Preparation and application of PET probe targeting chemokine receptor 4 (CXCR4)

Abstract

The present invention belongs to the field of cancer diagnosis drug development, and relates to a conjugate compound or a pharmaceutically acceptable salt thereof, the conjugate compound or the pharmaceutically acceptable salt thereof comprising a payload and two or more cell interaction molecules. The cell interaction molecule is a ligand capable of specifically binding to a cell surface receptor or a non-cell surface receptor. The payload of the present disclosure refers to a group capable of chelating with a radionuclide. The disclosure also provides markers of conjugate compounds and radionuclides, pharmaceutical compositions and their use in disease diagnosis and treatment.

Description

[0001] The present invention belongs to the field of cancer diagnosis drug development, and in particular relates to the application of a chemokine receptor 4 (CXCR4)-based polypeptide drug conjugate in breast cancer diagnosis. Background Art

[0002] Breast cancer is a common tumor among female tumors, and the incidence of breast cancer is increasing year by year worldwide. There are 1.2 million to 1.4 million new cases of breast cancer each year, and about 500,000 people die from the disease each year. Once breast cancer metastasizes to the bone, the median survival of the patient is 2 years, and the 5-year survival rate is about 10%. Tumor cells express a variety of specific receptor proteins on their surface. Chemokine receptor 4 (CXCR4), as a member of the chemokine receptor family, is highly expressed on the surface of a variety of tumor cells. Its expression level is directly related to the malignancy of the tumor and the patient's prognosis. The earliest studies on CXCR4 mainly explored its role in the pathological process of acquired immunodeficiency syndrome. Müller et al. reported that CXCR4 is highly expressed in human breast cancer cell lines and tissues, and studied the role of CXCR4 in the pathological development of tumors. The results showed that high expression of CXCR4 is related to tumor proliferation, angiogenesis, etc. Some studies have found that at least 23 different types of tumors, including hematological tumors and other solid tumors, highly express CXCR4. Therefore, blocking the binding of CXCR4 and CXCL12 and inhibiting their biological activity can inhibit tumor progression.

[0003] In recent years, tumor targeted therapy targeting CXCR4 has developed rapidly, and a variety of CXCR4 antagonists have been developed, including antibodies, peptides and small molecule compounds. Among them, the drug with a more significant therapeutic effect is AMD3100, which was approved for marketing by the FDA in 2008 and approved for marketing in China in 2018 for the treatment of multiple myeloma, non-Hodgkin's lymphoma, hematopoietic stem cell transplantation and lymphoma. However, other CXCR4 antagonists have not been approved for clinical cancer treatment. The main reason is that these CXCR4 antagonists have limited lethality to tumors, and antagonizing CXCR4 is not enough to achieve significant clinical therapeutic effects.

[0004] Since CXCR4 antagonists have good affinity to CXCR4, the radionuclide conjugates formed by coupling them with radionuclides have great potential in the field of PET imaging agents. At present, the radionuclide drug with the most clinical research for CXCR4-related diseases is Pentixafor. Pentixafor is the first PET imaging agent targeting CXCR4. It has high CXCR4 affinity, thus having high and sustained tumor uptake and suitable overall pharmacokinetics. Pentixafor has shown obvious lesion marking effects in the diagnosis of various diseases, including malignant tumors, atherosclerosis, fungal infections, systemic mastocytosis, etc. Among them, Pentixafor is the most widely used for tumor imaging. At present, it has successfully achieved imaging of tumors such as non-small cell lung cancer, lymphoma, breast cancer, esophageal cancer and adrenocortical carcinoma in clinical trials. Compared with the traditional imaging agent FDG, Pentixafor can more accurately locate tumor tissues with high expression of CXCR4. In addition to imaging diagnosis of cancer, Pentixather, which has a similar structure to Pentixafor, has also shown significant effects in the treatment of cancer. In a recent clinical study, three patients with advanced T-cell lymphoma received stem cell transplantation after injection of 90Y-Pentixather, and their survival time exceeded 54 months, indicating that CXCR4-targeted radiotherapy plays an important role in the treatment of cancers with high CXCR4 expression.

[0005] In general, the binding force between CXCR4 antagonists and CXCR4 can effectively promote drug targeting to lesion areas with high expression of CXCR4. Therefore, the research on diagnostic drugs for targeted tumors based on CXCR4 antagonists is a very promising research direction. Molecular imaging diagnostic methods targeting CXCR4 will be applied to precise and personalized targeted treatment of tumors. Summary of the invention

[0006] The present application relates to a conjugate compound or a pharmaceutically acceptable salt thereof, a pharmaceutical composition thereof and a method of using the same. More specifically, the present application relates to a multi-ligand drug conjugate, in particular a multi-ligand drug conjugate capable of inducing endocytosis and a pharmaceutical composition thereof, and a method of diagnosing a disease using the drug conjugate, the disease including but not limited to cancer.

[0007] One aspect of the present application discloses a conjugate compound or a pharmaceutically acceptable salt thereof, wherein the conjugate compound or a pharmaceutically acceptable salt thereof comprises a payload and two or more cell interaction molecules, wherein the payload is coupled to at least one of the cell interaction molecules. The cell interaction molecule comprises a first ligand capable of specifically binding to a first cell surface receptor, and a second ligand capable of specifically binding to a second cell surface receptor or a third ligand capable of specifically binding to a third non-cell surface receptor.

[0008] In some embodiments, the first cell surface receptor, the second cell surface receptor, and the third non-cell surface receptor are different from each other.

[0009] In some embodiments, the first cell surface receptor, the second cell surface receptor and the third cell surface receptor provided by the present application are independently selected from the following group: chemokine receptor 4 (CXCR4), folate receptor (FR), integrin receptor (α v β 3 ), fibroblast activation protein (FAP), albumin, etc.

[0010] In some embodiments, the first ligand, the second ligand and the third ligand are independently selected from the following group: cyclic peptide T140 and its analogs, folic acid, cyclic peptide RGD and its analogs, FAPI-04, albumin ligand.

