Integrin binding molecule and application thereof in targeted degradation of cell membrane protein or extracellular protein

Abstract

The invention relates to an integrin binding molecule and application of the integrin binding molecule in targeted degradation of cell membrane protein and extracellular protein, and particularly discloses a bifunctional small molecule compound which contains a small molecule compound Gua-Azide of an integrin recognition domain, and the stability is improved. Furthermore, a cell membrane protein or an extracellular protein is selected as a target protein, a small molecule compound with high affinity with the target protein is connected with a small molecule compound Gua-Azide capable of recognizing an integrin receptor alpha v beta 3 through a linker, and a compound capable of being used for cancer immunotherapy is constructed. The invention develops a small molecule compound Gua-Azide capable of replacing RGD cyclic peptide, and the small molecule compound Gua-Azide can be specifically combined with integrin, and meanwhile, the degradation during in-vivo circulation is avoided, and the stability is improved. On the basis, the invention successfully designs and synthesizes a bifunctional small molecule compound for targeted degradation of cell membrane PD-L1 and verifies the anti-tumor effect of the bifunctional small molecule compound.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of chemical biology, and in particular to an integrin-binding molecule and its application in targeted degradation of cell membrane proteins or extracellular proteins. BACKGROUND

[0002] Proteolysis targeting chimera (PROTAC) is a bifunctional molecule, one end of which can bind to a target protein, and the other end can bind to an E3 ubiquitin ligase, so that the target protein is close to the E3 ubiquitin ligase and is ubiquitinated, and then enters the proteasome for degradation, and has become one of the powerful means for drug development. With the proposal of LYTAC technology which can degrade extracellular proteins and cell membrane surface proteins, many researchers have also developed cell membrane protein degrading agents based on lysosomal degradation strategy. Lysosome targeting chimera (LYTAC) is also a bifunctional molecule, which has two binding domains, one end is an oligosaccharide peptide group that binds to the cell surface transmembrane receptor Cl-M6PR, and the other end is an antibody or small molecule that can bind to a target protein, which is endocytosed by forming a "Cl-M6PR-LYTAC-target protein" complex, and then transported to the lysosome for degradation. Compared with PROTAC, LYTAC is mainly used for degrading extracellular and membrane-associated proteins, as a supplement to intracellular protein degradation, LYTAC can be used for the treatment of autoimmune diseases, cancer and other diseases related to extracellular proteins and membrane proteins, and its application prospect is very promising. GlueTAC covalently modifies a single domain antibody and a cell membrane protein PD-L1 to form a covalent bond through an in situ cross-linking reaction, thereby reducing the off-target probability. At the same time, the study also couples a cell-penetrating peptide and a lysosomal sorting sequence to the covalent single domain antibody, which makes it possible to effectively promote the endocytosis and lysosomal transport of the "covalent single domain antibody-target membrane protein" complex without the help of specific cell surface receptors, and ultimately degrades the target protein in the lysosome. KineTAC is a kind of universal, modular and highly selective cytokine targeting chimera proposed by Wells' research group. KineTAC is mainly composed of two parts, i.e. a cytokine arm that can bind to a homologous cytokine receptor and a binding arm that targets a target protein, and it is a completely genetically encoded bispecific antibody that can target and degrade cell membrane proteins and extracellular proteins.

[0003] Previously, the inventors developed an integrin-facilitated lysosomal degradation (IFLD) strategy to combine a target protein-binding ligand with an integrin α vThe obtained bifunctional compound can form a ternary complex between the target protein and integrin, thereby inducing the endocytosis of extracellular or cell membrane proteins and entering the lysosome for degradation. Integrin is a transmembrane receptor that mediates the connection between cells and their external environment (such as extracellular matrix, ECM), which is formed by non-covalent combination of alpha subunit and beta subunit to form heterodimer. Integrin alpha v Beta 3 can recognize the amino acid binding motif Arg-Gly-Asp (RGD) in its endogenous ligand, and is overexpressed in various tumor cells, and is widely used in tumor targeted drug delivery, so the IFLD degradation strategy developed by us is suitable for degrading tumor-related membrane proteins. In previous studies, we selected RGD cyclic peptide as a ligand for recognizing integrin alpha v Beta 3, although the cyclic peptide is more stable than the linear peptide, it can still be degraded by proteases in vivo, which may affect its efficacy and in vivo half-life, and in addition, the production cost of the cyclic peptide is also relatively high. SUMMARY

[0004] In order to overcome the problem of insufficient anti-enzymatic stability of RGD cyclic peptide in the existing IFLD technology, a bifunctional small molecule compound is designed and synthesized, which contains a small molecule compound Gua-Azide that can recognize integrin, which improves the stability compared with the bifunctional compound developed based on RGD cyclic peptide in the previous stage. Further, in order to develop a PD-L1 targeted degradation agent, using the IFLD strategy, selecting PD-L1 as the target protein, connecting the small molecule compound BMS-8 with high affinity with the small molecule compound Gua-Azide that can recognize integrin receptor alpha v Beta 3 through a linker, a compound capable of being used for cancer immunotherapy is constructed.

[0005] To achieve the above-mentioned purpose, the first aspect of the present application provides a compound capable of recognizing integrin receptor alpha v Beta 3, which has the structural formula shown in formula I:

[0006]

[0007] Wherein m is an integer of 1-10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0008] R is an active group capable of reacting with other groups to form a coupling chemical bond.

[0009] Further, R group is selected from one or more of alkyl group, aromatic hydrocarbon group, heterocyclic aromatic hydrocarbon group, alkenyl group, alkynyl group, halogenated group, alcohol hydroxyl group, thiol group, aldehyde group, ketone group, carboxyl group, aldehyde group, amino group, enol group, azide group, maleimide, tetrazine group and its variants and alcohol hydroxyl group containing alpha hydrogen.

[0010] Further, m is an integer from 2 to 8.

[0011] The second aspect of the present application provides a bifunctional compound for degrading a target protein, which has a structural formula as shown in the following Formula II:

[0012]

[0013] In the formula, A is a compound capable of binding to a cell membrane protein or an extracellular protein, or is a compound capable of binding to a cell membrane protein or an extracellular protein connected by a linker.

[0014] Further, the bifunctional compound for degrading a target protein has a structural formula as shown in the following Formula III or IV:

[0015]

[0016] In the formula, A1 is a compound capable of binding to a cell membrane protein or an extracellular protein, or is a compound capable of binding to a cell membrane protein or an extracellular protein connected by a linker.

