Preparation and application of cxcr4 and folr1 based protein degradation drugs

Abstract

The application belongs to the field of cancer treatment drug development, and discloses preparation and application of a protein degradation drug based on CXCR4 and FOLR1. Specifically relates to a kind of conjugate compound or its pharmaceutically acceptable salt, the conjugate compound or its pharmaceutically acceptable salt includes linker and multiple cell interaction molecules.The cell interaction molecule is the ligand capable of specifically binding with cell surface lysosome targeting receptor or tumor cell surface disease-related protein.The present disclosure also provides conjugate compounds, pharmaceutical compositions and their use as disease treatment.

Description

Technical Field

[0001] This invention belongs to the field of cancer therapeutic drug development, specifically involving the application of protein degradation drugs based on chemokine receptor 4 (CXCR4) and folate receptor 1 (FOLR1) in cancer treatment. Background Technology

[0002] Lysosome-targeting chimeras (LYTACs) are an emerging targeted protein degradation technology. By simultaneously binding to a lysosome targeting receptor (LTR) and a target protein, they transport the target protein to lysosomes for degradation, thereby effectively regulating disease-related proteins. With in-depth research into the endosome-lysosome and autophagosome-lysosome degradation pathways, various targeted protein degradation (TPD) strategies via the lysosome have emerged in recent years, such as LYTAC, AbTAC, AUTAC, ATTAC, bispecific nucleic acid ligand chimeras, and AUTOTAC. Compared to proteasome-based PROTACs, which can only degrade intracellular proteins, lysosome-based targeted protein degraders (TPDs) can degrade not only intracellular proteins but also cell membrane proteins, extracellular proteins, and some damaged organelles. Membrane proteins and extracellular proteins, which account for 40% of all proteins, are key factors in neurodegenerative diseases, autoimmune diseases, and cancer. Therefore, lysosomal-based TPD can compensate for the shortcomings of PROTAC.

[0003] In 2020, Carolyn Bertozzi's team at Stanford University first reported LYTAC technology based on CI-M6PR. Their designed LYTAC molecules can simultaneously bind to cell membrane proteins or extracellular proteins and the cation-independent mannose-6-phosphate receptor (CI-M6PR) located on the cell surface, forming a ternary complex. This complex then enters the cell via lysosomal targeting of the receptor and is subsequently degraded via the endosome-lysosomal pathway. The low pH in the later stages of endosome formation leads to the dissociation of the target protein and CI-M6PR. The target protein is degraded by lysosomes, while CI-M6PR is transported to the Golgi apparatus and cell surface for recycling. Using this mechanism, the authors' LYTAC successfully degraded EGFR and PD-L1 on the cell membrane. This LYTAC strategy provides a novel tool for targeting the degradation of membrane and extracellular proteins, showing great potential, especially in the fields of cancer, neurodegenerative diseases, and autoimmune diseases. Despite challenges in endocytosis efficiency and delivery, with advancements in molecular design and delivery technologies, LYTAC holds promise as an important platform for next-generation precision medicine.

[0004] Both CXCR4 and FOLR1 are cell membrane surface receptors highly expressed in tumor cells. Both can mediate ligand internalization and lysosomal transport, and they have a synergistic effect. Therefore, constructing a dual-receptor lysosome-targeting chimera (DR-TAC) based on CXCR4 and FOLR1 can improve the degradation efficiency of LYTAC drugs on target proteins, thereby enhancing the therapeutic effect of the drug on the disease. Summary of the Invention

[0005] This application relates to conjugate compounds or pharmaceutically acceptable salts thereof, pharmaceutical compositions thereof, and methods of using thereof. More specifically, this application relates to multiligand drug conjugates, particularly multiligand drug conjugates capable of inducing endocytosis, pharmaceutical compositions thereof, and methods of treating diseases using said drug conjugates, including but not limited to cancer.

[0006] One aspect of this application discloses a conjugate compound or a pharmaceutically acceptable salt thereof, the conjugate compound or a pharmaceutically acceptable salt thereof comprising one or two molecules that interact with a cell membrane lysosomal targeting receptor, a molecule that interacts with a cell membrane target protein, and a linker for conjugating the molecules, wherein the molecule interacting with the cell membrane or extracellular target protein is conjugated to at least one of the molecules interacting with the cell membrane lysosomal targeting receptor via the linker. The molecule interacting with the cell membrane lysosomal targeting receptor comprises a first ligand capable of specifically binding to a first cell membrane lysosomal targeting receptor and a second ligand capable of specifically binding to a second cell membrane lysosomal targeting receptor. The molecule interacting with the cell membrane target protein comprises a ligand interacting with PD-L1 protein and a ligand interacting with EGFR protein.

