FGFR2-targeted polypeptide nuclide ligand, probe and application of FGFR2-targeted polypeptide nuclide ligand

Abstract

The invention belongs to the field of nuclear medicine, and particularly relates to a polypeptide nuclide ligand targeting FGFR2, a probe and application of the polypeptide nuclide ligand. The ligand has a structure as shown in a formula (1) or (2). A radioactive preparation obtained by labeling the FGFR2 with radionuclide has relatively high tumor uptake and relatively high tumor-muscle uptake ratio in FGFR2 overexpressed tumors, and has a good clinical application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of nuclear medicine, and relates to the field of radioactive chemistry and clinical nuclear medicine technology of radionuclide labeling, and specifically, to a polypeptide nuclide ligand and a probe targeting FGFR2 and applications thereof. Background Art

[0002] Cancer is a global public health challenge that seriously endangers human health. However, the current medical level is difficult to overcome advanced malignant tumors, and early detection and treatment are still the most effective means of treating malignant tumors. Therefore, early diagnosis of tumors and effective treatment of tumors are particularly important and urgent. For tumors, conventional imaging diagnostic technologies mainly include B-ultrasound, CT and MRI. These imaging diagnostic technologies achieve diagnostic results by showing functional changes in tissues and have good application value, but there are still certain deficiencies in differential diagnosis, systemic staging and early efficacy evaluation.

[0003] Theranostics has always been the main development direction in the field of nuclear medicine, and radiotherapy diagnosis is also the most mature and widespread clinical application in the field of theranostics. A significant advantage of radiotherapy diagnosis is that the treatment site is the diagnostic image of the patient's lesion, and imaging and therapeutic intervention are closely related.

[0004] Positron emission computed tomography (PET) and single photon emission computed tomography (SPECT) are two very important imaging technologies in the field of nuclear medicine. They are usually achieved by using probe molecules labeled with radionuclides (radioactive metal complexes are one type of probe molecules). After entering the body, the radioactive probe will accumulate specifically in the lesion area. By detecting the rays emitted by the decay of the nuclide, a three-dimensional image of the distribution of the probe molecule in the body can be obtained. Compared with traditional diagnostic methods, nuclear medicine imaging can reflect the location of lesions in the body non-invasively and accurately, and is safer and more reliable. If the above probes are labeled with β-particle therapeutic nuclides or α-particle therapeutic nuclides, corresponding radioactive therapeutic drugs can be obtained. Such drugs are enriched in targeted tissues and organs, and use the radiation particles released by the decay of the nuclides to cause irreversible damage to the DNA of the targeted cells, induce chromosome deletion and aberration, and induce apoptosis of diseased cells to achieve the purpose of treatment. Compared with traditional radiotherapy, radioactive therapeutic drugs cause less damage to healthy tissues.

[0005] Therefore, the development of specific imaging tracers with high affinity and specificity, low nonspecific uptake, sufficient retention and effective permeability has become a key research direction in the field of nuclear medicine.

[0006] FGFRs belong to the receptor tyrosine kinase (RTK) family in the human genome. They are a member of the tyrosine kinase family, including four receptor subtypes: FGFR1, FGFR2, FGFR3, and FGFR4. Their ligands include 23 subtypes, including FGF1, 4, 7, 8, 9, and 19. FGFRs are a class of single-chain glycoproteins, consisting of an extracellular region, a transmembrane region, and an intracellular region. The extracellular region of FGFRs includes three immunoglobulin-like domains (D1-D3), which are connected by a serine-rich region between the D1 and D2 structures, and the intracellular region is a tyrosine kinase domain.

[0007] In FGFR1, FGFR2 and FGFR3, all forms contain the first half of D3 (denoted as IIIa), but two alternative exons can be used for the second half of D3, resulting in IIIb and IIIc forms. For FGFR2, these forms are denoted as FGFR2-IIIb and FGFR2-IIIc (or just FGFR2b and FGFR2c), respectively; the corresponding β forms are denoted as FGFR2 (β) IIIb and FGFR2 (β) IIIc. The FGFR2-IIIb form of FGFR2 (also denoted as K-sam-II) is a high affinity receptor for FGF1 and KGF family members (FGF7, FGF10 and FGF22), while FGFR2-IIIc (also denoted as K-sam-I) fully binds to FGF1 and FGF2, but not to KGF family members (Miki et al., Proc. Natl. Acad. Sci. USA 89: 246, 1992).