[0017] Further, the cell membrane protein or extracellular protein is programmed cell death- ligand 1 (i.e., PD-L1), programmed death receptor 1 (i.e., PD-1), epidermal growth factor receptor (i.e., EGFR), human epidermal growth factor receptor-2 (i.e., HER2), G protein-coupled receptor (i.e., GPCR), fibroblast growth factor receptor (i.e., FGFRs), vascular endothelial growth factor receptor family (i.e., VEGFR, VEGF stands for vascular endothelial growth factor), cytotoxic T-lymphocyte-associated protein 4 (i.e., CTLA4 or CTLA-4), human interleukin 5 receptor alpha (IL-5Rα), apolipoprotein, apolipoprotein E4 (i.e., ApoE4), beta-amyloid, angiotensin-converting enzyme 2 (ACE2), sodium ion-taurocholate cotransporting polypeptide (NTCP), B7.1 and B7, TIFR1m, TNFR2, NADPH oxidase, Bcl IBax and other ligands in the apoptosis pathway, C5a receptor, HMG-CoA reductase, PDE V phosphodiesterase type, PDE IV phosphodiesterase type 4, PDE I, PDE II, PDE III, squalene cyclase inhibitors, CXCR1, CXCR2, nitric oxide synthase, cyclooxygenase 1, cyclooxygenase 2, 5HT receptors, dopamine receptors, G proteins, histamine receptors, 5-lipoxygenase, serine protease-like proteases, thymidylate synthetase, purine nucleoside phosphorylase, glyceraldehyde-3-phosphate dehydrogenase (i.e., GAPDH), glycogen phosphorylase, carbonic anhydrase, chemokine receptors, JAW STAT, RXR and analogs, HIV1 protease, HIV1 integrase, influenza neuraminidase, hepatitis B reverse transcriptase, sodium channels, protein P-glycoprotein, P glycoprotein and MRP metalloproteases, CD23, CD73, CD124, tyrosine kinase p561ck, CD4, CD5, IL-2 receptor, IL-1 receptor, TNF-alpha R, ICAM1, Ca2+ channels, VCAM, VLA-4 integrin, selectins, CD40 / CD40L, newokinins and receptors, inosine monophosphate dehydrogenase, p38 MAP kinase, Ras / Raf / MEW / ERK pathway, interleukin-1 converting enzyme, caspases, HCV, NS3 protease, HCV NS3 RNA helicase, glycinamide ribonucleotide formyltransferase, rhinovirus, 3C protease, herpes simplex virus-1, protease, cytomegalovirus protease, poly(ADP-ribose) polymerase, cyclin-dependent kinases, vascular endothelial growth factor, oxytocin receptor, microsomal transfer protein inhibitors, bile acid transport inhibitors, 5 alpha reductase inhibitors, angiotensin 11, glycine receptors, norepinephrine reuptake receptors, endothelin receptors, neuropeptide Y and receptors, adenosine receptors, adenosine kinase and AMP dehydrogenase, purinergic receptors, farnesyl transferase, geranyl transferase, TrkA receptor of NCF, tyrosine kinase Flk-II KDR, vitronectin receptors, integrin receptors, Her-21 nerve sheath, telomerase inhibition, cytosolic phosphoinositide A2 and EGF receptor tyrosine kinase, ecdysone 20-monooxygenase, GABA-gated chloride channels, acetylcholinesterase, voltage-sensitive sodium channel proteins, calcium release channels and chloride channels, acetyl-CoA carboxylase, adenylosuccinate synthetase, protoporphyrinogen oxidase and enolpyruvyl shikimate phosphate synthetase, and / or one or more of all variants, mutants, splice variants, insertions, deletions and fusions of the above proteins.

[0018] Further, the compound binding to the cell membrane protein or extracellular protein is BMS-8, Sul-acid, Biotin-NHS or PH-002.

[0019] Further, the structure of the bifunctional compound is shown as follows

[0020]

[0021] wherein m is an integer from 2 to 8, for example, 2, 3, 4, 5, 6, 7, 8; n is an integer from 1 to 6, for example, 1, 2, 3, 4, 5, 6.

[0022] The third aspect of the present application provides the above-mentioned compound capable of recognizing integrin receptor α v β3 in the preparation of a bifunctional compound degrading cell membrane protein or extracellular protein.

[0023] The fourth aspect of the present application provides the above-mentioned bifunctional compound degrading target protein in the preparation of a reagent degrading cell membrane protein or extracellular protein.

[0024] Further, the reagent is for non-diagnostic and therapeutic purposes.

[0025] Further, the reagent is for in vitro test.

[0026] The fifth aspect of the present application provides a method for degrading cell membrane protein in vitro, which comprises the steps of contacting and incubating the above-mentioned bifunctional compound degrading target protein with cells containing cell membrane protein or a mixture containing extracellular protein and cells.

[0027] The sixth aspect of the present application provides the use of the above-mentioned bifunctional compound degrading target protein in the preparation of a drug for diseases related to abnormal increase of cell membrane protein expression.

[0028] Further, the cell membrane protein or extracellular protein is programmed cell death- ligand 1 (i.e., PD-L1), programmed death receptor 1 (i.e., PD-1), epidermal growth factor receptor (i.e., EGFR), human epidermal growth factor receptor-2 (i.e., HER2), G protein-coupled receptor (i.e., GPCR), fibroblast growth factor receptor (i.e., FGFRs), vascular endothelial growth factor receptor family (i.e., VEGFR, VEGF stands for vascular endothelial growth factor), cytotoxic T-lymphocyte-associated protein 4 (i.e., CTLA4 or CTLA-4), human interleukin 5 receptor alpha (IL-5Ra), apolipoprotein, apolipoprotein E4 (i.e., ApoE4), beta-amyloid, angiotensin-converting enzyme 2 (ACE2), sodium ion-taurocholate cotransporting polypeptide (NTCP), B7.1 and B7, TIFR1m, TNFR2, NADPH oxidase, Bcl IBax and other ligands in the apoptosis pathway, C5a receptor, HMG-CoA reductase, PDE V phosphodiesterase type, PDE IV phosphodiesterase type 4, PDE I, PDE II, PDE III, squalene cyclase inhibitors, CXCR1, CXCR2, nitric oxide synthase, cyclooxygenase 1, cyclooxygenase 2, 5HT receptors, dopamine receptors, G proteins, histamine receptors, 5-lipoxygenase, serine protease-like proteases, thymidylate synthetase, purine nucleoside phosphorylase, glyceraldehyde-3-phosphate dehydrogenase (i.e., GAPDH), glycogen phosphorylase, carbonic anhydrase, chemokine receptors, JAW STAT, RXR and analogs, HIV1 protease, HIV1 integrase, influenza neuraminidase, hepatitis B reverse transcriptase, sodium channels, protein P-glycoprotein, P glycoprotein and MRP metalloproteases, CD23, CD73, CD124, tyrosine kinase p561ck, CD4, CD5, IL-2 receptor, IL-1 receptor, TNF-alpha R, ICAM1, Ca2+ channels, VCAM, VLA-4 integrin, selectins, CD40 / CD40L, newokinins and receptors, inosine monophosphate dehydrogenase, p38 MAP kinase, Ras / Raf / MEW / ERK pathway, interleukin-1 converting enzyme, caspases, HCV, NS3 protease, HCV NS3 RNA helicase, glycinamide ribonucleotide formyltransferase, rhinovirus, 3C protease, herpes simplex virus-1, protease, cytomegalovirus protease, poly(ADP-ribose) polymerase, cyclin-dependent kinases, vascular endothelial growth factor, oxytocin receptor, microsomal transfer protein inhibitors, bile acid transport inhibitors, 5 alpha reductase inhibitors, angiotensin 11, glycine receptors, norepinephrine reuptake receptors, endothelin receptors, neuropeptide Y and receptors, adenosine receptors, adenosine kinase and AMP dehydrogenase, purinergic receptors, farnesyl transferase, geranyl transferase, TrkA receptor of NCF, tyrosine kinase Flk-II KDR, vitronectin receptors, integrin receptors, Her-21 nerve sheath, telomerase inhibition, cytosolic phosphoinositide A2 and EGF receptor tyrosine kinase, ecdysone 20-monooxygenase, GABA-gated chloride channels, acetylcholinesterase, voltage-sensitive sodium channel proteins, calcium release channels and chloride channels, acetyl-CoA carboxylase, adenylosuccinate synthetase, protoporphyrinogen oxidase and enolpyruvyl shikimate phosphate synthetase, and / or one or more of all variants, mutants, splice variants, insertions, deletions and fusions of the above proteins.

[0029] Further, the disease associated with abnormal elevated expression of cell membrane proteins is selected from the group consisting of tumor, lowering blood lipid, and treating neurological diseases.

[0030] The seventh aspect of the present application provides a method for degrading a cell membrane protein or an extracellular protein, said method comprising the step of contacting the bifunctional compound as described above with a cell comprising the cell membrane protein, or said method comprising the step of incubating the bifunctional compound with the cell and the extracellular protein together;

[0031] The target protein-binding compound in the bifunctional compound is capable of specifically binding to the cell membrane protein to be degraded or the extracellular protein to be degraded;

[0032] Preferably, the method for degrading the cell membrane protein or the extracellular protein is carried out in vitro or in vivo.

[0033] Preferably, the cell membrane protein or the extracellular protein is degraded through the lysosomal pathway in the cell.

[0034] Preferably, the cell membrane protein or the extracellular protein enters the cell through endocytosis.

[0035] Preferably, the cell is a normal cell or a tumor cell.