[0007] In some implementations, the first cell membrane lysosome-targeting receptor, the second cell membrane lysosome-targeting receptor, and the cell membrane target protein are different from each other.

[0008] In some embodiments, the first cell membrane lysosome-targeting receptor, the second cell membrane lysosome-targeting receptor, and the cell membrane target protein provided in this application are independently selected from the group consisting of: chemokine receptor 4 (CXCR4), folate receptor 1 (FOLR1), programmed cell death-ligand 1 (PD-L1), and epidermal growth factor receptor (EGFR).

[0009] In some embodiments, the first ligand, the second ligand, and the third ligand are independently selected from the group consisting of: cyclic peptide T140 analogs, cyclic peptide CPCR4 analogs, folic acid (FA) and its analogs, ligand compound B of PD-L1, and ligand compound G of EGFR.

[0010] In some embodiments, the ligand comprises a cyclic peptide T140 analog with the amino acid sequence: NH2-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-(D-Lys)-Pro-Tyr-Arg-Cit-Cys-Arg-COOH (where two cysteine ​​residues form a disulfide bond), or an analog with substitutions, deletions, or insertions of 1 to 3 amino acids, including carboxyl-terminal amidation and L-Lys linkage to the D-Lys side. The second ligand cyclic peptide CPCR4 analog has the amino acid sequence: NH2-(D-Tyr)-(D-[NMe]Lys)-Arg-(2-Nal)-Gly-Arg-COOH, which undergoes initial and final amidation cyclization via terminal amino and terminal carboxyl groups.

[0011] In some embodiments, the molecules that interact with cell membrane lysosomal targeting receptors, as described in this application, are endocytic molecules capable of mediating endocytosis.

[0012] In some embodiments, the coupling compound or a pharmaceutically acceptable salt thereof comprises one or two molecules that interact with a cell membrane lysosomal targeting receptor and a molecule that interacts with a cell membrane target protein.

[0013] Another aspect of this application discloses a method for treating a disease characterized in a subject by overexpression of chemokine receptor 4 (CXCR4) or folate receptor 1 (FOLR1), the method comprising administering to the subject a therapeutically effective amount of the conjugate compound provided in this application or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition provided in this application. In some embodiments, the disease is cancer.

[0014] In some implementations, the cancer is selected from the group consisting of: lung cancer, breast cancer, prostate cancer, kidney cancer, ovarian cancer, stomach cancer, uterine cancer, endometrial cancer, liver cancer, thyroid cancer, pancreatic cancer, colon cancer, colorectal cancer, esophageal cancer, skin cancer, lymphoma, leukemia, and multiple myeloma.

[0015] In some embodiments, the coupling compound is selected from the following structures:

[0016] In the above structure, the linker C portion can be substituented accordingly. The substituents can be selected from the following non-hydrogen substituents: The C1-12 alkyl, C3-10 cycloalkyl, C1-12 alkoxy, C5-12 aryl, C5-12 heteroaryl, hydroxyl, halogen and C1-4 alkoxycarbonyl are substituted with at least one non-hydrogen substituent, and the aryl and heteroaryl groups are either unsubstituted or substituted with at least one non-hydrogen substituent selected from C1-4 alkyl, C1-4 alkoxy, hydroxyl and halogen.

[0017] As used herein, a "substituted" group refers to a group in which at least one hydrogen atom is replaced by at least one non-hydrogen group, provided that the group satisfies the valence requirement and that the substitution produces a chemically stable compound. In this specification, unless specifically described as "unsubstituted," all substituents may be substituted or unsubstituted.

[0018] "Alkyl" refers to a straight-chain or branched saturated hydrocarbon group, typically having a specific number of carbon atoms (e.g., 1 to 12 carbon atoms). Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, and n-heptyl. Alkyl groups can be attached to a parent group or matrix via any ring atom, unless such attachment would violate the valence requirement. Similarly, alkyl or alkenyl groups can contain at least one non-hydrogen substituent, unless such substitution would violate the valence requirement.

[0019] "Cycloalkyl" refers to a saturated monocyclic or polycyclic hydrocarbon ring, typically having a specific number of carbon atoms comprising the ring (e.g., C3-10 cycloalkyl refers to a cycloalkyl group having 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms as ring members). A cycloalkyl group can be attached to the parent or matrix via any ring atom unless such attachment would violate the valence requirement. Similarly, a cycloalkyl group can contain at least one non-hydrogen substituent unless such substitution would violate the valence requirement.