[0015] In some embodiments, the amino acid sequence of the cyclic peptide T140 and its analogs included in the ligand is: NH 2 -Arg-Arg-Nal-Cys-Tyr-Cit-Lys-(D-Lys)-Pro-Tyr-Arg-Cit-Cys-Arg-COOH (two cysteines in the sequence form a disulfide bond), or analogs with substitution, deletion, or insertion of 1 to 3 amino acids, including substitution of Lys with Arg in the sequence, acetylation of the amino terminus, amidation of the carboxyl terminus, and insertion of L-Lys into the D-Lys side chain.

[0011] In some embodiments, at least one cell interaction molecule as described herein is an endocytosis molecule capable of mediating endocytosis. In some embodiments, the endocytosis molecule can also specifically bind to a cell surface receptor.

[0012] In some embodiments, the ligand is selected from the group consisting of cyclic peptide T140 and its analogs, folic acid, FAPI-04, cyclic peptide RGD and its analogs.

[0013] In some embodiments, the conjugate compound or a pharmaceutically acceptable salt thereof comprises at least one payload.

[0014] In some embodiments, the payload is a radionuclide chelating group selected from:

[0015] In some embodiments, the radionuclide chelated by the chelating group is selected from 64 Cu.

[0016] Another aspect of the present application discloses a method for diagnosing or treating a disease characterized by overexpression of chemokine receptor 4 (CXCR4) in a subject, the method comprising administering to the subject a diagnostically effective amount of a conjugate compound provided herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition provided herein. In some embodiments, the disease is cancer.

[0017] In some embodiments, the cancer is selected from the group consisting of breast cancer, lung 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.

[0018] In some embodiments, the conjugate compound is selected from the following structures:

[0019] In the above structure, the linker C part may be substituted with corresponding substituents, and the substituents may be selected from the following non-hydrogen substituents: C1-12 alkyl, C3-10 cycloalkyl, C5-12 aryl, C5-12 heteroaryl, hydroxyl, halogen and C1-4 alkoxycarbonyl substituted with at least one non-hydrogen substituent of C1-12 alkyl, C3-10 cycloalkyl, C1-12 alkoxy, C5-12 aryl, C5-12 heteroaryl, hydroxyl and halogen, and the aryl and heteroaryl are unsubstituted or substituted with at least one non-hydrogen substituent selected from C1-4 alkyl, C1-4 alkoxy, hydroxyl and halogen.

[0020] 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 atom group, provided that the group satisfies the valence requirements and a chemically stable compound is produced by the substitution. In this specification, unless specifically described as "unsubstituted", it is understood that all substituents may be substituted or unsubstituted.

[0021] "Alkyl" refers to a saturated hydrocarbon radical, both straight and branched, 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. The alkyl group may be attached to the parent group or substrate via any ring atom, unless such attachment would violate the valence requirement. Similarly, an alkyl or alkenyl group may contain at least one non-hydrogen substituent, unless such substitution would violate the valence requirement.

[0022] "Cycloalkyl" refers to saturated monocyclic and polycyclic hydrocarbon rings, usually having a specific number of carbon atoms comprising the ring (e.g., C3-10 cycloalkyl refers to cycloalkyl having 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms as ring members). Cycloalkyl can be attached to the parent or substrate via any ring atom, unless such attachment would violate valence requirements. Likewise, cycloalkyl can contain at least one non-hydrogen substituent, unless such substitution would violate valence requirements.

[0023] "Aryl" refers to monovalent and divalent aromatic groups, including 5-membered and 6-membered monocyclic aromatic groups, respectively, and "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 aryl and heteroaryl groups include, but are not limited to, phenyl, pyridyl, furyl, pyrrolyl, thienyl, thiazolyl, isothiazolyl, imidazolyl, triazolyl, tetrazolyl, pyrazolyl, oxazolyl, isoxazolyl, pyrazinyl, pyridazinyl, pyrimidinyl, etc. Aryl and heteroaryl groups also include bicyclic groups, tricyclic groups, etc., including fused 5-membered and 6-membered rings mentioned above. Examples of polycyclic aryl and heteroaryl include, but are not limited to, isoquinolyl, naphthyl, biphenyl, anthracenyl, pyrenyl, carbazolyl, benzoxazolyl, benzodioxazolyl, benzothiazolyl, benzimidazolyl, benzothienyl, quinolyl, indolyl, benzofuranyl, purinyl, indolizinyl, etc. Aryl and heteroaryl can be connected to a parent group or substrate through any ring atom, unless such connection would destroy the valence requirement. Similarly, aryl and heteroaryl can contain at least one non-hydrogen substituent, unless such substitution would destroy the valence requirement. The non-hydrogen substituents of aryl and heteroaryl can also be substituted by other non-hydrogen substituents.

[0024] "Carbonyl" refers to -C(O)R'. As used herein, "(O)" refers to an oxygen connected to an atom such as carbon or sulfur by a double bond. Here, "R" refers to a non-hydrogen substituent such as a lower alkyl, a lower alkoxy, and the like. Examples of carbonyl include, but are not limited to, 2-methoxyoxyethyl, 3-methoxyoxypropyl, and the like. The carbonyl may be attached to the parent group or substrate via any ring atom unless such attachment would violate the valence requirement. Likewise, the carbonyl may contain at least one non-hydrogen substituent unless such substitution would violate the valence requirement.

[0025] "Alkoxy" refers to alkyl-O-. Here, alkyl is the same as defined above. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, and the like. Alkoxy may be attached to a parent group or substrate via any ring atom unless such attachment would violate valence requirements. Likewise, an alkoxy may contain at least one non-hydrogen substituent unless such substitution would violate valence requirements.

[0026] "Hydroxyl" refers to -OH, "halo" refers to fluoro, chloro, bromo and iodo, and "oxo" refers to =0.

[0027] The amino acids used in this patent are all L-amino acids except D-Lys.