[0036] The eighth aspect of the present application provides a design method of a bifunctional compound capable of degrading a cell membrane protein or an extracellular protein using the lysosome of a cell, said design method comprising the following steps:

[0037] S1) determining the target of the cell membrane protein or the extracellular protein to be degraded;

[0038] S2) obtaining a small molecule compound capable of specifically binding to the target of the cell membrane protein or the extracellular protein to be degraded;

[0039] S3) modifying the small molecule compound obtained in S2) or directly connecting it with a compound capable of recognizing integrin receptor α v β3 to obtain a bifunctional compound.

[0040] The ninth aspect of the present application provides a preparation method of a compound capable of recognizing integrin receptor α v β3, said preparation method comprising the following steps:

[0041] S11) coupling bromobenzaldehyde with 4-carboxyphenylboronic acid in the presence of a palladium catalyst and potassium carbonate to obtain compound Compound reacting with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide under alkaline conditions until complete, and continuing to react with NH2-(CH2) m -R under alkaline conditions until complete, and purifying to obtain compound Compound reacting with ethyl acetoacetate and piperidine together and purifying to obtain compound

[0042] S12) N,N'-di-Boc-1H-1-guanidinopyrazole reacts with 3-aminobenzoic acid in the presence of a basic organic reagent to obtain a compound Compound 9-fluorenylmethylhydrazinecarboxylate and HATU to obtain a compound

[0043] S13) removing the compound Fmoc protecting group, then acyl bond coupling with the compound , and finally removing the Boc protecting group and purifying to obtain a compound capable of recognizing integrin receptor α v β3

[0044]

[0045] wherein m is an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0046] R is an active group capable of reacting with other groups to form a coupling chemical bond.

[0047] Further, the R group is selected from one or more of an alkane group, an arene group, a heterocyclic arene group, an alkenyl group, an alkynyl group, a halogenated group, an alcoholic hydroxyl group, a thiol group, an aldehyde group, a ketone group, a carboxyl group, an aldehyde group, an amino group, an enol group, an azido group, a maleimide group, a tetrazine group, and variants thereof, and an alcoholic hydroxyl group containing an alpha hydrogen.

[0048] The tenth aspect of the present application provides a method for preparing a bifunctional compound degrading a target protein, the method comprising the steps of:

[0049] S1) modifying a compound capable of binding to a cell membrane protein or an extracellular protein to have a group capable of coupling with a compound capable of recognizing integrin receptor α v β3 ; or

[0050] obtaining a compound capable of binding to a cell membrane protein or an extracellular protein and having a group capable of coupling with a compound capable of recognizing integrin receptor α v β3 ;

[0051] S2) coupling a compound capable of recognizing integrin receptor α v β3 with the compound obtained in step S1) to obtain a bifunctional compound degrading a target protein.

[0052] Further, the compound capable of binding to a cell membrane protein is BMS-8, and the method for modification in step S1) is coupling modification of BMS-8 with pent-4-yn-1-amine hydrochloride.

[0053] Advantages

[0054] The present application develops a small molecule compound capable of replacing the RGD cyclic peptide, which can be specifically combined with integrin, while avoiding degradation in the body circulation, and improves the stability.

[0055] On this basis, the present application successfully designs and synthesizes a bifunctional small molecule compound targeting the degradation of cell membrane PD-L1.

[0056] The small molecule compound is confirmed in the biological activity research that it can effectively induce the endocytosis and lysosomal degradation of PD-L1 in an integrin-dependent manner. As a novel PD-L1 small molecule degrading agent, the compound has potential application prospect in the development of tumor immunotherapy drugs.

[0057] Meanwhile, it is expected that the bifunctional compound of the small molecule compound combined with other cell membrane proteins or extracellular protein targeting agents has similar effects, and realizes the targeted degradation of cell membrane proteins or extracellular proteins. BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1 The H NMR spectrum of compound 5. 1 H NMR spectrum.

[0059] Figure 2 The C NMR spectrum of compound 5. 13 C NMR spectrum.

[0060] Figure 3 The high-resolution mass spectrum of compound 5.

[0061] Figure 4 The H NMR spectrum of compound 6. 1 H NMR spectrum.

[0062] Figure 5 The C NMR spectrum of compound 6. 13 C NMR spectrum.

[0063] Figure 6 The high-resolution mass spectrum of compound 6.

[0064] Figure 7 The H NMR spectrum of compound 7. 1 H NMR spectrum.

[0065] Figure 8 The C NMR spectrum of compound 7. 13 C NMR spectrum.

[0066] Figure 9 The high-resolution mass spectrum of compound 7.

[0067] Figure 10 H NMR spectrum of Compound 10. 1 H NMR spectrum of Compound 10.

[0068] Figure 11 H NMR spectrum of Compound 10. 13 H NMR spectrum of Compound 10.

[0069] Figure 12 H NMR spectrum of Compound 11. 1 H NMR spectrum of Compound 11.

[0070] Figure 13 H NMR spectrum of Compound 11. 13 H NMR spectrum of Compound 11.

[0071] Figure 14 High resolution mass spectrum of Compound 11.

[0072] Figure 15 H NMR spectrum of Gua-Azide (1). 1 H NMR spectrum of Gua-Azide (1).

[0073] Figure 16 H NMR spectrum of Gua-Azide (1). 13 H NMR spectrum of Gua-Azide (1).

[0074] Figure 17 High resolution mass spectrum of Gua-Azide (1).

[0075] Figure 18 H NMR spectrum of Compound 13. 1 H NMR spectrum of Compound 13.

[0076] Figure 19 H NMR spectrum of Compound 13. 13 H NMR spectrum of Compound 13.

[0077] Figure 20 High resolution mass spectrum of Compound 13.

[0078] Figure 21 H NMR spectrum of BMS-Gua (2). 1 H NMR spectrum of BMS-Gua (2).

[0079] Figure 22 H NMR spectrum of BMS-Gua (2). 13 H NMR spectrum of BMS-Gua (2).

[0080] Figure 23 High resolution mass spectrum of BMS-Gua (2).

[0081] Figure 24 HPLC analysis of BMS-Gua (2).

[0082] Figure 25The figure shows the stability results of Gua-Azide and c(RGDyK) in artificial gastric fluid.

[0083] Figure 26 To determine the relationship between Gua-Azide and integrin α via ITC v The result of the interaction between β3 is shown in the figure.

[0084] Figure 27 Figure 1 shows the results of BMS-Gua-targeted degradation of PD-L1 protein on the cell membrane. (A) Western blot analysis of PD-L1 protein levels in MDA-MB-231 cells treated with BMS-Gua and BMS-L1-RGD (25 nM, 8 h). (B) Western blot analysis of BMS-Gua-mediated PD-L1 degradation at specified times and concentrations. (C) Western blot analysis of PD-L1 protein levels in MDA-MB-231 cells treated with 25 nM BMS-Gua, 100 μM Bafilomycin A1, or 5 μM MG132 for 24 h. (D) Western blot analysis of the effect of competitive inhibition of Gua-Azide binding to integrins on BMS-Gua-mediated PD-L1 degradation. (E) Immunofluorescence analysis of PD-L1 (red) degradation on the cell membrane. (F) Immunofluorescence analysis of the effect of competitive inhibition of Gua-Azide binding to integrins on BMS-Gua-mediated PD-L1 degradation. (G) Colocalization of PD-L1 (green) with early nuclear endosome marker (Rab5, red) in human cervical cancer (HeLa) cells. Nuclei were labeled with DAPI (blue) for (E), (F) and (G), scale bar, 10 μm. Detailed Implementation

[0085] The immune checkpoint PD-L1 can bind to the PD-1 receptor on T cells, thereby inhibiting T cell activity, weakening their attack on tumor cells, and promoting tumor cell immune escape. Studies have shown that this pathway can be blocked by degrading the PD-L1 protein. Therefore, this invention utilizes an integrin-mediated lysosomal degradation (IFLD) strategy, that is, by binding the target protein ligand to integrin α... v The bifunctional compound, coupled with the β3-recognizing ligand RGD cyclic peptide, can form a ternary complex with the target protein and integrin, thereby inducing the degradation of the target protein extracellularly or on the cell membrane via the endosome-lysosome pathway. Considering the potential poor enzymatic stability and high production cost of peptides, we designed and synthesized a small molecule compound, Gua-Azide, containing an integrin recognition domain, to replace the RGD cyclic peptide in the synthesis of IFLD degrading agents. The enzymatic stability of the small molecule Gua-Azide and its interaction with integrin α were determined.v After the affinity of β3 is better than RGD cyclic peptide, we replace Gua-Azide with RGD cyclic peptide, coupled with the binding molecule BMS-8 of PD-L1 through appropriate linker, and prove through biological activity research that the compound can promote the endocytosis and lysosomal pathway degradation of target protein PD-L1 in an integrin-dependent manner, providing a new compound for the development of cancer immunotherapy drugs.