[0008] FGFR2 is a therapeutic target across tumor types. FGFR2 protein is overexpressed in about 3% of breast cancers, including triple-negative breast cancer, and about 10% of gastric / esophageal cancers. FGFR2 overexpression has also been found in other cancers, including colon cancer, hepatocellular carcinoma, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, bladder cancer, lung cancer, colon cancer, glioma, and head and neck cancer.

[0009] CN117126237A discloses a CHO2 derivative containing an amino acid chain and a hydrazino-nicotinamide group and its application. The radioactive preparation obtained by labeling the CHO2 derivative with a radionuclide has a higher tumor uptake and a higher tumor-to-muscle uptake ratio in tumors with overexpression of FGFR2, and is a new tumor radiopharmaceutical with promotion significance.

[0010] There is still a significant unmet medical need for effective diagnostic, tracing and / or therapeutic agents / drugs in cancers expressing FGFR2, especially anti-cancer drugs specifically targeting the FGFR2-IIIc protein. Summary of the invention

[0011] The purpose of the present invention is to overcome the defects of the prior art and provide a polypeptide nuclide ligand, a probe and applications thereof targeting FGFR2.

[0012] The technical solution of the present invention is as follows: In a first aspect of the present invention, the present invention provides a polypeptide nuclide ligand targeting FGFR2, which has a structure as shown in general formula (1) or (2): XL 1 -SEQ-R 1 Formula (1) R 2 -SEQ-L 1 -L 2 -X Formula (2) in, SEQ is a polypeptide represented by the amino acid sequence DAMMFIYQYLNQLKH (SEQ ID No 1); X is a radionuclide complexing group, wherein X is one of the following radionuclide complexing groups: , , , , , , , , ; L 1 is the connection key or

[0013] m is an integer from 1 to 12; L 2 is a linker, lysine or arginine; R 1 is one of the following groups: -OH, -NH 2 、-NHMe、-NMe 2 , -NHEt; R 2 It is one of the following groups: -H, formyl, acetyl, propionyl.

[0014] In a preferred embodiment of the present invention, the polypeptide nuclide ligand has a structure as shown in general formula (3) or (4):

[0015] Formula (3)

[0016] Formula (4) in, X is a radionuclide complexing group; L 1 is the connection key or ; m is an integer from 1 to 12; L 2 is a linker, lysine or arginine; R 1 is one of the following groups: -OH, -NH 2 、-NHMe、-NMe 2 , -NHEt; R 2 It is one of the following groups: -H, formyl, acetyl, propionyl.

[0017] In a second aspect of the present invention, the present invention provides a polypeptide radionuclide probe targeting FGFR2, wherein the polypeptide radionuclide probe comprises the above-mentioned polypeptide radionuclide ligand labeled with a radionuclide.

[0018] In a third aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned polypeptide radionuclide ligand or polypeptide radionuclide probe and a pharmaceutically acceptable carrier or excipient.

[0019] In a fourth aspect of the present invention, the present invention provides a use of the above-mentioned polypeptide radionuclide ligand, polypeptide radionuclide probe or pharmaceutical composition in the preparation of a reagent for diagnosing, tracing or treating FGFR2-expressing tumors.

[0020] The present invention provides polypeptide nuclide ligands and polypeptide nuclide probes targeting FGFR2. Through cell uptake studies and receptor binding studies, it is found that the polypeptide nuclide ligands and polypeptide nuclide probes of the present invention have good binding affinity with FGFR2IIIc. The tumor imaging effect of the polypeptide nuclide probe of the present invention is investigated by small animal PET / CT. The probe of the present invention has high sensitivity, clear imaging, low non-specific tissue uptake, high tumor uptake and high tumor to muscle uptake ratio, and good in vivo metabolic performance. Compared with existing pathological detection, this technology has the characteristics of non-invasive, quantitative, real-time dynamic, high specificity and high sensitivity. It has good clinical application prospects.