[0028] The method for preparing the tumor-targeting polypeptide drug conjugate of the present invention comprises the following steps: a) synthesizing a T140 linear peptide or a T140 linear peptide connected to a second ligand or a third ligand, and a CXCR4 targeting linear peptide connected to a second ligand or a third ligand and a chelating group using an Fmoc solid phase synthesis strategy; b) subjecting the CXCR4 tumor-targeting linear polypeptide in a to a disulfide bond cyclization reaction to obtain a CXCR4-targeting cyclic peptide, and then connecting it with the second ligand or the third ligand through liquid phase condensation or click reaction to obtain a coupling intermediate; or directly subjecting the CXCR4-targeting linear polypeptide connected to the second ligand or the third ligand in a to a disulfide bond cyclization reaction to obtain a coupling intermediate; c) The coupling intermediate obtained in b is connected with a radionuclide chelating group through a condensation reaction and purified by HPLC to obtain a coupled compound; or the CXCR4 tumor-targeting linear polypeptide connected to the second ligand or the third ligand and the chelating group in a is directly subjected to a disulfide bond cyclization reaction to obtain a coupled compound.

[0029] The present invention obtains a conjugate compound with good tumor uptake effect by performing PET imaging tests on tumor-bearing mice. Therefore, the present invention provides an application of a polypeptide drug conjugate based on chemokine receptor protein CXCR4 in the preparation of tumor-targeted polypeptide-nuclide conjugate drugs. The tumor is preferably breast cancer and other tumors with abnormal CXCR4 expression.

[0030] Another aspect of the present invention provides a drug comprising the above tumor-targeting polypeptide drug conjugate or a pharmaceutically acceptable salt thereof as an active ingredient.

[0031] The pharmaceutical composition of the present invention comprises at least one pharmaceutically acceptable carrier in addition to the active ingredient. As used herein, "pharmaceutically acceptable carrier" refers to a known pharmaceutically acceptable excipient that can be used to formulate a pharmaceutically active compound for administration to a subject and is substantially non-toxic and non-irritating under the conditions of use. The exact amount of the excipient is determined by standard pharmaceutical practice and the solubility, chemical properties and selected route of administration of the active compound.

[0032] The pharmaceutical composition of the present invention may be formulated into an appropriate form for the desired method of administration using suitable and physiologically acceptable adjuvants (eg, excipients, disintegrants, sweeteners, binders, coating agents, expanders, lubricants, brighteners, flavoring agents, etc.).

[0033] The preparations of pharmaceutical compositions with polypeptide drug conjugates as active ingredients include freeze-dried powder injections, injections, tablets, liposomes, nanoformulations and other forms.