[0086]

[0087] Synthesis of small molecule compound Gua-Azide in Example 1

[0088]

[0089] Step 1. Synthesis of intermediate compound 7

[0090] In a reaction bottle, p-bromobenzaldehyde (3, 1.95 g, 10.54 mmol), 4-carboxyphenylboronic acid (4, 1.75 g, 10.55 mmol), tetrakis triphenylphosphine palladium (366 mg, 0.32 mmol) and potassium carbonate (3.6 g, 26.05 mmol) were dissolved in a mixed solution of 75 mL of methanol / water (volume ratio 2:1), protected by nitrogen, heated to reflux overnight, then the reaction mixture was cooled to room temperature, the catalyst was removed by filtration, the filtrate was collected, the pH was adjusted to acidic, a precipitate was separated out, filtered, and white solid 5 (2 g, 8.85 mmol, 84%) was obtained. 1 H NMR (400 MHz, DMSO) δ 10.07 (s, 1H), 8.07 (s, 1H), 8.05 (t, J = 3.8 Hz, 2H), 8.01 (s, 1H), 7.97 (d, J = 8.3 Hz, 2H), 7.89 (d, J = 8.5 Hz, 2H). Figure 1 ) 13 C NMR (101 MHz, DMSO) δ 192.77, 166.99, 144.64, 142.88, 135.63, 130.61, 130.19, 130.04, 127.73, 127.35. Figure 2 ) HRMS (ESI + )m / z: calcd. for C 14 H9O3[M-H] + 225.0557, found 225.0556. Figure 3 )

[0091] Compound 5 (945 mg, 4.18 mmol), l-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.6 g, 8.35 mmol) and N-hydroxysuccinimide (962 mg, 8.36 mmol) were dissolved in 10 mL of anhydrous DMF, stirred to dissolve, then triethylamine (1743 μL, 12.54 mmol) was added, stirred at room temperature overnight, then 6-azidohexan-l-amine (891 mg, 6.27 mmol) and triethylamine (4357 μL, 22.32 mmol) were added, stirred at room temperature overnight, after TLC monitoring reaction was completed, the reaction was concentrated in vacuo, the residue was dissolved with ethyl acetate and washed with saturated brine 2-3 times, the organic phase was dried with anhydrous Na2SO4, filtered, the filtrate was collected, concentrated in vacuo, then purified by column chromatography with PE:EA = 2: 1 to obtain white solid 6 (410 mg, 1.17 mmol, 28%). 1 H NMR (400 MHz, DMSO) δ 10.07 (s, 1H), 8.53 (t, J = 5.5 Hz, 1H), 8.03 (s, 1H), 8.01 (s, 1H), 7.97 (dd, J = 8.2, 4.4 Hz, 4H), 7.86 (d, J = 8.3 Hz, 2H), 3.33-3.30 (m, 2H), 3.30 - 3.26 (m, 2H), 1.58 - 1.51 (m, 4H), 1.38 - 1.32 (m, 4H). Figure 4 ) 13 C NMR (101 MHz, DMSO) δ 192.70, 165.52, 144.83, 141.06, 135.42, 134.48, 130.12, 127.91, 127.53, 126.94, 50.58, 28.93, 28.18, 25.99, 25.88. Figure 5 ) HRMS (ESI + )m / z: calcd. for C 20 H 23 N4O2[M+H] + 351.1816, found 351.1815. Figure 6 )

[0092] A mixture of compound 6 (410 mg, 1.17 mmol) and ethyl acetoacetate (304.8 mg, 2.34 mmol) was cooled to 0 °C, then piperidine (115.7 μL, 1.17 mmol) was added dropwise, the reaction was stirred at room temperature for 3 days, then the yellow mixture was added to an aqueous solution of sodium hydroxide (20 M) gradually, and stirred at 80 °C for 3 hours. After the reaction was completed, the reaction was cooled to room temperature, ice water was added and extracted with ethyl acetate, the aqueous phase was separated, the pH of the aqueous phase was adjusted to 1, and a precipitate was separated, then the precipitate was extracted with ethyl acetate, the organic phase was dried with anhydrous Na2SO4, filtered, the filtrate was collected, and concentrated under vacuum to obtain yellow solid 7 (456 mg, 1.01 mmol, 86%). 1 H NMR (400 MHz, DMSO) δ 12.10 (s, 2H), 8.46 (t, J = 5.6 Hz, 1H), 7.91 (d, J = 8.4 Hz, 2H), 7.74 (d, J = 8.4 Hz, 2H), 7.64 (d, J = 8.2 Hz, 2H), 7.38 (d, J = 8.3 Hz, 2H), 3.53 - 3.44 (m, 1H), 3.33 (d, J = 6.9 Hz, 2H), 3.30 - 3.26 (m, 2H), 2.69 (dd, J = 15.9, 6.2 Hz, 2H), 2.57 (dd, J = 15.8, 8.7 Hz, 2H), 1.54 (dd, J = 13.1, 6.4 Hz, 4H), 1.39 - 1.33 (m, 4H). Figure 7 ) 13 C NMR (101 MHz, DMSO) δ 172.75, 165.76, 143.36, 142.30, 137.33, 133.35, 128.16, 127.76, 126.65, 126.24, 50.60, 37.64, 28.98, 28.20, 26.01, 25.91. Figure 8 ) HRMS (ESI + )m / z: calcd. for C 24 H 29 N4O5[M+H] + 453.2133, found 453.2134. Figure 9 )

[0093]

[0094] Step 2. Synthesis of intermediate compound 11

[0095] In a reaction flask, N,N'-di-Boc-1H-1-guanidinopyrazole (compound 8, 2 g, 6.44 mmol), 3-aminobenzoic acid (compound 9, 1.77 g, 12.9 mmol) and triethylamine (2.7 mL, 19.33 mmol) were dissolved in 30 mL of methanol, the reaction was stirred at 40 °C overnight, after the reaction was completed by TLC monitoring, the reaction was concentrated under vacuum, the residue was extracted with ethyl acetate, the organic phase was dried with anhydrous Na2S04, filtered, the filtrate was collected, concentrated under vacuum and purified by column chromatography with DCM:MeOH = 100:1 (acid added) to obtain white solid 10 (1.6 g, 4.22 mmol, 67%). 1 H NMR (400 MHz, DMSO) δ 7.99 (s, 1H), 7.79 (d, J = 7.3 Hz, 1H), 7.71 (d, J = 7.8 Hz, 1H), 7.47 (t, J = 7.9 Hz, 1H), 1.45 (s, 18H). Figure 10 ) 13 C NMR (101 MHz, DMSO) δ 166.89, 131.37, 128.94, 127.12, 125.52, 123.48, 27.76. Figure 11 )

