected to molecular dynamics simulation at a constant temperature of 300K and 1 atm.

The results of molecular dynamics simulation calculations show that during the 100ns simulation calculation process, the complex structures of each small molecule and protein target gradually reached a stable state (Figure 2-Figure 10, series A). The binding energy of each small molecule and protein target calculated using the MMGPSA program of the Amber tool is shown in Table 1. By analyzing the energy contribution of the amino acid sites of each protein target to the binding of the ligand small molecule, it was found that I-1, I-4 and I-12 have similar binding sites in the same target (Figure 2-Figure 10, series B); however, the energy contribution of the same amino acid site to the binding of different small molecules is different, such as the energy contribution of LYS43 of TNIK to binding I-1, I-4 and I-12 is -0.39kJ/mol, -2.36kJ/mol and -0.58kJ/mol, respectively, which may be one of the reasons for the difference in sensitivity of TNIK to the three. Analysis of the complex structure after stabilization 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 the binding of the small molecules (binding energy contribution less than -0.5 kJ/mol) directly interacted with the small molecules mainly through hydrogen bonds and hydrophobic interactions (Figures 2-10, series C).

As shown in Figures 2-4, the A series of figures are the RMSD of the complexes of TNIK and I-1, I-4 and I-12 during the simulation process, which changes with the simulation time. The results show that TNIK can bind to I-1, I-4 and I-12, and the complex structure formed by the binding is in a stable binding state; the B series of figures are the energy decomposition diagrams of the interaction between each amino acid site of TNIK and I-1, I-4 and I-12, the x-axis is the amino acid sequence of TNIK, the y-axis is the energy value of the ligand binding contribution of each amino acid site, and the names of the amino acid sites that contribute less than -0.5 kJ/mol to the ligand binding energy have been marked. This result shows that the binding sites of TNIK and I-1, I-4 and I-12 are similar; further, the C series of figures are the complex structures of TNIK and I-1, I-4 and I-12 after molecular dynamics simulation stabilization. The protein is displayed as a cartoon model, and the color of each site indicates the energy value of its contribution to ligand binding. The small molecule and the main chain or side chain of the residues that interact with it are displayed as a stick model, the hydrogen bond interaction is displayed as a long dashed line, and the hydrophobic interaction is displayed as a short dashed line. This result shows that the active catalytic center of the kinase target bound by TNIK and I-1, I-4 and I-12 and the chemical basis of their interaction are clear, that is, the basis of chemical bond formation is consistent.

As shown in Figures 5 to 7, the A series of graphs are graphs showing the changes in RMSD of the complexes of PDGFRα with I-1, I-4 and I-12 over the simulation time during the simulation process. The results show that PDGFRα can bind to I-1, I-4 and I-12, and the complex structure formed by the binding is in a stable binding state; The B series of figures are energy decomposition diagrams of the interaction between each amino acid site of PDGFRα and I-1, I-4 and I-12. The x-axis is the amino acid sequence of PDGFRα, and the y-axis is the energy value of each amino acid site contributing to ligand binding. The names of amino acid sites that contribute less than -0.5 kJ/mol to ligand binding are marked. This result shows that the sites of PDGFRα binding to I-1, I-4 and I-12 are similar; further, the C series of figures are the complex structures of PDGFRα and I-1, I-4 and I-12 stabilized by molecular dynamics simulation. The protein is displayed as a cartoon model, and the color of each site indicates the energy value of its contribution to ligand binding. The main chain or side chain of small molecules and the residues that interact with it are displayed as stick models, hydrogen bond interactions are displayed as long dashed lines, and hydrophobic interactions are displayed as short dashed lines. This result shows that the active catalytic center of the kinase target bound by PDGFRα and I-1, I-4 and I-12 and the chemical basis of their interaction are clear, that is, the basis for chemical bond formation is consistent.