[0021] As used herein, "polypeptide" or "peptide" refers to a compound composed of a series of amino acid residues interconnected by amide (or peptide) bonds.

[0022] As used herein, "amino acid" includes both canonical amino acids (genetically encoded) and unnatural amino acids.

[0023] As used herein, the “twenty alpha amino acids that form a naturally encoded polypeptide” are understood in the art and refer to: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamic acid (Glu or E), glutamine (Gln or Q), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0024] As used herein, "pharmaceutically acceptable carrier or excipient" refers to any type of non-toxic, inert solid, semisolid or liquid filler, diluent, encapsulating material or formulation aid. Some examples of materials that can be used as pharmaceutically acceptable carriers are sugars, such as lactose, glucose and sucrose; cyclodextrins, such as alpha-(α), beta-(β) and gamma-(γ) cyclodextrins; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; chrysanthemum tea powder; malt; gelatin; talc; excipients, such as cocoa butter and suppository wax; peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil; Oils such as olive oil, corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol and phosphate buffer solutions, and other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as colorants, release agents, coating agents, sweeteners, flavorings and fragrances, preservatives and antioxidants, according to the judgment of the formulator. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the HPLC test result of compound 1; Figure 2 The LC-MS test results of compound 1, wherein Figure A is the liquid phase test result, and Figure B is the mass spectrometry test result; Figure 3 This is the HPLC test result of compound 2; Figure 4 The LC-MS test results of compound 2, wherein Figure A is the liquid phase test result, and Figure B is the mass spectrometry test result; Figure 5 Micro-PET / CT imaging and analysis of mice after injection of compound I; Figure 6 Micro-PET / CT imaging and analysis of mice after injection of compound II; Figure 7 This is the uptake analysis of compound II in tumor cells H716; Figure 8 Uptake analysis of compound II in tumor cells KATO III cells; Fig. 9 This is the uptake analysis of compound II in tumor cells SNU-16; Fig.10 Schematic diagram of the structure of compound I and compound II. DETAILED DESCRIPTION

[0026] In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be noted that the following embodiments do not limit the protection scope of the present invention.

[0027] As mentioned above, the first aspect of the present invention provides a polypeptide nuclide ligand targeting FGFR2, which has a structure as shown in general formula (1) or (2): XL 1 -SEQ-R 1 Formula (1) R 2 -SEQ-L 1 -L 2 -X Formula (2) in, SEQ is a polypeptide represented by the amino acid sequence DAMMFIYQYLNQLKH (SEQ ID No 1); X is a radionuclide complexing group, wherein X is one of the following radionuclide complexing groups: , , , , , , , , ; L 1 is the connection key or ; m is an integer from 1 to 12; L 2 is a linker, lysine or arginine; R 1 is one of the following groups: -OH, -NH 2、-NHMe、-NMe 2 , -NHEt; R 2 It is one of the following groups: -H, formyl, acetyl, propionyl.

[0028] In a preferred embodiment of the present invention, the polypeptide nuclide ligand has a structure as shown in general formula (3) or (4):

[0029] Formula (3)

[0030] Formula (4) In a preferred embodiment of the present invention, X is one of the following radionuclide complexing groups: , , , , ; In a preferred embodiment of the present invention, X is one of the following radionuclide complexing groups: , , , .

[0031] In a preferred embodiment of the present invention, X is one of the following radionuclide complexing groups: , .

[0032] In a preferred embodiment of the present invention, L 1 for , wherein m is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.

[0033] In a preferred embodiment of the present invention, L 1 for , m is 4, 6 or 8.

[0034] In a preferred embodiment of the present invention, L in the general formula (1) and (3) 1 for , m is an integer from 1 to 12; In a preferred embodiment of the present invention, L in the general formula (1) and (3) 1 for , m is 4, 6 or 8.

[0035] In a preferred embodiment of the present invention, L in the general formula (1) and (3)1 for , m is 6.