[0096] Compound 10 (1.6 g, 4.22 mmol), 9-fluorenylmethylhydrazinecarboxylate (1.29 g, 5.08 mmol) and HATU (1.9 g, 5.00 mmol) were dissolved in 10 mL of DMF, stirred and dissolved, then DIEA (2 mL, 12.66 mmol) was added, the reaction was stirred at room temperature for 2 hours. After the reaction was completed by TLC monitoring, the reaction was concentrated under vacuum, the residue was extracted with ethyl acetate, the organic phase was dried with anhydrous Na2S04, filtered, the filtrate was collected, concentrated under vacuum and purified by column chromatography with PE:EA = 2:1 to obtain white solid 11 (1.4 g, 2.28 mmol, 54%). 1 H NMR (400 MHz, DMSO) δ 11.41 (s, 1H), 10.39 (s, 1H), 10.13 (s, 1H), 9.47 (s, 1H), 7.96 - 7.85 (m, 4H), 7.77 (d, J = 7.3 Hz, 2H), 7.66 (d, J = 7.5 Hz, 1H), 7.53 - 7.47 (m, 1H), 7.44 (t, J = 7.3 Hz, 2H), 7.36 (t, J = 7.1 Hz, 2H), 4.40 (d, J = 6.7 Hz, 2H), 4.33 - 4.26 (m, 1H), 1.52 (s, 9H), 1.40 (s, 9H). Figure 12 ) 13C NMR (101 MHz, DMSO) δ 165.53, 162.55, 156.28, 152.86, 152.10, 143.66, 140.77, 137.06, 132.94, 128.96, 127.70, 127.14, 126.25, 125.29, 123.61, 121.87, 120.16, 83.42, 78.96, 66.20, 46.53, 27.88, 27.65. Figure 13 ) HRMS (ESI + )m / z: calcd. for C 33 H 38 N5O7[M+H] + 616.2766, found 616.2768. Figure 14

[0097]

[0098] Step 3. Synthesis of compound Gua-Azide (1)

[0099] Compound 11 (100 mg, 0.16 mmol) was dissolved in 10 mL of diethylamine / dichloromethane (1:1 by volume) and stirred at room temperature for 1 h. After completion of the reaction, the reaction mixture was concentrated under vacuum to get the crude deprotected amine which was dissolved in 1 mL of DMF. To this, compound 7 (172.5 mg, 0.38 mmol), HATU (72.5 mg, 0.19 mmol) and DIEA (112.87 μί, 0.64 mmol) were added and the reaction mixture was stirred at room temperature for 2 h. After completion of the reaction as monitored by TLC, the reaction mixture was concentrated under vacuum. The residue was extracted with ethyl acetate and the organic phase was dried over anhydrous Na2S04, filtered, and the filtrate was concentrated under vacuum. To the residue, 20% TFA in DCM was added at ice bath temperature and then allowed to warm to room temperature and stirred for 2 h. The progress of the reaction was monitored by analytical HPLC. The reaction mixture was concentrated under vacuum and purified by preparative HPLC. The white powder obtained after lyophilization was 1, i.e., Gua-Azide (50.25 mg, 0.08 mmol, 63%). 1 ​H NMR (400 MHz, DMSO) δ 12.11 (s, 1H), 10.39 (s, 1H), 10.12 (s, 1H), 10.04 (s, 1H), 8.52 (t, J = 5.0 Hz, 1H), 7.92 (d, J = 8.2 Hz, 2H), 7.75 (t, J = 7.4 Hz, 3H), 7.70 (s, 1H), 7.65 (d, J = 8.3 Hz, 2H), 7.62 (s, 2H), 7.54 (t, J = 7.9 Hz, 1H), 7.43 (d, J = 7.5 Hz, 1H), 7.39 (d, J = 8.3 Hz, 2H), 3.61 - 3.53 (m, 1H), 3.34 - 3.23 (m, 5H), 2.82 (dd, J = 15.9, 5.2 Hz, 1H), 2.65 - 2.55 (m, 2H), 1.58 - 1.48 (m, 4H), 1.38 - 1.30 (m, 4H). Figure 15 ) 13 C NMR (101 MHz, DMSO) δ 172.90, 169.77, 165.77, 164.67, 142.33, 137.33, 135.64, 133.85, 133.35, 129.94, 128.16, 127.84, 127.65, 126.76, 126.28, 125.26, 50.62, 37.81, 29.02, 28.24, 26.05, 25.95. Figure 16 ) HRMS (ESI + )m / z: calcd. for C 32 H 38 N9O5[M+H] + 628.2990, found 628.2993. Figure 17 )

[0100] Enzymatic stability study of small molecule compound Gua-Azide of Example 2

[0101] To explore the stability of compound Gua-Azide and cRGD, the stability of the two compounds in artificial gastric juice for 72 hours was detected. 0.08 mg of small molecule Gua-Azide was weighed in a 2 mL centrifuge tube, first dissolved with 5% DMSO, then 1641 μL of artificial gastric juice was added, and after fully mixed, it was placed in a 37°C constant temperature shaker for oscillation. At the same time, 0.22 mg of cRGD was weighed in a 2 mL centrifuge tube, and the same was first dissolved with 5% DMSO, then 1539 μL of artificial gastric juice was added, and after fully mixed, it was placed in a 37°C constant temperature shaker for oscillation. Then at 0, 4, 8, 12, 24, 48, 72 hours, 40 μL of solution was removed, 90 μL of acetonitrile was added to terminate the reaction and precipitate the protein, centrifuged for 10 minutes, 90 μL of supernatant was taken into a sample bottle, and 50 μL of sample was analyzed by HPLC. Each time period took three groups.

[0102] The experimental results are shown in Table 1. Figure 25 Under the action of pepsin, the half-life of small molecule Gua-Azide was significantly prolonged compared with c(RGDyK), which showed that it was more stable than c(RGDyK).

[0103] Example 3 Study on the affinity of small molecule compound Gua-Azide and integrin α v β3

[0104] To study the interaction between compound Gua-Azide and integrin receptor α v β3, isothermal titration calorimetry was used for analysis. The test concentration of small molecule Gua-Azide was 0.05 mM, and the test concentration of integrin receptor αvβ3 protein was 0.00016 mM. The initial volume of DNA solution added to the sample cell was 300 μL, and the initial volume of small molecule solution titrated was 50 μL, which was sucked into a syringe, and the titration experiment was carried out in an environment with a temperature of 25°C. 25 drops, 2 μL per drop, titration interval 120 s, stirring speed 350 r / min. The experimental group was small molecule Gua-Azide titrating α v β3 protein, and the control group was small molecule titrating blank solvent (10% DMSO in PBS solution).

[0105] The experimental results are shown in Table 2. Figure 26 The experimental results show that there is a certain heat change (exothermic) during the titration of small molecule Gua-Azide with protein, indicating that there is a combination between the two. Based on the Independent model fitting, the corresponding dissociation constant K d , stoichiometric ratio n, enthalpy change, entropy change and other parameters are shown in Table 2. Figure 26

[0106] Example 4 Synthesis of bifunctional small molecule compound BMS-Gua​

[0107]

[0108] Step 1. Synthesis of intermediate compound 13

[0109] In a 10 mL reaction tube, was added compound 12 (50 mg, 0.10 mmol), pent-4-yn-1-amine hydrochloride (18.1 mg, 0.15 mmol), HATU (57.7 mg, 0.15 mmol) and 1.5 mL DMF, stirred to dissolve, followed by the addition of DIEA (83.57 μL, 0.65 mmol) and reacted at room temperature for 2 hours, the reaction progress was followed using analytical HPLC, the reaction mixture was directly purified by preparative HPLC to give 13 (55.43 mg, 0.10 mmol, 98%) as a white powder after lyophilization. 1 HNMR (400 MHz, DMSO) δ 8.94 (s, 1H), 7.72 (d, J = 1.9 Hz, 1H), 7.54 (d, J = 6.9 Hz, 1H), 7.49 - 7.42 (m, 3H), 7.42 - 7.35 (m, 2H), 7.34 - 7.28 (m, 3H), 7.22 (d, J = 6.8 Hz, 1H), 5.29 (s, 2H), 4.25 (d, J = 13.0 Hz, 1H), 4.08 (dd, J = 13.0, 6.3 Hz, 1H), 3.78 (t, J = 10.4 Hz, 1H), 3.37 (s, 2H), 3.32 - 3.21 (m, 3H), 3.03 - 2.93 (m, 1H), 2.82 (t, J = 2.6 Hz, 1H), 2.27 - 2.22 (m, 4H), 2.09 (d, J = 13.1 Hz, 1H), 1.77 - 1.62 (m, 5H), 1.47 - 1.32 (m, 1H). Figure 18 ) 13 C NMR (101 MHz, DMSO) δ 168.03, 155.61, 142.40, 141.43, 136.19, 135.13, 134.03, 132.70, 129.95, 129.36, 128.46, 127.77, 127.20, 125.78, 113.99, 111.25, 83.91, 71.83, 69.66, 64.97, 57.17, 50.95, 38.12, 28.69, 27.80, 21.97, 16.05, 15.57. Figure 19 ) HRMS (ESI + )m / z: calcd for C 32 H 36 BrN2O2[M+H] +559.1955, found 559.1957. Figure 20 )