[0020] "Aryl" refers to monovalent and divalent aromatic groups, including 5-membered and 6-membered monocyclic aromatic groups, respectively. "Heteroaryl" refers to monovalent and divalent aromatic groups, including 5-membered and 6-membered monocyclic aromatic groups containing 1 to 4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, respectively. Examples of monocyclic and heteroaryl groups include, but are not limited to, phenyl, pyridyl, furanyl, pyrroleyl, thiopheneyl, thiazolyl, isothiazolyl, imidazolyl, triazolyl, tetrazolyl, pyrazolyl, oxazolyl, isoxazolyl, pyrazinyl, pyridazinyl, and pyrimidinyl. Aryl and heteroaryl groups also include bicyclic and tricyclic groups, including fused 5-membered and 6-membered rings as described above. Examples of polycyclic aryl and heteroaryl groups include, but are not limited to, isoquinolinyl, naphthyl, biphenyl, anthraceneyl, pyrene, carbazole, benzoxazolyl, benzodioxazolyl, benzothiazolyl, benzimidazolyl, benzothiophene, quinolinyl, indolyl, benzofuranyl, purine, indoleazinyl, etc. Aryl and heteroaryl groups can be attached to the parent group or matrix via any ring atom, unless such attachment would violate the valence requirement. Similarly, aryl and heteroaryl groups can contain at least one non-hydrogen substituent, unless such substitution would violate the valence requirement. The non-hydrogen substituents of aryl and heteroaryl groups can also be substituted by other non-hydrogen substituents.

[0021] "Carbonyl" refers to -C(O)R'. As used herein, "(O)" refers to oxygen bonded to an atom such as carbon or sulfur via a double bond. Here, "R" refers to a non-hydrogen substituent such as a lower alkyl group, lower alkoxy group, etc. Examples of carbonyl groups include, but are not limited to, 2-methoxyoxoethyl, 3-methoxyoxopropyl, etc. A carbonyl group can be attached to a parent group or matrix via any ring atom, unless such attachment would violate the valence requirement. Similarly, a carbonyl group can contain at least one non-hydrogen substituent, unless such substitution would violate the valence requirement.

[0022] "Alkoxy" refers to an alkyl group with an -O- alkyl group. Here, alkyl is the same as defined above. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, etc. An alkoxy group can be attached to a parent group or matrix via any ring atom unless such attachment would violate the valence requirement. Similarly, an alkoxy group can contain at least one non-hydrogen substituent unless such substitution would violate the valence requirement.

[0023] "Hydroxyl" refers to -OH, "halogen" refers to fluorine, chlorine, bromine and iodine, and "oxo" refers to =O.

[0024] The preparation method of the conjugate drug for protein degradation in this invention includes the following steps: a) The T140 analog linear peptide, CPCR4 analog linear peptide, azido-modified FA analog FA-1, and azido-modified FA analog FA-2 were synthesized using the Fmoc solid-phase synthesis strategy. b) The T140 linear peptide linked to FA in a is cyclized with a disulfide bond to obtain intermediate compound T140-c; ​​then the CPCR4 analog linear peptide in a is cyclized with a disulfide amide to obtain cyclic peptide CPCR4; then the cyclic peptide CPCR4 is condensed with FA-1 in a to obtain intermediate compound CF; CPCR4 is condensed with NHS azidoacetate to obtain azide-modified CPCR4 analog CPCR4-1; c) The ligand compound B of PD-L1 is coupled with propargyl-PEG(n+1)-carboxylic acid (n = 1, 2, 3, 4, 5, 6, 7) via a click reaction to obtain the alkynyl-modified intermediate compound Ln-B. At the same time, the ligand compound G of EGFR is coupled with propargyl-PEG6-carboxylic acid via a click reaction to obtain the alkynyl-modified intermediate compound L5-G. d) The T140-c obtained in b and the Ln-B obtained in c are coupled by a click reaction to obtain the conjugate compound TF-Ln-B (n = 1, 2, 3, 4, 5, 6, 7); the CF obtained in b and the L5-B obtained in c are coupled by a click reaction to obtain the conjugate compound CF-L5-B; the CF obtained in b and the L5-G obtained in c are coupled by a click reaction to obtain the conjugate compound CF-L5-G; the CPCR4-1 obtained in b and the L5-G obtained in c are coupled by a click reaction to obtain the conjugate compound C-L5-G; the FA-2 obtained in a and the L5-G obtained in c are coupled by a click reaction to obtain the conjugate compound F-L5-G.

[0025] This invention evaluates the degradation activity of cell membrane proteins in different types of cancer cells, obtaining protein-degrading drugs with good tumor-associated membrane protein degradation activity. Furthermore, this invention evaluates the in vivo activity of the protein-degrading drugs in H1975 tumor-bearing mice, obtaining protein-degrading drugs with good tumor-suppressive effects. Therefore, this invention provides a method for preparing a conjugate drug for protein degradation based on chemokine receptor 4 (CXCR4) and folate receptor 1 (FOLR1), and its application in tumor-targeted therapy. The tumors preferentially selected are lung cancer, and other tumors with abnormal expression of CXCR4 or FOLR1.