[0036] In a preferred embodiment of the present invention, L in the general formula (2) and (4) 1 for , m is an integer from 1 to 12; L 2 is lysine or arginine; X and L 2 Side chain connected.

[0037] In a preferred embodiment of the present invention, L in the general formula (2) and (4) 1 for , m is 4, 6 or 8; L 2 is lysine; X and L 2 Side chain connected.

[0038] In a preferred embodiment of the present invention, L in the general formula (2) and (4) 1 for , m is 6; L 2 is lysine; X and L 2 Side chain connected.

[0039] In a preferred embodiment of the present invention, the polypeptide nuclide ligand is one of the following structures:

[0040] Compound 1,

[0041] Compound 2,

[0042] Compound 3,

[0043] Compound 4

[0044] Compound 5

[0045] Compound 6

[0046] Compound 7.

[0047] In a preferred embodiment of the present invention, the polypeptide nuclide ligand is Compound 1 or Compound 2.

[0048] The second aspect of the present invention provides a polypeptide radionuclide probe targeting FGFR2, wherein the polypeptide radionuclide probe comprises the above-mentioned polypeptide radionuclide ligand labeled with a radionuclide.

[0049] The polypeptide nuclide probe targeting FGFR2 of the present invention can be prepared by labeling the polypeptide nuclide ligand targeting FGFR2 with radioactive nuclides. Specifically, the ligand is dissolved in a radioactive labeling buffer, and then different radioactive nuclides are added to react to obtain the corresponding molecular probe.

[0050] In a preferred embodiment of the present invention, the radionuclide is selected from a diagnostic radionuclide or a therapeutic radionuclide, wherein: The diagnostic radionuclide is selected from 68 Ga, 64 Cu, 18 F. 99m Tc, 123 I. 125 I. 124 One of I; The therapeutic radionuclide is selected from 177 Lu, 125 I. 131 I. 67 Cu, 225 One of Ac.

[0051] In a preferred embodiment of the present invention, the diagnostic radionuclide is selected from 68 Ga, 64 Cu, 18 F. 99m One of Tc.

[0052] In a preferred embodiment of the present invention, the diagnostic radionuclide is selected from 68 Ga or 99m Tc.

[0053] In a preferred embodiment of the present invention, the therapeutic radionuclide is 177 Lu.

[0054] In a preferred embodiment of the present invention, the polypeptide radionuclide probe targeting FGFR2 is obtained by one of Compounds 1 to 7 labeled with a radionuclide.

[0055] In a preferred embodiment of the present invention, the polypeptide radionuclide probe targeting FGFR2 is obtained by compound 1 or compound 2 labeled with a radionuclide.

[0056] In some specific embodiments, the polypeptide nuclide probe targeting FGFR2 is one of the following structures:

[0057] Compound I

[0058] Compound II.

[0059] The third aspect of the present invention provides a pharmaceutical composition, which comprises the above-mentioned polypeptide nuclide ligand or polypeptide nuclide probe and a pharmaceutically acceptable carrier or excipient.

[0060] In a fourth aspect, the present invention provides a use of the above-mentioned polypeptide radionuclide ligand, polypeptide radionuclide probe or pharmaceutical composition in the preparation of a reagent for diagnosing, tracing or treating FGFR2-expressing tumors.

[0061] In a preferred embodiment of the present invention, the tumor diagnosis agent is at least one of a SPECT imaging agent and a PET imaging agent.

[0062] In a preferred embodiment of the present invention, the FGFR2-expressing tumor is selected from any one or more of colorectal cancer, gastric cancer, endometrial cancer, breast cancer, and ovarian tumor.

[0063] The present invention will be described in detail below by way of examples.