As shown in Figures 8 to 10, the A series of graphs are the RMSD of the complexes of CLK4 and I-1, I-4 and I-12 during the simulation process, which changes with the simulation time. The results show that CLK4 can bind to I-1, I-4 and I-12, and the complex structure formed by the binding is in a stable binding state; the B series of graphs are the energy decomposition diagrams of the interaction between each amino acid site of CLK4 and I-1, I-4 and I-12, the x-axis is the amino acid sequence of CLK4, the y-axis is the energy value of the ligand binding contribution of each amino acid site, and the names of the amino acid sites that contribute to the ligand binding energy less than -0.5 kJ/mol have been marked. This result shows that the binding sites of CLK4 and I-1, I-4 and I-12 are similar; further, the C series of graphs are the complex structures of CLK4 and I-1, I-4 and I-12 after molecular dynamics simulation stabilization. The protein is displayed as a cartoon model, and the color of each site indicates the energy value of its contribution to ligand binding. The small molecule and the main chain or side chain of the residues that interact with it are displayed as a stick model, the hydrogen bond interaction is displayed as a long dashed line, and the hydrophobic interaction is displayed as a short dashed line. This result shows that the active catalytic center of the kinase target bound by CLK4 and I-1, I-4 and I-12 and the chemical basis of their interaction are clear, that is, the basis of chemical bond formation is consistent.

As shown in Figures 11 and 12, the A series of graphs are the RMSD of the complex of JNK1 with I-1 and I-4 during the simulation process, and the results show that JNK1 can bind to both I-1 and I-4, and the complex structure formed by the binding is in a stable binding state; the B series of graphs are the energy decomposition diagrams of the interaction between each amino acid site of JNK1 and I-1 and I-4, the x-axis is the amino acid sequence of JNK1, the y-axis is the energy value of each amino acid site contributing to ligand binding, and the names of amino acid sites that contribute ligand binding energy less than -0.5kJ/mol have been marked. This result shows that the binding sites of JNK1 and I-1 and I-4 are similar; further, the C series of graphs are the complex structures formed by JNK1 and I-1 and I-4 after molecular dynamics simulation stabilization. The protein is displayed as a cartoon model, and the color of each site indicates the energy value of its contribution to ligand binding. The main chain or side chain of the small molecule and the residue interacting with it are displayed as a stick model, the hydrogen bond interaction is displayed as a long dashed line, and the hydrophobic interaction is displayed as a short dashed line. This result indicates that the chemical basis of the active catalytic center of the kinase targets bound by JNK1 and I-1 and I-4 and their interactions is clear, that is, the basis for chemical bond formation is consistent.

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.

Example 2 Biochemical screening of biphenyl derivatives and target kinases

According to the results in Example 1, the biphenyl derivatives in the present application were screened for in vitro kinase spectrum, so as to further confirm the actual function of the biphenyl derivatives in the present application with the target kinase. By screening the inhibitory effect of activity in 330 kinases, during the kinase activity test, the kinase (15-50nM, 2.5μL, prepared in test buffer) and the biphenyl derivative I-1 (10mM, 25nL, prepared in DMSO) were first mixed and incubated at 25°C for 10 minutes, and then the kinase peptide substrate (0.2mg/ml; supplier: GenScript) was added to the mixed system. ATP (10-60 μM; supplier: Promega; product number: V915B) mixture (total 2.5 μL, prepared in assay buffer), incubate at 25°C for 60-120 minutes, and finally quantify the reaction product using HTRF or ADP-Glo method. The assay buffer composition is: 50 mM HEPES (supplier: Thermo Fisher; product number: 15630080), 10 mM MgCl ₂ (supplier: Sigma; product number: M1028), 0.01% Brij35 (supplier: Millipore; product number: 1018940100), 1 mM EGTA (supplier: Sigma; product number: E3889), 2 mM DTT (supplier: MCE; product number: HY-15917). During the kinase activity test, the consumption of each substrate was less than 10%. Two technical replicates were set for each kinase activity test. The results show that the biphenyl derivative I-1 (Figures 13-14) can significantly inhibit multiple target kinases. This result further confirms the calculation and prediction in Example 1.

Example 3 IC ₅₀ screening of biphenyl derivatives and target kinases

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. In this experiment, IC ₅₀ represents the concentration of drug or inhibitor required to inhibit the activity of a specified kinase by half. In pharmacy, it is used to characterize the antagonistic ability of antagonists in in vitro experiments. The specific operation steps of IC ₅₀ refer to "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).

First, 2×ATP and substrate mixed solution and 2×TNIK and MgCl2 mixed solution were prepared using test buffer respectively; then 40nL of small molecules and 2μL of 2×TNIK and MgCl2 mixed solution were transferred to a 384-well plate, mixed at room temperature and incubated for 10 minutes; then 2μL of 2×ATP and substrate mixed solution was transferred to a 384-well plate and reacted at room temperature for 60 minutes; 4μL of ADP-Glo reagent (supplier: Promega; product number: V9103) was added to the reaction system and incubated at room temperature for 40 minutes; finally, 8μL of kinase detection reagent (supplier: Promega; product number: V9103) was added to the reaction system, and after incubation at room temperature for 40 minutes, the fluorescence signal was read using an enzyme 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.