[0026] Another aspect of the present invention provides a medicine comprising the above-described conjugate drug or its pharmaceutically acceptable salt as an active ingredient.

[0027] In addition to the active ingredient, the pharmaceutical compositions of the present invention comprise at least one pharmaceutically acceptable carrier. As used in the present invention, a "pharmaceutically acceptable carrier" refers to a known pharmaceutically acceptable excipient that can be used to formulate a pharmaceutically active compound for application to a target, and is substantially non-toxic and non-irritating under the conditions of use. The accurate amount of the excipient is determined through standard pharmaceutical practice, as well as the solubility, chemical properties, and chosen route of administration of the active compound.

[0028] The pharmaceutical compositions of the present invention can be formulated into suitable forms for use in the desired method of administration using suitable and physiologically acceptable excipients (e.g., excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants, brighteners, flavoring agents, etc.).

[0029] Pharmaceutical compositions with conjugate drugs as active ingredients include various forms such as lyophilized powder for injection, injection solution, tablets, liposomes, and nano-formulations.

[0030] The beneficial effects of the present invention are as follows: The conjugate drug provided by the present invention links disease-related cell membrane protein ligands with CXCR4 targeting ligands and FOLR1 targeting ligands. By utilizing the lysosomal targeting function of CXCR4 and FOLR1, the target protein is internalized in a mediated manner and eventually reaches lysosomal degradation. By affecting the expression level of disease-related proteins, it achieves the effect of precision tumor treatment. Attached Figure Description To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Figure 1 HRMS analysis spectrum of TF-L1-B. Figure 2 HRMS analysis spectrum of TF-L2-B. Figure 3 HRMS analysis spectrum of TF-L3-B. Figure 4 HRMS analysis spectrum of TF-L4-B. Figure 5 HRMS analysis spectrum of TF-L5-B. Figure 6 HRMS analysis spectrum of TF-L6-B. Figure 7 HRMS analysis spectrum of TF-L7-B. Figure 8 HRMS analysis spectrum of CF-L5-B. Figure 9 HRMS analysis of CF-L5-G spectrum. Figure 10 HRMS analysis of C-L5-G spectra. Figure 11 HRMS analysis spectrum of F-L5-G. Figure 12 The graph shows the degradation results of PD-L1 by TF-Ln-B (n=1, 2, 3, 4, 5, 6, 7). Figure 13 The graph shows the degradation results of PD-L1 by CF-L5-B. Figure 14 The graph shows the degradation results of EGFR by CF-L5-G, C-L5-G and F-L5-G. Figure 15 The figure shows the experimental results of the inhibitory activity of CF-L5-G on H1975 xenografts. Detailed Implementation To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention. To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention. In some embodiments of the present invention, the cells that may be used include: Human triple-negative breast cancer cells: MDA-MB-231; Human lung adenocarcinoma cells: H1975; All of the above cells can be obtained commercially, for example, purchased from the Cell Bank of the Chinese Academy of Sciences. Example 1 Chemical Synthesis (1) Synthesis of intermediate Ln-B (n=1, 2, 3, 4, 5, 6, 7): The specific method is as follows: PD-L1 ligand compound B (20 mg, 0.028 mmol), propargyl-PEG(n+1)-carboxylic acid (n = 1, 2, 3, 4, 5, 6, 7) (0.028 mmol), HATU (11 mg, 0.028 mmol) and HOAT (4 mg, 0.028 mmol) were added to a round-bottom flask, dissolved in 3 mL of DMF, and then DIEA (10 μL, 0.057 mmol) was added. The mixture was stirred at room temperature for 2 h and concentrated under reduced pressure. Compounds L1-B (13.7 mg, 0.016 mmol, 57%), L2-B (9.5 mg, 0.010 mmol, 36%), L3-B (11.4 mg, 0.012 mmol, 43%), L4-B (10 mg, 0.010 mmol, 36%), L5-B (10 mg, 0.010 mmol, 36%), L6-B (11 mg, 0.010 mmol, 36%), and L7-B (12 mg, 0.011 mmol, 38%) were prepared by HPLC and lyophilized. (2) Synthesis of TF-Ln-B (n=1, 2, 3, 4, 5, 6, 7): The specific method is as follows: T140-c (11 mg, 0.004 mmol), Ln-B (4 mg, 0.004 mmol), CuSO4·5H2O (2 mg, 0.008 mmol), and VcNa (3.2 mg, 0.016 mmol) were added to a round-bottom flask, along with H2O / DMF solvent (0.4 mL: 1.6 mL). The mixture was stirred overnight at room temperature, concentrated under reduced pressure, and prepared by HPLC. The final product was lyophilized to obtain TF-L1-B (6.8 mg, 0.0019 mmol, 48%). , TF-L2-B (7.4mg, 0.002mmol, 52%), TF-L3-B (8mg, 0.0022mmol, 55%), TF-L4-B (7.3mg, 0.002mmol, 50%), T F-L5-B (8.9 mg, 0.0024 mmol, 60%), TF-L6-B (8.5 mg, 0.0023 mmol, 57%), TF-L7-B (7.6 mg, 0.002 mmol, 50%). HRMS (ESI) of TF-L1-B: C 166 H 224 ClN 49 O 33 S2[M+H] 6+ Predicted value: 589.6159, Actual value: 589.6125. Purity: >99.00%. HRMS (ESI) of TF-L2-B: C168 H 228 ClN 49 O 34 S2[M+H] 6+ Predicted value: 596.9536, Actual value: 596.9549. Purity: >99.00%. HRMS (ESI) of TF-L3-B: C 170 H 232 ClN 49 O 35 S2[M+H] 6+ Predicted value: 604.2913, Actual value: 604.2914. Purity: >99.00%. HRMS (ESI) of TF-L4-B: C 172 H 236 ClN 49 O 36 S2[M+H] 6+ Predicted value: 611.6290, Actual value: 611.6287. Purity: >99.00%. HRMS (ESI) of TF-L5-B: C 174 H 240 ClN 49 O 37 S2[M+H] 6+ Predicted value: 618.9667, Actual value: 618.9667. Purity: >99.00%. HRMS (ESI) of TF-L6-B: C 176 H 244 ClN 49 O 38 S2[M+H] 6+ Predicted value: 626.3044, Actual value: 626.3043. Purity: >99.00%. HRMS (ESI) of TF-L7-B: C 178 H 248 ClN 49 O 39 S2[M+H] 6+ Predicted value: 633.6422, Actual value: 633.6421. Purity: >99.00%. (6) Synthesis of CF-L5-B: The specific method is as follows: Synthesis of cPCR4: 2-CTC resin (459 mg, 0.5 mmol) was swollen in 10 mL DCM for 30 min, washed with DMF, 3 × 10 mL × 2 min. Fmoc-Arg(pbf)-OH was added, blocked with 10 mL methanol for 15 min, the peptide was washed, Fmoc was removed, the peptide was washed again, and an indene test was performed. Subsequently, Gly-(2-Nal)-Arg-(D-[NMe]Lys)-(D-Tyr) was added sequentially. The peptide was cleaved with DCM / hexafluoroisopropanol (HFIP) (8 mL: 2 mL), retaining the side chain protecting groups. 