[0034] Beneficial effects of the present invention: The polypeptide drug conjugate provided by the present invention connects the radionuclide to the CXCR4 targeting polypeptide through a chelating group, thereby achieving high-concentration enrichment and long-term retention of the radionuclide in the tumor tissue site, allowing the radionuclide to accurately target tumor cells, thereby achieving a more accurate diagnosis of CXCR4-highly expressed tumors. BRIEF DESCRIPTION OF THE DRAWINGS In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work. Figure 1 This is the HLK-01 HRMS analysis spectrum. Figure 2 This is the HLK-02 HRMS analysis spectrum. Figure 3 This is the HLK-03 HRMS analysis spectrum. Figure 4 This is the HLK-04 HRMS analysis spectrum. Figure 5 This is the HLK-06 HRMS analysis spectrum. Figure 6 This is the HLK-07 HRMS analysis spectrum. Figure 7 This is the HLK-08 HRMS analysis spectrum. Figure 8 This is the HLK-09 HRMS analysis spectrum. Fig. 9 This is the HLK-10 HRMS analysis spectrum. Fig.10 This is the HLK-11 HRMS analysis spectrum. Fig.11 This is the HLK-12 HRMS analysis spectrum. Fig.12 This is the HLK-13 HRMS analysis spectrum. Fig.13 This is the HLK-14 HRMS analysis spectrum. Fig.14 This is the HLK-15 HRMS analysis spectrum. Fig.15 for[ 64 PET / CT imaging results of MCF-7 tumor-bearing mice at different time points after Cu]HLK-01 injection. Fig.16 for[ 64 Cu]HLK-01 uptake in tumor, liver, and kidney of MCF-7 tumor-bearing mice at different time points after injection. Fig.17 for[ 64 Cu]HLK-02, 64 Cu]HLK-03, 64 PET / CT imaging results of MCF-7 tumor-bearing mice 1 h after Cu]HLK-04 injection. Fig.18 for[ 64 Cu]HLK-01, 64 Cu]HLK-02, 64 Cu]HLK-03, 64 Cu]HLK-04 uptake values ​​in tumor, liver and kidney of MCF-7 tumor-bearing mice 1 h after injection. Fig.19 for[ 64 Cu]HLK-10, 64 Cu]HLK-11, [ 64 PET / CT imaging results of Daudi tumor-bearing mice 1 h after Cu]HLK-12 injection. Fig. 20 for[ 64 Cu]HLK-13, [ 64 Cu]HLK-14, [ 64 PET / CT imaging results of Daudi tumor-bearing mice 1 h after Cu]HLK-15 injection. Fig.21 for[ 64 Cu]HLK-01, 64 Cu]HLK-10, 64 Cu]HLK-11, [64 Cu]HLK-12, [ 64 Cu]HLK-13, [ 64 Cu]HLK-14, [ 64 Cu]HLK-15, 64 Cu]T140-DOTA uptake values ​​in tumor, liver and kidney of Daudi tumor-bearing mice 1 h after injection. Fig. 22 for[ 64 Cu]HLK-01, 64 Cu]HLK-10, 64 Cu]HLK-11, [ 64 Cu]HLK-12, [ 64 Cu]HLK-13, [ 64 Cu]HLK-14, [ 64 Cu]HLK-15, 64 Cu]T140-DOTA uptake ratio between tumor and liver and kidney in Daudi tumor-bearing mice 1 h after injection. DETAILED DESCRIPTION To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. In certain embodiments of the present invention, the cells that can be used include: Human breast cancer cells: MCF-7; Human lymphoma cells: Daudi; The above cells can be obtained commercially, for example, from the Cell Bank of the Chinese Academy of Sciences. Example 1 Chemical Synthesis (1) Synthesize T140 linear peptides, T140 linear peptides connected with functional groups, and T140 linear peptides connected with functional groups and chelating groups (the R groups in the structures include folic acid, ibuprofen, 4-methylphenylbutyric acid, 4-methoxyphenylbutyric acid, 4-chlorophenylbutyric acid, dodecanoic acid, and hexadecanoic acid), the structures of which are as follows: The synthetic route is as follows: The specific method is as follows: Peptide elution method: DMF (3×10 mL×2 min). Method for removing Fmoc protecting group: 3% piperidine + 3% DBU + 94% DMF (3×10 mL×5 min). Method for removing Dde protecting group: 2% hydrazine hydrate + 98% DMF (3×10mL×3min) Ninhydrin test: Add the prepared ninhydrin, phenol, and pyridine in the amount of 1 drop: 1 drop: 2 drops, heat at 100℃ for 30s-90s. If there are exposed amino groups, the system will be blue or light brown; if there are no exposed amino groups, the system will be colorless. The method for connecting the next amino acid is as follows: 3eq amino acid + 3eq HBTU (different condensing agents can be selected according to different situations) + 4eq HOBT + 6eq DIEA, DMF 10mL, and oscillate at room temperature for 1 to 4h. Peptide cleavage: 95% TFA + 2% H2O + 2% TIPs + 1% 2-Mercaptoethanol, shake for 3 hours. Peptide extraction, HPLC preparation and freeze-drying: 10 mL of icy ether, 2 x 10 mL of water, collect the aqueous phase, HPLC preparation: acetonitrile / water, acetonitrile: 20% to 100%. Freeze-drying. Synthesis of series A compounds: (434 mg, 0.2 mmol) MBHA resin was swollen in 10 mL DCM for 30 min, and washed with DMF for 3×10 ml×2 min. Fmoc-Arg(pbf)-OH was added, peptide was washed, Fmoc was removed, peptide was washed, indene was tested, and then Cys-Cit-Arg-Tyr-Pro-(D-Lys)-Lys-Cit-Tyr-Cys-Nal-Arg-Arg (D-Lys is Fmoc-D-Lys(Boc)-OH) was added in sequence (amino acid added→indene test→Fmoc removal→peptide washing→indene test→next amino acid added), the terminal Fmoc protecting group was removed, Fmoc-L-Lys(Dde)-OH was added, the Fmoc protecting group was removed, tert-butyl protected DOTA (compound 1) was added, and finally Dde protection was removed. The base was connected with different functional groups (folic acid, ibuprofen, 4-methylphenylbutyric acid, 4-methoxyphenylbutyric acid), peptide cleavage, peptide extraction, HPLC preparation: 0.1% TFA acetonitrile / 0.1% TFA water, 0-90min acetonitrile 10%-100%, flow rate: 8ml / min, Rt=20-25min, and freeze-dried to obtain products A-1 (0.051mmol, 152mg, yield: 26%), A-2 (0.046mmol, 127mg, yield: 23%), A-3 (0.050mmol, 135mg, yield: 25%), A-4 (0.054mmol, 147mg, yield: 27%). The structure of the intermediate compound of series A is as follows: Synthesis of series B intermediate compounds: (434 mg, 0.2 mmol) MBHA resin was swollen in 10 mL DCM for 30 min, and washed with DMF for 3×10 ml×2 min. Fmoc-Arg(pbf)-OH was added, the peptide was washed, Fmoc was removed, the peptide was washed, indene was tested, and then Cys-Cit-Arg-Tyr-Pro-(D-Lys)-Lys-Cit-Tyr-Cys-Nal-Arg-Arg (D-Lys is Fmoc-D-Lys(Dde)-OH, and the terminal Arg is Boc-Arg(pbf)-OH) was added in sequence (adding amino acid→indene test→removing Fmoc→peptide washing→indene test→adding the next amino acid), the Dde protecting group on the side chain Lys was removed, Fmoc-L-Lys(Dde)-OH