The inhibition rate % was calculated as follows: inhibition rate % = 100% - (small molecule reading - positive control reading) / (negative control reading - positive control reading) * 100%. The IC ₅₀ of the small molecule was calculated by substituting the inhibition rate % and the Log value of the inhibitor concentration into the nonlinear regression equation: Y = Bottom + (Top-Bottom) / (1 + 10^((LogIC ₅₀ -X)*hillslope)), X is the Log value of the inhibitor concentration; Y is the inhibition rate %. As shown in FIG. 15 , the biphenyl derivatives I-1, I-4 and I-12 all showed the ability to bind to the target kinase and showed significant antagonism to the target kinase.

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

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

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. In order to further verify whether biphenyl derivatives can initiate cell reprogramming, this application also uses CHIR99021 for control experiments.

Human mesenchymal cells were cultured in T25, and cells were inoculated at 4x10 ⁵ in serum-free Dulbecco's modified Eagle's medium (DMEM-F12 medium), in which 20uM of the above-mentioned biphenyl derivatives were added for culture, and the culture conditions were 37°C and 5% carbon dioxide. On the third day, total RNA was extracted using RNeasy Mini or Micro Kit (QIAGEN), and 1mg RNA was synthesized into cDNA using SuperScript III First-Strand Synthesis System (Invitrogen). SYBR Premix Ex Taq (TaKaRa) and Thermal Cycler Dice Real Time System (TaKaRa) were used for Quantitative PCR labeling and reaction, and beta-Actin was used as an internal reference. All data were analyzed using the delta-Ct method. Three groups of experiments were used for each group of experiments, 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 are shown in Figure 16. Compared with the blank control group (CK) without the use of biphenyl derivative small molecules and the control group with CHIR99021 added alone, only biphenyl derivatives can simultaneously induce the expression of Oct4, Lin28A, and c-Myc. Therefore, the use of biphenyl derivatives alone can significantly increase the expression of multiple reprogramming core genes. The simultaneous expression of Oct4, Lin28A, and c-Myc, a group of reprogramming core genes, is a necessary condition for the reprogramming of multiple somatic cells.

Table 2. QPCR primer sequences for compound-responsive genes

The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described 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.

The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description and drawings may be used to interpret the contents of the claims.

Claims (15)

A biphenyl derivative, 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;
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; ^R1 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. The biphenyl derivative according to claim 1, characterized in that the R ¹ is selected from one of the following structures:
Wherein, “*” indicates the connection site. The biphenyl derivative according to claim 2, characterized in that the R ¹ is selected from one of the following structures:
Wherein, “*” indicates the connection site. The biphenyl derivative according to claim 1, characterized in that the A ring is selected from one of the following structures:
Wherein, X is CR ³ R ⁴ or NR ⁵ , and R ² to R ⁵ are independently selected from -H, -CH ₃ or -NH ₂ ; “*” indicates the attachment site. The biphenyl derivative according to claim 3, characterized in that the A ring is selected from one of the following structures:
Wherein, “*” indicates the connection site. The biphenyl derivative according to claim 3, characterized in that the A ring is selected from one of the following structures:
Wherein, “*” indicates the connection site. The biphenyl derivative according to claim 1, characterized in that 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;

A pharmaceutical composition comprising the biphenyl derivative according to any one of claims 1 to 7 and at least one pharmaceutically acceptable carrier. 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. A method for cell reprogramming, comprising the following steps: The cells are contacted with the biphenyl derivative according to any one of claims 1 to 7 or the pharmaceutical composition according to claim 8. The method according to claim 10, characterized in that, during the contact treatment, the concentration of the biphenyl derivative is 1 μM to 50 μM. 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. The use according to claim 12, characterized in that the kinase comprises one or more of PDGFRα, TNIK, CLK4 and JNK1. A method for inhibiting 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; The kinases include one or more of the TK kinase family, the STE kinase family and the CMGC kinase family. The method according to claim 14, characterized in that the kinase comprises one or more of PDGFRα, TNIK, CLK4 and JNK1.