[0064] The polypeptide compound and its derivative provided by the present invention adopt a solid phase synthesis method to synthesize its linear precursor, and the crude peptide obtained after cleavage is directly purified to obtain the target compound. The synthetic carrier is Rink Amide-AM Resin or Wang resin. During the synthesis process, the resin is first fully swollen in N,N-dimethylformamide (DMF), and the solid phase carrier and the activated amino acid derivative are repeatedly condensed → washed → Fmoc protection → washed → the next round of amino acid condensation to achieve the desired length of the synthesized polypeptide chain, and then DOTA is coupled to the N-terminal or C-terminal lysine side chain of the solid phase carrier, and finally a mixed solution of trifluoroacetic acid: water: triisopropylsilane (95:2.5:2.5, v:v:v) is reacted with the resin to cleave the polypeptide from the solid phase carrier, and then the solid crude product of the linear precursor is obtained after precipitation by frozen methyl tert-butyl ether. The crude linear precursor after cleavage is subjected to disulfide bond oxidation in a neutral solution to obtain the crude target polypeptide. The solid crude product or the oxidized crude polypeptide product is purified and separated by a C18 reverse phase preparative chromatography column in a system of 0.1% trifluoroacetic acid in acetonitrile / water to obtain pure polypeptides and their derivatives.

[0065] The experimental reagents used in the present invention are shown in Table 1.

[0066]

[0067] Example 1 Preparation of Compound 1

[0068] Compound 1

[0069] Step 1: Synthesis of linear precursor peptide chain The linear precursor peptide chain of compound 1: {PEG6}-DAMMFIYQYLNQLKH.

[0070] 294 mg (0.2 mmol) of Rink Amide-AM Resin was fully swollen in DMF for 1 h. Then the linear precursor sequence was synthesized from the carboxyl end to the amino end. Each coupling cycle was performed as follows: Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min; Wash the resin with DMF 6-8 times until the pH is neutral; Dissolve 1.0 mmol Fmoc-AA, 1.0 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 2 mmol 4-methylmorpholine (NMM) in DMF, add the resin and react at room temperature for 1 h; The resin was washed 4-6 times with DMF before coupling the next amino acid.

[0071] After the linear peptide synthesis, the resin was washed 5 times with DMF.

[0072] Step 2: N-terminal coupling with DOTA Weigh 1.0mmol tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid and 1.0mmol ethyl 2-oxime cyanoacetate and dissolve them in 8mL DMF, then add 160µL DIC to pre-activate for 3 min, then add the mixed solution to the resin obtained in the previous step and shake for 16h. After the reaction, drain the reaction solution, wash with DMF 4-5 times, and wash with DCM 5 times.

[0073] Step 3: Cleavage of the linear precursor peptide chain Add freshly prepared trifluoroacetic acid: water: triisopropylsilane (95:2.5:2.5, v:v:v; 10 mL) to the resin obtained in step 2 and shake at room temperature for 2 hours. After the reaction is completed, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate with 4 times the volume of cold MTBE to obtain a crude product. Wash the crude product with MTBE 3 times and dry it in a vacuum.

[0074] Step 4: Purification and preparation of peptide nuclide ligand The crude polypeptide nuclide ligand obtained in step 3 was dissolved in 20% acetonitrile aqueous solution, filtered through a 0.45 um membrane, and separated using a reversed-phase high-performance liquid chromatography system, with buffers A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column, and the chromatograph detection wavelength was set to 230 nm during the purification process, the flow rate was 15 mL / min, and the gradient was 30-50% acetonitrile in 40 min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions >95% were combined and freeze-dried to obtain the pure polypeptide nuclide ligand.

[0075] Step 5: Detection and characterization methods The purified peptide nuclide ligand in step 4 was subjected to analytical HPLC and LC / MS to determine the purity and completion of the N-terminal coupling of the compound with DOTA. Figure 1 , Figure 2 shown.

[0076] Example 2 Preparation of Compound I

[0077] Compound I

[0078] The germanium gallium generator was eluted in sections with hydrochloric acid solution, and the eluent with the highest activity was taken for ligand compound 1 68 Ga labeling. According to the activity calculation, the amount of compound 1 to be added was mixed with the eluent, and the reaction system was adjusted to pH 3-4 using a metal-free 1M sodium acetate buffer at pH 7. The reaction temperature was set at 95°C and the reaction time was 10 min. After the reaction was completed, the reaction solution was purified using a pre-activated C18 column to obtain the final product. Unlabeled 68 Ga ions, and then eluted with ethanol solution to obtain compound I. The eluent was diluted with physiological saline and sterile filtered to obtain the injection of compound I for experimental use.