[0110]

[0111] Step 2. Synthesis of compound BMS-Gua

[0112] Compound Gua-Azide (1) (20 mg, 0.03 mmol), compound 13 (21.36 mg, 0.04 mmol), copper sulfate pentahydrate (4.78 mg, 0.02 mmol) and sodium ascorbate (25.27 mg, 0.13 mmol) were dissolved in 1 mL of mixed solution of DMF / H2O (1:1 by volume), the solution was stirred at room temperature for 2 hours and the reaction progress was monitored by HPLC, the mixture was directly purified by preparative HPLC, and white powder BMS-Gua (2) was obtained after freeze-drying (tR= 14.072 min, 19.28 mg, 0.02 mmol, 51%). R = 14.072 min, 19.28 mg, 0.02 mmol, 51%). Figure 24 ). 1 H NMR (400 MHz, DMSO) δ 10.40 (s, 1H), 10.16 (s, 1H), 10.06 (s, 1H), 9.04 (t, J = 5.2 Hz, 1H), 8.53 (t, J = 5.4 Hz, 1H), 7.91 (t, J = 4.1 Hz, 3H), 7.82 - 7.73 (m, 2H), 7.71 (dd, J = 5.5, 3.6 Hz, 3H), 7.65 (d, J = 8.1 Hz, 4H), 7.60 - 7.49 (m, 2H), 7.46 (dd, J = 9.5, 5.0 Hz, 3H), 7.43 - 7.35 (m, 4H), 7.30 (dd, J = 6.9, 5.6 Hz, 3H), 7.21 (d, J = 7.1 Hz, 1H), 5.34 - 5.22 (m, 2H), 4.34 - 4.20 (m, 3H), 4.09 (d, J = 9.4 Hz, 1H), 3.80 (d, J = 8.5 Hz, 3H), 3.23 (d, J = 5.4 Hz, 5H), 2.98 (s, 1H), 2.82 (dd, J = 15.8, 5.0 Hz, 1H), 2.71 - 2.63 (m, 2H), 2.63 - 2.54 (m, 2H), 2.22 (s, 2H), 2.14 - 1.96 (m, 2H), 1.86 - 1.61 (m, 7H), 1.50 (dd, J = 13.8, 6.9 Hz, 2H), 1.41 - 1.20 (m, 6H). Figure 21 ) 13C NMR (101 MHz, DMSO) δ 172.94, 169.82, 167.87, 165.82, 164.74, 156.02, 155.45, 146.03, 143.58, 142.37, 142.25, 141.28, 137.34, 136.07, 135.66, 134.99, 133.91, 133.88, 133.34, 132.61, 129.99, 129.84, 129.23, 128.34, 128.19, 127.88, 127.66, 127.08, 126.79, 126.30, 125.67, 125.30, 123.30, 122.87, 121.98, 113.82, 111.09, 69.51, 64.87, 56.95, 50.78, 49.25, 38.40, 37.80, 29.74, 29.00, 28.54, 25.92, 25.67, 22.48, 21.84, 21.18, 15.93. Figure 22 )HRMS (ESI + )m / z: calcd for C 64 H 72 N 11 O7Br[M+H] + 1186.4872, found 1186.4877.( Figure 23 )

[0113] Example 5 Application of IFLD strategy to degrade PD-L1 protein on the membrane to evaluate the performance of BMS-RGD series compounds in vitro

[0114] Immunoblotting was used to analyze PD-L1 levels. MDA-MB-231 cells were cultured in 12-well plates until the cell density reached 70-80%. To compare the degradation of PD-L1 by BMS-Gua and BMS-L1-RGD, BMS-Gua was diluted to 25 nM with DMEM and incubated with cells for 8 hours. To determine the optimal concentration of BMS-Gua, it was diluted to 12.5, 25, 50, 100, 200 nM with DMEM and incubated with cells for 24 hours. BMS-Gua was diluted to 25 nM at different time gradients and added to cells at the appropriate time. To verify the degradation pathway, Bafilomycin Al (100 nM) was incubated with cells for 2 hours before BMS-Gua was added, and the cells were incubated at 37°C for another 24 hours. In another well, MG132 (5 μM) and BMS-Gua were added, and the cells were incubated at 37°C for 24 hours. In the competition experiment, Gua-Azide (5 μM) was incubated with cells at 4°C for 1 hour, BMS-Gua was added, and the cells were incubated at 37°C for another 24 hours. Then the cells were washed with PBS 3 times, and 80-100 μL of SDS lysis buffer containing phosphatase and protease inhibitors was added. After centrifugation at 14000 rpm for 5 minutes, the protein sample was boiled in 100°C boiling water for 20 minutes, and then SDS-loading (5x) was added and boiled for another 10 minutes. The protein sample was electrophoretically separated by 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and then transferred to a 0.22 μm PVDF membrane. Then, the membrane was blocked with 5% skim milk in PBST buffer (PBS + 0.1% Tween-20) at room temperature for 2 hours with gentle shaking, and after blocking, the membrane was washed with PBST 3 times for 5 minutes each time. The membrane was incubated with primary antibody (PD-L1 antibody, rabbit, 1:3000; GAPDH antibody, rabbit, 1:5000) at 4°C overnight with gentle shaking. After incubation, the membrane was washed with PBST buffer 3 times for 5 minutes each time. The membrane was incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody (1:5000 dilution) at room temperature for 1 hour. Finally, the membrane was washed with PBST buffer 3 times for 5 minutes each time, and chemiluminescence liquid was used to detect the protein band.

[0115] Immunofluorescence experiments. HeLa cells stably expressing PD-L1 were seeded on coverslips in 24-well plates to a density of about 40-50%. In the membrane protein PD-L1 degradation experiment, BMS-8, Gua-Azide, BMS-Gua were diluted to 25 nM in culture medium and then added to the well plates, and the cells were incubated at 37 °C for 8 hours. In the competition experiment, a high concentration (5 mM) of Gua-Azide was added to the well plates and incubated at 4 °C for 1 hour, and then BMS-Gua was added to the well and incubated at 37 °C for 8 hours. The cells on the coverslips were fixed with 4% PFA for 15 minutes and then washed with PBS (10 mM, pH 7.4) for 3 times, 5 minutes each time. Then, the cells were blocked with 3% BSA for 30 minutes, and incubated with the indicated primary and secondary antibodies at room temperature for 2 hours and 1 hour, respectively. After this step, the nuclei were stained with DAPI in the dark for 15 minutes. After staining, the cells were washed with PBS for 3 times, 5 minutes each time. Fluorescence images were taken by Nikon ECLIPSE Ti2 super-resolution confocal fluorescence microscope.

[0116] Co-localization experiment. Co-localization of PD-L1 with early endosome marker (Rab5). HeLa cells stably expressing PD-L1 were seeded on coverslips in 24-well plates, and when the cell confluence reached about 60-70%, Rab5-RFP plasmid was transfected into HeLa cells stably expressing PD-L1 using MegaTran 2.0 transfection reagent. After transfection, the cells were incubated for 24 hours to allow Rab5-RFP to be expressed in early endosomes. During this period, BMS-Gua was diluted to 25 nM in culture medium and added to the 24-well plates and incubated at 37 °C for 4 hours. The remaining steps were performed according to the above fluorescence staining method.