10 mL of ice-cold diethyl ether was added, and the mixture was filtered to obtain a precipitate of crude peptide (665 mg). The crude peptide (114 mg, 0.1 mmol), diphenyl azidophosphate (83 mg, 0.3 mmol), and NaHCO3 (42 mg, 0.5 mmol) were added to a round-bottom flask, dissolved in 20 mL DMF, stirred at room temperature for 4 h, and concentrated under reduced pressure. Add 5 mL of TFA, stir at room temperature for 1 h, and concentrate under reduced pressure. Prepare by HPLC, and lyophilize to obtain compound CPCR4 (46 mg, 0.065 mmol). HRMS (ESI): C 37 H 49 N9O6[M+H] + Predicted value: 716.3879, Actual value: 716.3867. Purity: 95.41%. Synthesis of FA-1: 2-CTC resin (459 mg, 0.5 mmol) was swollen in 10 mL DCM for 30 min, washed with DMF, 3 × 10 mL × 2 min. Fmoc-Lys(Dde)-OH was inoculated, blocked with 10 mL methanol for 15 min, peptide was washed, Fmoc was removed, peptide was washed again, indene was detected, azidoacetic acid was inoculated, Dde was removed, folic acid was inoculated, TFA was used to cleave the peptide, and it was extracted with ice-cold ether and deionized water. The aqueous phase was collected and lyophilized. HPLC was used to prepare compound FA-1 (52 mg, 0.08 mmol, 16%), which was then lyophilized. Synthesis of CF: CPCR4 (20 mg, 0.028 mmol), FA-1 (18 mg, 0.028 mmol), HATU (11 mg, 0.028 mmol), and HOAT (4 mg, 0.028 mmol) were added to a round-bottom flask, dissolved in 3 mL of LDM, and then DIEA (10 μL, 0.057 mmol) was added. The mixture was stirred at room temperature for 2 h and concentrated under reduced pressure. The solution was prepared by HPLC and lyophilized to obtain compound CF (15 mg, 0.011 mmol, 39%). HRMS (ESI): C 64 H 79 N 21 O 13 [M+H] 2+ Predicted value: 675.8156, Actual value: 675.8151. Purity: 95.30%. CF (10 mg, 0.0074 mmol), L5-B (8 mg, 0.0074 mmol), CuSO4·5H2O (4 mg, 0.015 mmol), and VcNa (6 mg, 0.03 mmol) were added to a round-bottom flask, along with H2O / DMF solvent (0.4 mL: 1.6 mL). The mixture was stirred overnight at room temperature, concentrated under reduced pressure, and prepared by HPLC. The lyophilized product was then obtained as CF-L5-B (8 mg, 0.0034 mmol, 45%). HRMS (ESI): C 121 H 148 ClN 25 O 25 [M+H] 4+ Predicted value: 597.7773, Actual value: 597.7764. Purity: >99.00%. (7) Synthesis of L5-G: The specific method is as follows: Compound G (100 mg, 0.224 mmol), propargyl-PEG6-carboxylic acid (78 mg, 0.224 mmol), HATU (85 mg, 0.224 mmol), and HOAT (30 mg, 0.224 mmol) were added to a round-bottom flask, dissolved in 10 mL of DMF, and then DIEA (78 μL, 0.448 mmol) was added. The mixture was stirred at room temperature for 2 h and concentrated under reduced pressure. The solution was prepared by HPLC and lyophilized to give compound L5-G (123 mg, 0.158 mmol, 71%). (8) Synthesis of CF-L5-G: The specific method is as follows: CF (10 mg, 0.0074 mmol), L5-G (6 mg, 0.0074 mmol), CuSO4·5H2O (4 mg, 0.015 mmol), and VcNa (6 mg, 0.03 mmol) were added to a round-bottom flask, and H2O / DMF (0.4 mL: 1.6 mL) solvent was added. The mixture was stirred overnight at room temperature, concentrated under reduced pressure, and prepared by HPLC. The lyophilized product was then obtained as CF-L5-G (7 mg, 0.0033 mmol, 45%). HRMS (ESI): C 102 H 130 ClFN 26 O 22 [M+H] 3+ Predicted value: 709.6592, Actual value: 709.6586. Purity: >99.00%. (9) Synthesis of C-L5-G: The specific method is as follows: First, CPCR4 (20 mg, 0.028 mmol) and NHS azidoacetate (6 mg, 0.028 mmol) were added to a round-bottom flask, dissolved in 2 mL of DMF, and then DIEA (10 μL, 0.056 mmol) was added. The mixture was stirred at room temperature for 2 h and concentrated under reduced pressure. The solution was prepared by HPLC and lyophilized to obtain compound CPCR4-1 (12 mg, 0.015 mmol, 53%). Then, CPCR4-1 (10 mg, 0.013 mmol), L5-G (10 mg, 0.013 mmol), CuSO4·5H2O (6 mg, 0.025 mmol), and VcNa (10 mg, 0.05 mmol) were added to a round-bottom flask, and H2O / DMF (0.4 mL: 1.6 mL) was added. The mixture was stirred at room temperature overnight, concentrated under reduced pressure, prepared by HPLC, and lyophilized to obtain C-L5-G (4 mg, 0.0025 mmol, 20%). HRMS (ESI): C 77 H 101 ClFN 17 O 16 [M+H] 3+ Predicted value: 525.5834, Actual value: 525.5853. Purity: >99.00%. (10) Synthesis of F-L5-G: The specific method is as follows: MBHA resin (434 mg, 0.2 mmol) was swollen in 10 mL DCM for 30 min, washed with DMF (3 × 10 mL × 2 min). Fmoc-Lys(Dde)-OH was inoculated, blocked with 10 mL methanol for 15 min, peptides were washed, Fmoc was removed, peptides were washed again, indene was detected, azidoacetic acid was inoculated, Dde was removed, folic