was added, the Fmoc protecting group was removed, and tert-butyl protected DOTA (compound) was added. The Dde protecting group was removed, and different functional groups (folic acid, ibuprofen, 4-methylphenylbutyric acid, 4-methoxyphenylbutyric acid) were added. The peptides were cleaved and extracted. HPLC preparation: 0.1% TFA acetonitrile / 0.1% TFA water, 0-90min acetonitrile 10%-100%, flow rate: 8ml / min, Rt=20-25min, and freeze-dried to obtain products B-1 (0.054mmol, 160mg, yield: 27%), B-2 (0.067mmol, 180mg, yield: 33%), B-3 (0.061mmol, 165mg, yield: 30%), and B-4 (0.052mmol, 141mg, yield: 26%). The structures of the intermediate compounds of series B are as follows: Synthesis of C series intermediate compounds: (434 mg, 0.2 mmol) MBHA resin was swollen in 10 mL DCM for 30 min, and washed with DMF for 3×10 ml×2 min. Fmoc-Arg(pbf)-OH was added, peptide was washed, Fmoc was removed, peptide was washed, indene was tested, and then Cys-Cit-Arg-Tyr-Pro-(D-Lys)-Arg-Cit-Tyr-Cys-Nal-Arg-Arg-Ac (D-Lys is Fmoc-D-Lys(Dde)-OH) was added in sequence (amino acid added→indene test→Fmoc removal→peptide washing→indene test→next amino acid added). Dde protecting group on side chain Lys was removed, and Fmoc-L-Lys(Dde)-OH was added. H, then remove the Fmoc protecting group, connect tert-butyl protected DOTA (compound 1), finally remove the Dde protecting group, connect different functional groups (dodecanoic acid, hexadecanoic acid), cleave the peptide, extract the peptide, HPLC preparation: 0.1% TFA acetonitrile / 0.1% TFA water, 0-90min acetonitrile 10%-100%, flow rate: 8ml / min, Rt=20-25min, freeze-dried to obtain the products C-1 (0.043mmol, 122mg, yield: 22%), C-2 (0.050mmol, 143mg, yield: 25%). The structures of the C series intermediate compounds are as follows: Synthesis of compound D: (434 mg, 0.2 mmol) MBHA resin was swollen in 10 mL DCM for 30 min, and washed with DMF for 3×10 ml×2 min. Fmoc-Arg(pbf)-OH was added, peptide was washed, Fmoc was removed, peptide was washed, indene was tested, and then Cys-Cit-Arg-Tyr-Pro-(D-Lys)-Arg-Cit-Tyr-Cys-Nal-Arg-Arg-Ac (D-Lys is Fmoc-D-Lys(Dde)-OH) was added in sequence (amino acid added→indene test→Fmoc removal→peptide washing→indene test→next amino acid added). The Dde protecting group on the side chain Lys was connected to Fmoc-L-Lys(Dde)-OH, and then the Fmoc protecting group was removed, azidoacetic acid was connected, and finally the Dde protecting group was removed, the peptide was cleaved, and the peptide was extracted. HPLC preparation: 0.1% TFA acetonitrile / 0.1% TFA water, acetonitrile 10%-100% from 0 to 90 min, flow rate: 8 ml / min, Rt=20 min, and lyophilization gave the product D (0.078 mmol, 180 mg, yield: 39%). (2) Synthesis of HLK-01 The specific method is as follows: (50 mg, 0.017 mmol) of intermediate compound B-1 was added to a round-bottom flask, and 50 mL of 20% DMSO aqueous solution was added, followed by (40 mg, 0.5 mmol) of NH 4 HCO 3 , open round-bottom flask, stirred at room temperature for 24 h, freeze-dried, HPLC preparation: 0.1% TFA acetonitrile / 0.1% TFA water, 0-90 min acetonitrile 10%-100%, flow rate: 8 ml / min, Rt=18 min, freeze-dried to obtain disulfide cyclization product HLK-01 (22 mg, 0.007 mmol, yield 44%). HRMS (ESI): C 131 H 197 N 47 O 30 S 2 [M+H] 4+ calcd:744.3773,found:744.3756. Purity: >99.00%. (3) Synthesis of HLK-02 The specific method is as follows: (50 mg, 0.018 mmol) of intermediate compound B-3 was added to a round-bottom flask, and 50 mL of 20% DMSO aqueous solution was added, followed by (40 mg, 0.5 mmol) of NH 4 HCO 3 , open round-bottom flask, stirred at room temperature for 24 h, freeze-dried, HPLC preparation: 0.1% TFA acetonitrile / 0.1% TFA water, acetonitrile 10%-100% from 0 to 90 min, flow rate: 8 ml / min, Rt = 22 min, freeze-dried to obtain the disulfide cyclization product HLK-02 (24 mg, 0.009 mmol, yield 49%). 123 H 192 N 40 O 26 S 2 [M+H] 3+ calcd:904.4873,found:904.4864. Purity: >99.00%. (4) Synthesis of HLK-03 The specific method is as follows: The intermediate compound D (50 mg, 0.022 mmol) was added to a round-bottom flask, and 50 mL of 20% DMSO solution was added, followed by NH 4 HCO3 (40 mg, 0.5 mmol), open round-bottom flask, stirred at room temperature for 24 h, freeze-dried, prepared by HPLC: 0.1% TFA acetonitrile / 0.1% TFA water, 0-90 min acetonitrile 10%-100%, flow rate: 8 ml / min, Rt=20 min, freeze-dried to obtain the disulfide cyclization product T140-D (32 mg, 0.014 mmol, yield 64%). RGD-1 (50 mg, 0.083 mmol) and Alkyne NHS ester (906564-59-8) (17 mg, 0.083 mmol) were added to a round-bottom flask, dissolved in 3 mL DMF, and then added with DIEA (29 μL, 0.166 mmol), stirred at room temperature for 2 h, and concentrated under reduced pressure. Dissolved in acetonitrile / water, HPLC preparation: 0.1% TFA acetonitrile / 0.1% TFA water, 0-90 min acetonitrile 10%-100%, flow rate: 8 ml / min, Rt=25 min, and freeze-dried to obtain compound RGD-2 (40 mg, 0.057 mmol, yield 69%). T140-D (30 mg, 0.013 mmol), RGD-2 (9 mg, 0.013 mmol) and CuSO 4 ·5H 2 O (3.2 mg, 0.013 mmol) and VcNa (2.6 mg, 0.013 mmol) were added to a round-bottom flask, and solvent H 2 O / DMF (0.4mL: 1.6mL), stirred at room temperature overnight, concentrated under reduced pressure, prepared by HPLC: 0.1% TFA acetonitrile / 0.1% TFA water, acetonitrile 10%-100% from 0 to 90 min, flow rate: 8 ml / min, Rt=22 min, and lyophilized to obtain T140-D-RGD (23 mg, 0.007 mmol, yield 57%). T140-D-RGD (10 mg, 0.003 mmol) and compound 2 (1.8 mg, 0.003 mmol) were added to a round-bottom flask, dissolved in 1 mL DMF, and then added with DIEA (1.2 μL, 0.006 mmol), stirred at room temperature for 2 h, and concentrated under reduced pressure. Dissolved in acetonitrile / water, prepared by HPLC: 0.1% TFA acetonitrile / 0.1% TFA water, acetonitrile 10%-100% from 0 to 90 min, flow rate: 8 ml / min, Rt=18 min, and freeze-dried to obtain compound RGD-2 (5 mg, 0.001 mmol, yield 42%). 