[0117] The experimental results are shown in 27. First, the degradation effect of compound BMS-Gua and BMS-L1-RGD on target protein PD-L1 was analyzed by Western blot experiment. The experimental results showed that both BMS-Gua and BMS-L1-RGD could effectively degrade PD-L1 protein, and the effect of BMS-Gua was better than that of BMS-L1-RGD. Then, by using different concentrations of BMS-Gua to co-incubate with cells, the results showed that BMS-Gua had different degrees of degradation activity to PD-L1 at the concentration of 12.5-200 nM under the same treatment time, and 25 nM concentration showed significant degradation effect. Further, the cells were treated with 25 nM concentration of drug at different times, and it was observed that BMS-Gua caused the protein level of PD-L1 to decrease quickly, and the degradation effect was significant at 24 hours. Subsequently, in order to verify the degradation mechanism of PD-L1, MDA-MB-231 cells were co-incubated with BMS-Gua in the presence or absence of Bafilomycin A1 (a lysosome inhibitor) or MG132 (a proteasome inhibitor), and the change of protein level was evaluated by Western blot experiment. The experimental results showed that in the presence of lysosome inhibitor, the degradation of PD-L1 promoted by BMS-Gua was significantly inhibited, while the presence of proteasome inhibitor had little effect on the degradation of PD-L1, proving that the degradation of PD-L1 by BMS-Gua depends on the lysosome pathway. At the same time, in order to detect whether the degradation of PD-L1 is dependent on integrin αvβ3, the MDA-MB-231 cells were pretreated with excess Gua-Azide to make them fully bind to the integrin on the cell surface, and then incubated with BMS-Gua. The experimental results showed that under this condition, the activity of BMS-Gua to induce the degradation of PD-L1 was significantly inhibited. The above results showed that the degradation of PD-L1 induced by BMS-Gua was carried out through integrin-mediated lysosome pathway.

[0118] To further investigate the degradation effect of BMS-Gua on PD-L1, HeLa cells stably expressing PD-L1 were treated with BMS-Gua, and the immunofluorescence results showed that the cell surface PD-L1 level was significantly reduced, while the cells treated with BMS-8, Gua-Azide alone or in combination did not reduce PD-L1. Consistent with Western blot analysis, pretreatment of cells with excess Gua-Azide significantly blocked the degradation of PD-L1 by BMS-Gua. In addition, the results of colocalization experiments showed that after BMS-Gua treatment, the PD-L1 protein on the cell membrane was significantly reduced, and the colocalization of PD-L1 with Rab5 in the cell was significantly increased, indicating that the PD-L1 on the cell membrane was endocytosed into the endosome, and then degraded through the endosome-lysosome pathway. In summary, BMS-Gua can promote the endocytosis of PD-L1 through integrin-dependent manner, and degrade through lysosome pathway( Figure 27 ).

Claims

1. A compound identifiable to an integrin receptor α v β3, characterized in that, It has a structural formula shown in formula I: Wherein, m is an integer from 1 to 10; R is an active group capable of reacting with other groups to form a coupling chemical bond; Preferably, the R group is selected from one or more of alkane group, arene group, heterocyclic arene group, alkenyl group, alkynyl group, halogenated group, alcohol hydroxyl group, thiol group, aldehyde group, ketone group, carboxyl group, aldehyde group, amino group, enol group, azide group, maleimide, tetrazine group and its variants and alcohol hydroxyl group containing alpha hydrogen.

2. A bifunctional compound that degrades a target protein, characterized in that, It has a structural formula shown in formula II as follows: Wherein, m is an integer from 1 to 10; A is a compound capable of binding to cell membrane protein or extracellular protein, or is a compound capable of binding to cell membrane protein or extracellular protein connected by a linker; Preferably, the bifunctional compound for degrading target protein has a structural formula shown in formula III or IV as follows: Wherein, A1 is a compound capable of binding to cell membrane protein or extracellular protein, or is a compound capable of binding to cell membrane protein or extracellular protein connected by a linker; Preferably, the cell membrane or extracellular protein is programmed cell death-ligand 1 (i.e. PD-L1), programmed death receptor 1 (i.e. PD-1), epidermal growth factor receptor (i.e. EGFR), human epidermal growth factor receptor-2 (i.e. HER2), G protein-coupled receptor (i.e. GPCR), fibroblast growth factor receptor (i.e. FGFRs), vascular endothelial growth factor receptor family (i.e. VEGFR, VEGF stands for vascular endothelial growth factor), cytotoxic T-lymphocyte-associated protein 4 (i.e. CTLA4 or CTLA-4), human interleukin 5 receptor alpha (IL-5Ra), apolipoprotein, apolipoprotein E4 (i.e. ApoE4), beta-amyloid, angiotensin-converting enzyme 2 (ACE2), sodium ion-taurocholate co-transporting polypeptide (NTCP), B7.1 and B7, T1 FR1m, TNFR2, NADPH oxidase, Bcl IBax and other ligands in the apoptosis pathway, C5a receptor, HMG-CoA reductase, PDE V phosphodiesterase type, PDE IV phosphodiesterase type 4, PDE I, PDE II, PDE III, squalene cyclase inhibitors, CXCR1, CXCR2, nitric oxide synthase, cyclooxygenase 1, cyclooxygenase 2, 5HT receptors, dopamine receptors, G proteins, histamine receptors, 5-lipoxygenase, serine protease-like proteases, thymidylate synthetase, purine nucleoside phosphorylase, glyceraldehyde-3-phosphate dehydrogenase (i.e., GAPDH), glycogen phosphorylase, carbonic anhydrase, chemokine receptors, JAW STAT, RXR and analogs, HIV1 protease, HIV1 integrase, influenza neuraminidase, hepatitis B reverse transcriptase, sodium channels, protein P-glycoprotein, P glycoprotein and MRP metalloproteases, CD23, CD73, CD124, tyrosine kinase p561ck, CD4, CD5, IL-2 receptor, IL-1 receptor, TNF-alpha R, ICAM1, Ca2+ channels, VCAM, VLA-4 integrin, selectins, CD40 / CD40L, newokinins and receptors, inosine monophosphate dehydrogenase, p38 MAP kinase, Ras / Raf / MEW / ERK pathway, interleukin-1 converting enzyme, caspases, HCV, NS3 protease, HCV NS3 RNA helicase, glycinamide ribonucleotide formyltransferase, rhinovirus, 3C protease, herpes simplex virus-1, protease, cytomegalovirus protease, poly(ADP-ribose) polymerase, cyclin-dependent kinases, vascular endothelial growth factor, oxytocin receptor, microsomal transfer protein inhibitors, bile acid transport inhibitors, 5 alpha reductase inhibitors, angiotensin 11, glycine receptors, norepinephrine reuptake receptors, endothelin receptors, neuropeptide Y and receptors, adenosine receptors, adenosine kinase and AMP dehydrogenase, purinergic receptors, farnesyl transferase, geranyl transferase, TrkA receptor of NCF, tyrosine kinase Flk-II KDR, vitronectin receptors, integrin receptors, Her-21 neural sheath, telomerase inhibition, cytosolic phosphoinositide A2 and EGF receptor tyrosine kinase, ecdysone 20-monooxygenase, GABA-gated chloride channels, acetylcholinesterase, voltage-sensitive sodium channel proteins, calcium release channels and chloride channels, acetyl-CoA carboxylase, adenylosuccinate synthetase, protoporphyrinogen oxidase and enolpyruvyl shikimate phosphate synthetase, and / or one or more of all variants, mutants, splice variants, insertions, deletions and fusions of the aforementioned proteins. More preferably, the compound capable of binding to cell membrane protein or extracellular protein is BMS-8, Sul-acid, Biotin-NHS or PH-002; Preferably, the structural formula of the bifunctional compound is shown in the following structural formula Wherein, m is an integer from 2 to 8; n is an integer from 1 to 6.

3. The identifiable integrin receptor alpha of claim 1 v Use of a compound of Formula (I) in the manufacture of a bifunctional compound for degrading a cell membrane protein or an extracellular protein.