acid was inoculated, TFA was used to cleave the peptide, and extraction was performed with ice-cold ether and deionized water. The aqueous phase was collected and lyophilized. HPLC was used to prepare compound FA-2 (26 mg, 0.04 mmol, 20%), which was then lyophilized. Then, FA-2 (10 mg, 0.015 mmol), L5-G (12 mg, 0.015 mmol), CuSO4·5H2O (8 mg, 0.031 mmol), and VcNa (12 mg, 0.061 mmol) were added to a round-bottom flask, along with the solvent H2O / DMF (0.4 mL: 1.6 mL). The mixture was stirred overnight at room temperature, concentrated under reduced pressure, and prepared by HPLC. The resulting product was lyophilized to obtain F-L5-G (13 mg, 0.009 mmol, 61%). HRMS (ESI): C 65 H 84 ClFN 18 O 16 [M+H]2+ Predicted value: 714.3065, Actual value: 714.3058. Purity: >99.00%. Example 2: Testing the protein degradation activity of the protein-degrading drug (1) The activity of TF-Ln-B in degrading PD-L1 was evaluated. The specific operation procedure is as follows: MDA-MB-231 cells, which highly express PD-L1, were seeded into 6-well cell culture plates, with a total of 8 wells, including one control group and 7 treatment groups. 5 × 10⁶ cells were seeded per well. 5 Cells were cultured for 8 hours. After the cells adhered to the well, 100 nM of TF-Ln-B composed of different linkers (n = 1, 2, 3, 4, 5, 6, 7) was added to each well. After culturing for 8 hours, the cells were collected for Western blot experiments. The PD-L1 protein bands were imaged and the results were analyzed using ImageJ software. Figure 12 The results showed that TF-L1-B, TF-L2-B, TF-L3-B, TF-L4-B, TF-L5-B, TF-L6-B and TF-L7-B all exhibited significant degradation activity against PD-L1, with TF-L5-B showing the best degradation effect, reaching a degradation rate of 86%. (2) The activity of CF-L5-B in degrading PD-L1 was evaluated. The specific operation procedure is as follows: MDA-MB-231 cells, which highly express PD-L1, were seeded into 6-well cell culture plates, with a total of 6 wells, including one control group and 5 drug-treated groups. Each well was seeded with 5 × 10⁶ cells. 5 Cells were cultured for 8 hours. After cell adhesion, different concentrations of CF-L5-B were added to each well. After further culture, cells were collected for Western blot experiments. The PD-L1 protein bands were imaged and the results were analyzed using ImageJ software. Figure 13 The results showed that the higher the concentration of CF-L5-B, the better the degradation effect of PD-L1. CF-L5-B can degrade 74% of PD-L1 at a concentration of 150 nM. (3) The activities of CF-L5-G, C-L5-G and F-L5-G in degrading EGFR were evaluated. The specific operation procedure is as follows: H1975 cells expressing high levels of EGFR were seeded into 6-well cell culture plates, with a total of 4 wells, including one control group and 3 treatment groups. Each well was seeded with 5 × 10⁶ cells. 5After cells adhered to the culture vessel, 200 nM of CF-L5-G, C-L5-G, and F-L5-G were added to each well. After culturing for 48 h, cells were collected for Western blot experiments. The EGFR protein bands were imaged and the results were analyzed using ImageJ software. Figure 14 The results showed that CF-L5-G, C-L5-G and F-L5-G could all mediate the degradation of EGFR, among which CF-L5-G had the most significant effect on the degradation of EGFR, with a degradation rate of 78%. Example 3: Testing the antitumor activity of EGFR-degrading drugs H1975 cells were inoculated into the right axilla of Balb / c nude mice, with 5 × 10⁶ cells per mouse. 6 Each cell, until the tumor grows to 90-110 mm 3 Nude mice were randomly divided into 5 groups: 1 control group and 4 treatment groups (20 mg / kg Gefitinib, 20 mg / kg Gefitinib + 20 mg / kg CF, 10 mg / kg CF-L5-G, 20 mg / kg CF-L5-G). The treatment was started on the same day and administered intraperitoneally. The mice were then given the drug every two days. The weight and tumor volume of the nude mice were recorded. Tumor volume calculation method: V (mm) 3 )=(L×W 2 / 2), L: major axis; W: minor axis. The average tumor volume of the blank control group should reach 1000 mm. 3 On the left and right sides, the mice were euthanized, the tumors were removed, and their weight was measured. Figure 15 The results showed that 10 mg / kg CF-L5-G had better inhibitory activity against H1975 xenografts than 20 mg / kg Gefitinib and 20 mg / kg Gefitinib + 20 mg / kg CF. Furthermore, 20 mg / kg CF-L5-G had an inhibition rate of 68.5% against the tumor, while 20 mg / kg Gefitinib had an inhibition rate of 37.3%, demonstrating that CF-L5-G had significantly better inhibitory activity against H1975 xenografts than Gefitinib. Western blot and immunohistochemical experiments on tumor tissue revealed that CF-L5-G can degrade EGFR in transplanted tumor tissue. Immunofluorescence experiments showed that CF-L5-G induced apoptosis and inhibited proliferation of tumor cells, demonstrating that CF-L5-G overcomes the resistance of H1975 transplanted tumors to Gefitinib by degrading EGFR. The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A coupling compound or a pharmaceutically acceptable salt thereof, said coupling compound or a pharmaceutically acceptable salt thereof comprising a linker and a plurality of cell-interacting molecules, wherein each coupling compound comprises at least two different cell-interacting molecules.