[0079] Example 3 Preparation of Compound 2

[0080] Compound 2

[0081] Step 1: Coupling of the first amino acid Fmoc-Lys(Mtt)-OH 177 mg (0.2 mmol) Wang resin was fully swelled in DCM for 1 h. To prepare the amino acid solution, 0.16 mmol Fmoc-Lys(Mtt)-OH and 0.16 mmol HOBt were weighed and dissolved in 10-15 mL DCM, and 0.32 mmol DIC was added. After the resin was swollen, the DCM was drained, the prepared amino acid solution was added, and 0.02 mmol DMAP was added, and the reaction was carried out at room temperature for 15 h. After the reaction was completed, the blocking solution (10 mL) DCM: methanol: DIEA (85:10:5, v:v:v) was added at room temperature for 10 min for blocking. The blocked resin was washed 5 times with DCM and 5 times with DMF.

[0082] Step 2: Synthesis of linear precursor peptide chain The linear precursor peptide chain of compound 2: DAMMFIYQYLNQLKH-{PEG6}-{K(Mtt)}.

[0083] The resin obtained in step 1 was fully swollen in DMF for 1 h, and then the linear precursor sequence was synthesized from the second position of the carboxyl terminal {PEG6} to the amino terminal. Each coupling cycle was performed as follows: Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min; Wash the resin with DMF 6-8 times until the pH is neutral; Dissolve 1 mmol Fmoc-AA, 1 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 2 mmol 4-methylmorpholine (NMM) in DMF, add the resin and react at room temperature for 1 hour; The resin was washed 4-6 times with DMF before coupling one amino acid.

[0084] After the linear peptide synthesis, the resin was washed 5 times with DMF and 5 times with DCM. The resin was dried in vacuo.

[0085] Step 3: N-terminal acetylation Prepare 10 mL of acetylation reagent: Dissolve 500 µL of acetic anhydride and 500 µL of DIEA in 9 mL of DMF. Add 10 mL of the prepared acetylation reagent to the resin obtained in step 2, shake well, and oscillate for 10 min. After the reaction, drain the reaction solution, rinse the resin with DMF 6-8 times, and rinse the resin with DCM 5 times.

[0086] Step 4: Coupling of DOTA to the C-terminal Lysine Side Chain Removal of the Mtt protecting group of the lysine side chain: After swelling the resin with DCM for 1 h, add a mixed solution of hexafluoroisopropanol / dichloromethane (30% v / v, 10 mL) to the resin, oscillate the reaction at room temperature for 45 minutes and then remove the solution. Repeat the operation once. After the reaction, rinse the resin with DCM 5 times and DMF 6 times.

[0087] Lysine side chain coupling DOTA: Weigh 1.0mmol tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid and 1.0mmol ethyl 2-oximecyanoacetate and dissolve them in 8mL DMF, then add 160µL DIC to pre-activate for 3 min, then add the mixed solution to the resin obtained in the previous step and shake for 16h. After the reaction, drain the reaction solution, wash with DMF 4-5 times, and wash with DCM 5 times.

[0088] Step 5: Linear precursor peptide chain cleavage Add freshly prepared trifluoroacetic acid: water: triisopropylsilane (95:2.5:2.5, v:v:v; 10 mL) to the resin obtained in step 4 and shake at room temperature for 2 hours. After the reaction is completed, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate with 4 times the volume of cold MTBE to obtain a crude product. Wash the crude product with MTBE 3 times and dry it in a vacuum.

[0089] Step 6: Purification and preparation of peptide nuclide ligands The crude polypeptide nuclide ligand obtained in step 5 was dissolved in 20% acetonitrile aqueous solution, filtered through a 0.45 um membrane, and separated using a reversed-phase high-performance liquid chromatography system, with buffers A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column, and the chromatograph detection wavelength was set to 230 nm during the purification process, the flow rate was 15 mL / min, and the gradient was 30-50% acetonitrile in 40 min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions >95% were combined and freeze-dried to obtain the pure polypeptide nuclide ligand.