4. Use of the bifunctional compound for degrading target protein according to claim 2 in the preparation of a reagent for degrading cell membrane protein or extracellular protein.

5. A method of degrading a cell membrane protein in vitro, characterized by, The method comprises the steps of contacting and incubating the bifunctional compound for degrading target protein according to claim 2 with cells containing cell membrane protein or a mixture containing extracellular protein and cells.

6. Use of the bifunctional compound for degrading target protein according to claim 2 in the preparation of a drug for a disease related to abnormally elevated expression of cell membrane protein or extracellular protein; Preferably, the cell membrane or extracellular protein is programmed cell death-ligand 1 (i.e. PD-L1), programmed death receptor 1 (i.e. PD-1), epidermal growth factor receptor (i.e. EGFR), human epidermal growth factor receptor-2 (i.e. HER2), G protein-coupled receptor (i.e. GPCR), fibroblast growth factor receptor (i.e. FGFRs), vascular endothelial growth factor receptor family (i.e. VEGFR, VEGF stands for vascular endothelial growth factor), cytotoxic T-lymphocyte-associated protein 4 (i.e. CTLA4 or CTLA-4), human interleukin 5 receptor alpha (IL-5Ra), apolipoprotein, apolipoprotein E4 (i.e. ApoE4), beta-amyloid, angiotensin-converting enzyme 2 (ACE2), sodium ion-taurocholate co-transporting polypeptide (NTCP), B7.1 and B7, T1 FR1m, TNFR2, NADPH oxidase, Bcl IBax and other ligands in the apoptosis pathway, C5a receptor, HMG-CoA reductase, PDE V phosphodiesterase type, PDE IV phosphodiesterase type 4, PDE I, PDE II, PDE III, squalene cyclase inhibitors, CXCR1, CXCR2, nitric oxide synthase, cyclooxygenase 1, cyclooxygenase 2, 5HT receptors, dopamine receptors, G proteins, histamine receptors, 5-lipoxygenase, serine protease-like proteases, thymidylate synthetase, purine nucleoside phosphorylase, glyceraldehyde-3-phosphate dehydrogenase (i.e., GAPDH), glycogen phosphorylase, carbonic anhydrase, chemokine receptors, JAW STAT, RXR and analogs, HIV1 protease, HIV1 integrase, influenza neuraminidase, hepatitis B reverse transcriptase, sodium channels, protein P-glycoprotein, P glycoprotein and MRP metalloproteases, CD23, CD73, CD124, tyrosine kinase p561ck, CD4, CD5, IL-2 receptor, IL-1 receptor, TNF-alpha R, ICAM1, Ca2+ channels, VCAM, VLA-4 integrin, selectins, CD40 / CD40L, newokinins and receptors, inosine monophosphate dehydrogenase, p38 MAP kinase, Ras / Raf / MEW / ERK pathway, interleukin-1 converting enzyme, caspases, HCV, NS3 protease, HCV NS3 RNA helicase, glycinamide ribonucleotide formyltransferase, rhinovirus, 3C protease, herpes simplex virus-1, protease, cytomegalovirus protease, poly(ADP-ribose) polymerase, cyclin-dependent kinases, vascular endothelial growth factor, oxytocin receptor, microsomal transfer protein inhibitors, bile acid transport inhibitors, 5 alpha reductase inhibitors, angiotensin 11, glycine receptors, norepinephrine reuptake receptors, endothelin receptors, neuropeptide Y and receptors, adenosine receptors, adenosine kinase and AMP dehydrogenase, purinergic receptors, farnesyl transferase, geranyl transferase, TrkA receptor of NCF, tyrosine kinase Flk-II KDR, vitronectin receptors, integrin receptors, Her-21 neural sheath, telomerase inhibition, cytosolic phosphoinositide A2 and EGF receptor tyrosine kinase, ecdysone 20-monooxygenase, GABA-gated chloride channels, acetylcholinesterase, voltage-sensitive sodium channel proteins, calcium release channels and chloride channels, acetyl-CoA carboxylase, adenylosuccinate synthetase, protoporphyrinogen oxidase and enolpyruvyl shikimate phosphate synthetase, and / or one or more of all variants, mutants, splice variants, insertions, deletions and fusions of the aforementioned proteins. Preferably, the disease related to abnormally elevated expression of cell membrane protein or extracellular protein is selected from tumor, lipid-lowering, treatment of neurological diseases.

7. A method for degrading a cell membrane protein or an extracellular protein, characterized by, The method comprises the step of contacting the bifunctional compound according to claim 2 with cells containing cell membrane protein, or the method comprises the step of co-incubating the bifunctional compound with cells and extracellular protein; The target protein binding compound in the bifunctional compound is capable of specifically binding to the cell membrane protein to be degraded or the extracellular protein to be degraded; Preferably, the method for degrading cell membrane protein or extracellular protein is carried out in vitro or in vivo; Preferably, the cell membrane protein or extracellular protein is degraded by intracellular lysosomal pathway; Preferably, the cell membrane protein or extracellular protein enters the cell by endocytosis; Preferably, the cell is a normal cell or a tumor cell.

8. A method for designing a bifunctional compound capable of degrading cell membrane proteins or extracellular proteins using lysosomes, characterized in that, The design method comprises the following steps: S1) determining the target of cell membrane protein or extracellular protein to be degraded; S2) obtaining a small molecule compound capable of specifically binding to the target of cell membrane protein or extracellular protein to be degraded; S3) modifying the small molecule compound obtained in S2) or directly linking the small molecule compound to the integrin receptor alpha v β3-recognizing compound of claim 1 to obtain a bifunctional compound.

9. The identifiable integrin receptor alpha of claim 1 v a method of preparing a compound of Formula (I): ###0002### Formula (I) comprising the steps of: S11) coupling of 4-bromobenzaldehyde with 4-carboxyphenylboronic acid in the presence of a palladium catalyst and potassium carbonate to give the compound Compound with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N- hydroxysuccinimide under basic conditions to completion and continued reaction with NH2-(CH2) m -R to completion and purification to give the compound Compound with acetylacetate ethyl ester and piperidine and after purification to give the compound S12) N,N'-di-Boc-1H-1-guanidinopyrazole is reacted with 3-aminobenzoic acid in the presence of a basic organic reagent to obtain the compound Compound 9-fluorenylmethylhydrazinecarboxylate and HATU to obtain the compound S13) removing the compound Fmoc protecting group, followed by amide bond coupling, and finally removing the Boc protecting group, and purification to obtain a compound that can recognize integrin receptor α Fmoc protecting group, followed by amide bond coupling, and finally removing the Boc protecting group, and purification to obtain a compound that can recognize integrin receptor α v β3 Wherein, m is an integer from 1 to 10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; R is an active group capable of reacting with other groups to form a coupling chemical bond; Preferably, the R group is selected from one or more of an alkane group, an arene group, a heterocyclic arene group, an alkenyl group, an alkynyl group, a halo group, an alcoholic hydroxyl group, a thiol group, an aldehyde group, a ketone group, a carboxyl group, an aldehyde group, an amino group, an enolic group, an azido group, a maleimide, a tetrazine group, and variants thereof, and an alcoholic hydroxyl group containing an alpha hydrogen.

10. A method of preparing a bifunctional compound of claim 2 that degrades a target protein, the method comprising the steps of: S1) modifying the compound which binds to a cell membrane protein or an extracellular protein so that it has a group which is capable of coupling to a compound which can be recognized by an integrin receptor α v β3; and S2) coupling the compound which binds to a cell membrane protein or an extracellular protein to the compound which can be recognized by an integrin receptor α or obtained which have a group capable of coupling to a compound that can bind to a cell membrane protein or an extracellular protein that can bind to an identifiable integrin receptor α v β3 S2) the integrin receptor alpha identifiable of claim 1 v coupling of the compound obtained in step S1) with a compound of formula S2) Preferably, the compound that binds to a cell membrane protein is BMS-8 and the method of modification in step S1) is the coupling modification of BMS-8 with pent-4-yn-1-amine hydrochloride.

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