2. The conjugate compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein the conjugate compound or a pharmaceutically acceptable salt thereof comprises a first ligand specifically binding to a first cell membrane lysosomal targeting receptor, a second ligand specifically binding to a second cell membrane lysosomal targeting receptor, and a third ligand specifically binding to a cell membrane target protein; wherein a portion of the conjugate compound or a pharmaceutically acceptable salt thereof comprises a first ligand specifically binding to a first cell membrane lysosomal targeting receptor or a second ligand specifically binding to a second cell membrane lysosomal targeting receptor, and a third ligand specifically binding to a cell membrane target protein; wherein the first cell membrane lysosomal targeting receptor is chemokine receptor 4CXCR4, the first ligand is selected from cyclic peptide T140 analogs or cyclic peptide CPCR4 analogs; the second cell membrane lysosomal targeting receptor is folic acid receptor 1FOLR1, the second ligand is selected from folic acid and its analogs; the cell membrane target protein is programmed cell death-ligand 1PD-L1 or epidermal growth factor receptor EGFR, and the third ligand is selected from ligand compound B of PD-L1 or ligand compound G of EGFR.

3. The conjugate compound according to any one of claims 1-2, or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence of the first ligand cyclic peptide T140 analog is: NH2-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-(D-Lys)-Pro-Tyr-Arg-Cit-Cys-Arg-COOH (where two cysteine ​​residues form a disulfide bond), or analogs with substitution, deletion, or insertion of 1 to 3 amino acids, including amidation of the carboxyl terminus and L-Lys linkage to the D-Lys side. The amino acid sequence of the second ligand cyclic peptide CPCR4 analog is: NH2-(D-Tyr)-(D-[NMe]Lys)-Arg-(2-Nal)-Gly-Arg-COOH, which is cycloidized at both ends via a terminal amino group and a terminal carboxyl group.