[0090] Step 6: Detection and characterization methods The purified peptide nuclide ligand in step 5 was subjected to analytical HPLC and LC / MS to determine the purity and compound to complete the coupling of C-terminal lysine side chain with DOTA. Figure 3 , Figure 4 shown.

[0091] Example 4 Preparation of Compound II

[0092] Compound II

[0093] The germanium gallium generator was eluted in sections with hydrochloric acid solution, and the eluent with the highest activity was used to precipitate compound 2. 68 Ga labeling. According to the activity calculation, the amount of compound 2 to be added, the compound 2 solution was mixed with the eluent, and the reaction system was adjusted to pH 3-4 using a metal-free 1M sodium acetate buffer at pH = 7. The reaction temperature was set at 95°C and the reaction time was 10 min. After the reaction was completed, the reaction solution was purified using a pre-activated C18 column to obtain the final product compound II. The unlabeled 68 Ga ions, and then eluted with ethanol solution to obtain compound II. The eluent was diluted with physiological saline and sterile filtered to obtain the injection of compound II for experimental use.

[0094] Other polypeptide nuclide ligands and polypeptide nuclide probes of the present invention can be synthesized by referring to the synthesis methods of Examples 1 to 4.

[0095] Test Example 1 SPR test of affinity (KD) between peptide nuclide ligand and FGFR2IIIC 1. Materials and Methods (1) Experimental materials The experimental materials are shown in Table 2.

[0096] Table 2 Experimental materials

[0097] (2) Experimental methods

[0098] Biacore T200 was used to test the affinity of peptides with FGFR2IIIb and FGFR2IIIc. ProteinA chip was used to capture FGFR2IIIb and FGFR2IIIc proteins at about 1800RU and 1300RU at 25°C. 1×HBS-EP+, pH 7.4 was used as running buffer. Binding experiments were performed at 25°C. The peptide analysis flow rate was 30μL / min, Association 120 s, Dissociation 600s. Single-cycle multi-concentration detection of peptide samples and FGFR2IIIb and FGFR2IIIc proteins was selected. Gly-HCl pH = 1.5, 30 μL / min, 30 s was used for chip regeneration, and the data were fitted with a 1:1 binding model.

[0099] 2. Experimental results SPR single cycle kinetic analysis showed that the polypeptide nuclide ligand of the present invention had good binding affinity to FGFR2IIIc. The binding affinity of compound 1 and compound 2 to FGFR2IIIc is shown in Table 3.

[0100] Table 3 Affinity of compounds binding to FGFR2IIIc

[0101] Test Example 2: PET-CT imaging of tumor animal model Experimental steps: Follow the Micro-PET / CT operating procedures. The probe obtained after drug labeling in the above-mentioned example steps was diluted to 1 mCi / mL with normal saline, and a certain amount was extracted for injection. For the randomly grouped mice, the tumor-bearing mice were anesthetized with isoflurane, and the probe was injected into the tail vein after the righting reflex of the tumor-bearing mice disappeared. PET scans and whole-body CT scans were performed 30 minutes after injection. The uptake in the tumor area was quantified, and the uptake in the contralateral muscle, heart and its contents, and liver was quantified.

[0102] The experimental results are shown in Figure 5 , Figure 6 , Figure 5 and Figure 6 The Micro PET maximum intensity projection images of H716 tumor-bearing mice, KATO III tumor-bearing mice, and SNU-16 tumor-bearing mice 30 minutes after intravenous injection of compound I and compound II, respectively. Figure 5 and Figure 6 It can be seen that at the time point of image acquisition, the tumor is clearly visible with good tissue contrast. In addition, the tumor uptake of compound II is higher than that of compound I. Both compound II and compound I have obvious tumor targeting and can be used as potential molecules for diagnosis and treatment of tumors with high expression of FGFR2IIIC. Compound II may have a better effect.