157 H 236 N 54 O 37 S 3 [M+H] 5+calcd:714.1554,found:714.1565. Purity: >99.00%. (5) Synthesis of HLK-04 The specific method is as follows: FAPI-04 (50 mg, 0.103 mmol) and propyne-monoethylene glycol-propionic acid (1859379-85-3) (18 mg, 0.103 mmol) were added to a round-bottom flask, dissolved in 3 mL DMF, and then DIEA (36 μL, 0.206 mmol) was added, stirred at room temperature for 2 h, and concentrated under reduced pressure. Dissolved in acetonitrile / water, HPLC preparation: 0.1% TFA acetonitrile / 0.1% TFA water, 0-90 min acetonitrile 10%-100%, flow rate: 8 ml / min, Rt=25 min, freeze-dried to obtain compound 3 (40 mg, 0.063 mmol, yield 61%). T140-D (30 mg, 0.013 mmol), compound 3 (8 mg, 0.013 mmol) and CuSO 4 ·5H 2 O (3.2 mg, 0.013 mmol) and VcNa (2.6 mg, 0.013 mmol) were added to a round-bottom flask, and solvent H 2 O / DMF (0.4mL: 1.6mL), stirred at room temperature overnight, concentrated under reduced pressure, prepared by HPLC: 0.1% TFA acetonitrile / 0.1% TFA water, acetonitrile 10%-100% from 0 to 90 min, flow rate: 8 ml / min, Rt=22 min, and lyophilized to obtain T140-D-FAPI-04 (25 mg, 0.008 mmol, yield 65%). T140-D-FAPI-04 (10 mg, 0.003 mmol) and compound 2 (1.8 mg, 0.003 mmol) were added to a round-bottom flask, dissolved in 1 mL of DMF, and then added with DIEA (1.2 μL, 0.006 mmol), stirred at room temperature for 2 h, and concentrated under reduced pressure. Dissolved in acetonitrile / water, HPLC preparation: 0.1% TFA acetonitrile / 0.1% TFA water, 0-90 min acetonitrile 10%-100%, flow rate: 8 ml / min, Rt=20 min, freeze-dried to obtain compound HLK-04 (4 mg, 0.001 mmol, yield 32%). 156 H 227 F 2 N 51 O 35 S 3 [M+H] 3+calcd:1170.5633,found:1170.5647. Purity: >99.00%. (6) Synthesis of HLK-06 The specific method is as follows: (50 mg, 0.017 mmol) of intermediate compound A-1 was added to a round-bottom flask, and 50 mL of 20% DMSO solution was added, followed by (40 mg, 0.5 mmol) of NH 4 HCO 3 , open the round bottom flask, stir at room temperature for 24h, freeze-dry, HPLC preparation: 0.1% TFA acetonitrile / 0.1% TFA water, 0-90min acetonitrile 10%-100%, flow rate: 8ml / min, Rt=18min, freeze-dried to obtain the disulfide cyclization product HLK-06 (24mg, 0.008mmol, yield 47%). 131 H 197 N 47 O 30 S 2 [M+H] 4+ calcd:744.3773,found:744.3760. Purity: >99.00%. (7) Synthesis of HLK-07 The specific method is as follows: (50 mg, 0.018 mmol) of intermediate compound A-2 was added to a round-bottom flask, and 50 mL of 20% DMSO aqueous solution was added, followed by (40 mg, 0.5 mmol) of NH 4 HCO 3 , open the round bottom flask, stir at room temperature for 24h, freeze-dry, HPLC preparation: 0.1% TFA acetonitrile / 0.1% TFA water, 0-90min acetonitrile 10%-100%, flow rate: 8ml / min, Rt=25min, freeze-dry to obtain the disulfide cyclization product HLK-07 (24mg, 0.009mmol, yield 48%). 125 H 196 N 40 O 26 S 2 [M+H] 4+ calcd:685.6251,found:685.6258. Purity: >99.00%. (8) Synthesis of HLK-08 The specific method is as follows: (50 mg, 0.018 mmol) of intermediate compound A-3 was added to a round-bottom flask, and 50 mL of 20% DMSO solution was added, followed by (40 mg, 0.5 mmol) of NH 4 HCO 3 , open round-bottom flask, stirred at room temperature for 24 h, freeze-dried, HPLC preparation: 0.1% TFA acetonitrile / 0.1% TFA water, acetonitrile 10%-100% from 0 to 90 min, flow rate: 8 ml / min, Rt = 23 min, freeze-dried to obtain the disulfide cyclization product HLK-08 (22 mg, 0.008 mmol, yield 45%). 123 H 192 N 40 O 26 S 2 [M+H] 3+ calcd:904.4873,found:904.4863. Purity: >99.00%. (9) Synthesis of HLK-09 The specific method is as follows: (50 mg, 0.018 mmol) of intermediate compound A-4 was added to a round-bottom flask, and 50 mL of 20% DMSO aqueous solution was added, followed by (40 mg, 0.5 mmol) of NH 4 HCO 3 , open round-bottom flask, stirred at room temperature for 24 h, freeze-dried, HPLC preparation: 0.1% TFA acetonitrile / 0.1% TFA water, acetonitrile 10%-100% from 0 to 90 min, flow rate: 8 ml / min, Rt = 23 min, freeze-dried to obtain the disulfide cyclization product HLK-09 (26 mg, 0.010 mmol, yield 53%). 123 H 192 N 40 O 27 S 2 [M+H] 4+ calcd:682.6160,found:682.6183. Purity: >99.00%. (10) Synthesis of HLK-10 The specific method is as follows: (50 mg, 0.018 mmol) of intermediate compound A-2 was added to a round-bottom flask, and 50 mL of 20% DMSO aqueous solution was added, followed by (40 mg, 0.5 mmol) of NH 4 HCO 3, open the round bottom flask, stir at room temperature for 24h, freeze-dry, HPLC preparation: 0.1% TFA acetonitrile / 0.1% TFA water, 0-90min acetonitrile 10%-100%, flow rate: 8ml / min, Rt=25min, freeze-dry to obtain the disulfide cyclization product HLK-10 (23mg, 0.010mmol, yield 47%). 125 H 196 N 40 O 26 S 2 [M+H] 4+ calcd:685.6251,found:685.6227·. Purity: >99.00%. (11) Synthesis of HLK-11 The specific method is as follows: (50 mg, 0.018 mmol) of intermediate compound A-2 was added to a round-bottom flask, and 50 mL of 20% DMSO aqueous solution was added, followed by (40 mg, 0.5 mmol) of NH 4 HCO 3 , open round-bottom flask, stirred at room temperature for 24 h, freeze-dried, HPLC preparation: 0.1% TFA acetonitrile / 0.1% TFA water, acetonitrile 10%-100% from 0 to 90 min, flow rate: 8 ml / min, Rt = 22 min, freeze-dried to obtain the disulfide cyclization product HLK-11 (22 mg, 0.008 mmol, yield 44%). 123 H 192 N 40 O 27 S 2 [M+H] 4+ calcd:682.6160,found:682.6188. Purity: >99.00%. (12) Synthesis of HLK-12 The specific method is as follows: (50 mg, 0.018 mmol) of intermediate compound C-1 was added to a round-bottom flask, and 50 mL of 20% DMSO solution was added, followed by (40 mg, 0.5 mmol) of NH 4 HCO 3, open round-bottom flask, stirred at room temperature for 24 h, freeze-dried, HPLC preparation: 0.1% TFA acetonitrile / 0.1% TFA water, acetonitrile 10%-100% from 0 to 90 min, flow rate: 8 ml / min, Rt = 24 min, freeze-dried to obtain the disulfide cyclization product HLK-12 (25 mg, 0.009 mmol, yield 50%). 126 H 203 N 41 O 28 S 2 [M+H] 3+ calcd:935.1794,found:935.1785. Purity: >99.00%. (13) Synthesis of HLK-13 The specific method is as follows: (50 mg, 0.017 mmol) of intermediate compound C-2 was added to a round-bottom flask, and 50 mL of 20% DMSO aqueous solution was added, followed by (40 mg, 0.5 mmol) of NH 4 HCO 3 , open the round bottom flask, stir at room temperature for 24h, freeze-dry, HPLC preparation: 0.1% TFA acetonitrile / 0.1% TFA water, 0-90min acetonitrile 10%-100%, flow rate: 8ml / min, Rt=25min, freeze-dry to obtain the disulfide cyclization product HLK-13 (25mg, 0.009mmol, yield 50%). 