4. The coupling compound according to any one of claims 1-3, or a pharmaceutically acceptable salt thereof, wherein the folic acid FA, The structures of compounds B and G are as follows.

5. The coupling compound according to any one of claims 1-4 or a pharmaceutically acceptable salt thereof, wherein the linker is selected from propargyl-PEG(n+1)-carboxylic acid, wherein n = 1, 2, 3, 4, 5, 6, 7, and has the following structure.

6. The coupling compound according to any one of claims 1-5 or a pharmaceutically acceptable salt thereof, selected from the following structures.

7. The method for preparing the coupling compound according to any one of claims 1-6, comprising the following steps. a) The T140 analog linear peptide, CPCR4 analog linear peptide, azido-modified FA analog FA-1, and azido-modified FA analog FA-2 were synthesized using the Fmoc solid-phase synthesis strategy. b) The T140 linear peptide linked to FA in a is cyclized via a disulfide bond to obtain intermediate compound T140-c; ​​then the CPCR4 analog linear peptide in a is cyclized via a disulfide bond to obtain cyclic peptide CPCR4; then the cyclic peptide CPCR4 is condensed with FA-1 in a to obtain intermediate compound CF; and CPCR4 is condensed with NHS azidoacetate to obtain a azido-modified CPCR4 analog CPCR4-1. c) The ligand compound B of PD-L1 is coupled with propargyl-PEG(n+1)-carboxylic acid (n = 1, 2, 3, 4, 5, 6, 7) via a click reaction to obtain the alkynyl-modified intermediate compound Ln-B. At the same time, the ligand compound G of EGFR is coupled with propargyl-PEG6-carboxylic acid via a click reaction to obtain the alkynyl-modified intermediate compound L5-G. d) The T140-c obtained in b and the Ln-B obtained in c are coupled by a click reaction to obtain the conjugate compound TF-Ln-B, where n = 1, 2, 3, 4, 5, 6, 7; the CF obtained in b and the L5-B obtained in c are coupled by a click reaction to obtain the conjugate compound CF-L5-B; the CF obtained in b and the L5-G obtained in c are coupled by a click reaction to obtain the conjugate compound CF-L5-G; the CPCR4-1 obtained in b and the L5-G obtained in c are coupled by a click reaction to obtain the conjugate compound C-L5-G; the FA-2 obtained in a and the L5-G obtained in c are coupled by a click reaction to obtain the conjugate compound F-L5-G.

8. A pharmaceutical composition comprising a conjugate compound according to any one of claims 1-6 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

9. Use of the conjugate compound of any one of claims 1-6 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 8 in the preparation of an agent for treating a disease characterized in a subject by overexpression of chemokine receptor 4 (CXCR4) or folate receptor 1 (FOLR1).

10. The use according to claim 9, wherein the disease is selected from cancer.

11. The use according to claim 10, wherein the cancer is selected from: lung cancer, breast cancer, prostate cancer, kidney cancer, leukemia, ovarian cancer, stomach cancer, uterine cancer, endometrial cancer, liver cancer, colon cancer, thyroid cancer, pancreatic cancer, colorectal cancer, esophageal cancer, skin cancer, lymphoma, and multiple myeloma.

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