[0103] Test Example 3 Cellular uptake and inhibition experiments

[0104] Experimental steps: H716 cells, KATO III cells, and SNU-16 cells were plated in 6-well plates and cultured in an incubator. After overnight culture, the culture medium was removed and the cells were washed once or twice. 68 Ga-labeled probe was used as the experimental group, and the above cells were added with a certain amount of 68The Ga-labeled probe was incubated at 37°C for 15min, 30min, 60min, and 120min, and an unlabeled ligand compound was used to block cell uptake at 60min. After the incubation, the incubation solution was aspirated and washed three times with 4°C pre-cooled phosphate buffer to terminate cell uptake. All cells were lysed with lysis buffer, and the lysate was collected for radioactivity counting. After attenuation correction and reference standard conversion of the radioactivity count, the probe cell uptake rate was calculated, and the cell uptake of the probe and the change in uptake rate over time were analyzed to determine the probe binding specificity and probe distribution ability. The experimental results are as follows: Figure 7-9 shown.

[0105] According to the results of the cell uptake experiment, compound II has a high uptake in H716 cells, KATO III cells, and SNU-16 cells in vitro, and the uptake increases with the incubation time. The significant difference in cell uptake between the unlabeled ligand compound and the unlabeled ligand compound shows that compound II specifically binds to FGFR2, indicating that the new FGFR2 targeting probe maintains the same specificity as the ligand compound while taking into account more types of radionuclide labeling.

[0106] The above description is only a preferred embodiment of the present invention, and therefore cannot limit the scope of implementation of the present invention. That is, equivalent changes and modifications made according to the patent scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A polypeptide nuclide ligand targeting FGFR2, characterized in that: The polypeptide nuclide ligand has a structure as shown in general formula (1) or (2): X-L1-SEQ-R1 Formula (1) R2-SEQ-L1-L2-X Formula (2) in, SEQ is a polypeptide represented by the amino acid sequence DAMMFIYQYLNQLKH (SEQ ID No. 1); X is a radionuclide complexing group, wherein X is one of the following radionuclide complexing groups: 、 、 、 、 、 、 、 ； L1 is a connection key or ; m is an integer from 1 to 12; L2 is a linker, lysine or arginine; R1 is one of the following groups: -OH, -NH2, -NHMe, -NMe2, -NHEt; R2 is one of the following groups: -H, formyl, acetyl, propionyl.

2. The polypeptide nuclide ligand according to claim 1, characterized in that: The polypeptide nuclide ligand has a structure as shown in general formula (3) or (4): Formula (3) Formula (4).

3. The polypeptide nuclide ligand according to claim 1, characterized in that: The m is 4, 6 or 8.

4. The polypeptide nuclide ligand according to claim 1, characterized in that: The polypeptide nuclide ligand is one of the following structures: Compound 1, Compound 2, Compound 3, Compound 4 Compound 5 Compound 6 Compound 7.

5. A polypeptide radionuclide probe targeting FGFR2, characterized in that: The polypeptide nuclide probe comprises the polypeptide nuclide ligand according to any one of claims 1 to 4 labeled with a radionuclide.

6. The polypeptide nuclide probe according to claim 5, characterized in that: The radionuclide is selected from diagnostic radionuclides or therapeutic radionuclides, wherein: The diagnostic radionuclide is selected from 68 Ga, 64 Cu, 18 F. 99m Tc, 123 I. 125 I. 124 One of I; The therapeutic radionuclide is selected from 177 Lu, 125 I. 131 I. 67 Cu, 225 One of Ac.

7. A pharmaceutical composition, characterized in that The invention comprises the polypeptide nuclide ligand according to any one of claims 1 to 4 or the polypeptide nuclide probe according to claim 5 or 6 and a pharmaceutically acceptable carrier or excipient.

8. Use of the polypeptide radionuclide ligand according to any one of claims 1 to 4, the polypeptide radionuclide probe according to claim 5 or 6, or the pharmaceutical composition according to claim 7 in the preparation of reagents / drugs for diagnosis, tracing and / or treatment of FGFR2-expressing tumors.

9. The use according to claim 8, characterized in that The FGFR2-expressing tumor is selected from any one or more of colorectal cancer, gastric cancer, endometrial cancer, breast cancer, and ovarian cancer.

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