130 H 211 N 41 O 28 S 2 [M+H] 3+ calcd:953.8669,found:953.8682. Purity: >99.00%. (14) Synthesis of HLK-14 The specific method is as follows: Compound 5 (50 mg, 0.085 mmol) and propyne-monoethylene glycol-propionic acid (1859379-85-3) (15 mg, 0.085 mmol) were added to a round-bottom flask, dissolved in 3 mL DMF, and then DIEA (30 μL, 0.17 mmol) was added, stirred at room temperature for 2 h, and concentrated under reduced pressure. Dissolved in acetonitrile / water, HPLC preparation: 0.1% TFA acetonitrile / 0.1% TFA water, 0-90 min acetonitrile 10%-100%, flow rate: 8 ml / min, Rt=26 min, freeze-dried to obtain compound 6 (44 mg, 0.062 mmol, yield 73%). T140-D (30 mg, 0.013 mmol), compound 6 (9 mg, 0.013 mmol) and CuSO 4 ·5H 2 O (3.2 mg, 0.013 mmol) and VcNa (2.6 mg, 0.013 mmol) were added to a round-bottom flask, and solvent H 2 O / DMF (0.4mL: 1.6mL), stirred at room temperature overnight, concentrated under reduced pressure, added 2mL 20% TFA / DCM to a round-bottom flask, stirred at room temperature for 1h, concentrated under reduced pressure, HPLC preparation: 0.1% TFA acetonitrile / 0.1% TFA water, 0-90min acetonitrile 10%-100%, flow rate: 8ml / min, Rt=24min, freeze-dried to obtain T140-D-3 (16mg, 0.005mmol, yield 41%). T140-D-3 (10 mg, 0.003 mmol) and compound 2 (1.8 mg, 0.003 mmol) were added to a round-bottom flask, dissolved in 1 mL DMF, and then added with DIEA (1.2 μL, 0.006 mmol), stirred at room temperature for 2 h, and concentrated under reduced pressure. HPLC preparation: 0.1% TFA acetonitrile / 0.1% TFA water, acetonitrile 10%-100% from 0 to 90 min, flow rate: 8 ml / min, Rt=23 min, freeze-dried to obtain compound HLK-14 (6 mg, 0.002 mmol, yield 51%). 152 H 229 IN 48 O 36 S 3 [M+H] 3+ calcd:1176.1996,found:1176.1973. Purity: >99.00%. (15) Synthesis of HLK-15 The specific method is as follows: Compound 7 (50 mg, 0.076 mmol) and propyne-monoethylene glycol-propionic acid (1859379-85-3) (13 mg, 0.076 mmol) were added to a round-bottom flask, dissolved in 3 mL of DMF, and then DIEA (27 μL, 0.152 mmol) was added, stirred at room temperature for 2 h, and concentrated under reduced pressure. HPLC preparation: 0.1% TFA acetonitrile / 0.1% TFA water, 0-90 min acetonitrile 10%-100%, flow rate: 8 ml / min, Rt=28 min, freeze-dried to obtain compound 8 (40 mg, 0.051 mmol, yield 67%). T140-D (30 mg, 0.013 mmol), compound 8 (10 mg, 0.013 mmol) and CuSO 4 ·5H 2 O (3.2 mg, 0.013 mmol) and VcNa (2.6 mg, 0.013 mmol) were added to a round-bottom flask, and solvent H 2 O / DMF (0.4mL: 1.6mL), stirred at room temperature overnight, concentrated under reduced pressure, added 2mL 20% TFA / DCM to a round-bottom flask, stirred at room temperature for 1h, concentrated under reduced pressure, HPLC preparation: 0.1% TFA acetonitrile / 0.1% TFA water, 0-90min acetonitrile 10%-100%, flow rate: 8ml / min, Rt=25min, lyophilized to obtain T140-D-4 (20mg, 0.006mmol, yield 50%). T140-D-4 (10 mg, 0.003 mmol) and compound 2 (1.8 mg, 0.003 mmol) were added to a round-bottom flask, dissolved in 1 mL DMF, and then added with DIEA (1.2 μL, 0.006 mmol), stirred at room temperature for 2 h, and concentrated under reduced pressure. HPLC preparation: 0.1% TFA acetonitrile / 0.1% TFA water, acetonitrile 10%-100% from 0 to 90 min, flow rate: 8 ml / min, Rt=23 min, and freeze-dried to obtain compound HLK-15 (3 mg, 0.001 mmol, yield 26%). 160 H 228 N 50 O 40 S 5 [M+H] 3+ calcd:1217.5389,found:1217.5388. Purity: >99.00%. Example 2 PET / CT imaging of a CXCR4-overexpressing tumor-bearing mouse model using a peptide-drug conjugate 20 μg of HLK-01, HLK-02, HLK-03, HLK-04, HLK-10, HLK-11, HLK-12, HLK-13, HLK-14, HLK-15 and 64 CuCl 2 (500-1000 μCi, 0.1 mol / L NaOAc, pH=4.1) were mixed, and the reaction mixture was heated to 80°C for 10 min. Get the marked [ 64 Cu]HLK-01, 64 Cu]HLK-02, 64 Cu]HLK-03, 64 Cu]HLK-04,64 Cu]HLK-10, 64 Cu]HLK-11, 64 Cu]HLK-12, [ 64 Cu]HLK-13, [ 64 Cu]HLK-14, [ 64 Cu]HLK-15 was diluted with saline, and 200 μCi of each labeled peptide-drug conjugate was injected into the mouse tail vein. PET / CT scanning imaging was performed at 1 hour or other time points, and the PET / CT image processing software was used to reconstruct the PET / CT imaging results. The uptake value of the labeled peptide-nuclide conjugate in the mouse organ was obtained by processing the PET / CT imaging results, and the unit was %ID / cc (the ratio of radioactive counts per cubic centimeter of organ to the total injected radioactive counts) Fig.15 , 16 , 17 and 18 show that [ 64 Cu]HLK-01, 64 Cu]HLK-02, 64 Cu]HLK-03, 64 Cu]HLK-04 was significantly taken up in MCF-7 tumors, and [ 64 Cu]HLK-01 can be enriched in tumors for a long time, and obvious tumor uptake is still observed 20 h after injection. Fig.19 , 20 , 21 and 22 show that [ 64 Cu]HLK-10, 64 Cu]HLK-11, [ 64 Cu]HLK-12, [ 64 Cu]HLK-13, [ 64 Cu]HLK-14, [ 64 Cu]HLK-15 was significantly taken up in Daudi tumors, and the uptake of most peptide-nuclides in tumors was higher than that of known compounds [ 64 Cu]T140-DOTA has more advantages in diagnosing tumors with high CXCR4 expression; 64 Cu]HLK-01 and [ 64 The uptake ratio of Cu]HLK-15 in tumor and liver and kidney was significantly higher than that of other compounds. The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A conjugate compound or a pharmaceutically acceptable salt thereof, The conjugate compound or a pharmaceutically acceptable salt thereof comprises a payload and two or more cell-interacting molecules, wherein the payload is conjugated to at least one of the 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 capable of specifically binding to a first cell surface receptor, and a second ligand capable of specifically binding to a second cell surface receptor or a third ligand capable of specifically binding to a third non-cell surface receptor, wherein the first cell surface receptor is chemokine receptor 4 (CXCR4), the first ligand is selected from the cyclic peptide T140 and its analogs; the second ligand is selected from folic acid, cyclic peptide RGD and its analogs and FAPI-04; the third ligand is selected from albumin ligands.

3. The conjugate compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence of the first ligand cyclic peptide T140 and its analogs is: NH 2 -Arg-Arg-Nal-Cys-Tyr-Cit-Lys-(D-Lys)-Pro-Tyr-Arg-Cit-Cys-Arg-COOH (two cysteines in the sequence form a disulfide bond), or analogs with substitution, deletion, or insertion of 1 to 3 amino acids, including substitution of Lys with Arg in the sequence, acetylation of the amino terminus, amidation of the carboxyl terminus, and insertion of L-Lys into the D-Lys side chain.

4. The conjugate compound according to claim 1-2 or a pharmaceutically acceptable salt thereof, wherein the structures of folic acid and FAPI-04 are as follows:

5. The conjugate compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the cyclic peptide RGD and its analogs are selected from:

6. The conjugate compound or a pharmaceutically acceptable salt thereof according to claim 1-2, wherein the albumin ligand is selected from: ibuprofen, 4-methylphenylbutyric acid, 4-methoxyphenylbutyric acid, 4-chlorophenylbutyric acid, truncated Evans blue, dodecanoic acid, hexadecanoic acid, 4-iodophenyl terminal group. The albumin ligand structure is as follows:

7. The conjugate compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, wherein the effective load is a radionuclide chelating group and a radionuclide, and the chelating group is selected from:

8. The conjugate compound according to any one of claims 1-7, or a pharmaceutically acceptable salt thereof, wherein the radionuclide is selected from 177 Lu, 64 Cu, 67 Cu, 68 Ga, 67 Ga, 18 F, 51 Cr, 111 In, 99 mTc, 186 Re, 188 Re, 139 La, 140 La, 175 Yb, 15 3Sm, 166 Ho, 86 Y, 88 Y, 90 Y, 149 Pm, 165 Dy, 169 Er, 47 Sc, 142 Pr, 159 Gd, 212 Bi, 72 As, 72 Se, 97 Ru, 109 Pd, 105 Ph, 101m Ph, 119 Sb, 128 Ba, 123 I, 124 I, 131 I, 197 Hg, 211 At, 151 Eu, 153 Eu, 169 Eu, 201 T1, 203 Pb, 212 Pb, 188 Re, 186 Re, 198 Au, 225 Ac, 227 Th and 199 Ag.

9. The conjugate compound according to claim 1-12 or a pharmaceutically acceptable salt thereof, selected from the following structures:

10. A method for preparing a coupled compound according to claims 1-9, comprising the following steps: a) synthesizing a T140 linear peptide, a T140 linear peptide connected to a second ligand or a third ligand, and a T140 linear peptide connected to a second ligand or a third ligand and a chelating group using an Fmoc solid phase synthesis strategy; b) subjecting the T140 linear peptide in a to a disulfide cyclization reaction to obtain a T140 cyclic peptide, and then connecting it with the second ligand or the third ligand through liquid phase condensation or click reaction to obtain a coupling intermediate; or subjecting the T140 linear peptide connected to the second ligand or the third ligand in a to a disulfide cyclization reaction to obtain a coupling intermediate; c) The coupling intermediate obtained in b is connected with a radionuclide chelating group through a condensation reaction and purified by HPLC to obtain a coupled compound; or the T140 linear peptide connecting the second ligand or the third ligand and the chelating group in a is directly subjected to a disulfide bond cyclization reaction to obtain a coupled compound.

11. A pharmaceutical composition comprising the conjugate compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

12. Use of the conjugate according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition according to claim 11 in the preparation of an agent for diagnosing or treating a disease characterized by overexpression of chemokine receptor 4 (CXCR4) in a subject.

13. The use according to claim 12, wherein the disease is selected from cancer.

14. The use according to claim 13, wherein the cancer is selected from the group consisting of breast cancer, lung 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.

15. Use of the conjugate compound or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a tumor diagnosis product.

16. The use according to claim 15, It is characterized in that The tumor is selected from breast cancer or a tumor with abnormal expression of CXCR4.

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