A polypeptide radionuclide ligand targeting FGFR2, a probe thereof, and applications thereof

Abstract

The present invention belongs to the field of nuclear medicine, and particularly relates to a polypeptide radionuclide ligand targeting FGFR2, a probe and their applications. The ligand has the structure shown in formula (1) or (2). The radioactive preparation obtained by labeling it with a radionuclide has high tumor uptake and a high tumor-to-muscle uptake ratio in tumors with overexpression of FGFR2, and has good clinical application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of nuclear medicine, and relates to the fields of radiochemistry and clinical nuclear medicine labeled with radionuclides. Specifically, it relates to a polypeptide radionuclide ligand targeting FGFR2, a probe and their applications. Background Art

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

[0003] Theranostics has always been the main development direction in the field of nuclear medicine. At the same time, radiotherapy diagnostics is also the most mature and widely used clinical application in the field of theranostics. A significant advantage of radiotherapy diagnostics 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 techniques in the field of nuclear medicine, which are usually realized by relying on probe molecules labeled with radionuclides (radioactive metal complexes belong to one of the probe molecules). After the radioactive probe enters the body, it will specifically accumulate in the lesion area. By detecting the rays emitted by the decay of the radionuclide, 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 non-invasively and accurately reflect the location of lesions in the body, and is safer and more reliable. If the above probe is labeled with a β-particle therapeutic radionuclide or an α-particle therapeutic radionuclide, the corresponding radioactive therapeutic drug can be obtained. Such drugs accumulate in the targeted tissues and organs, and use the radiation particles released by the decay of the radionuclide to cause irreversible damage to the DNA of targeted cells, induce chromosome deletions and aberrations, and induce apoptosis of diseased cells to achieve the therapeutic purpose. 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 non-specific 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 and are members of the tyrosine kinase family, including four receptor subtypes: FGFR1, FGFR2, FGFR3, and FGFR4. Their ligands include 23 subtypes such as FGF1, 4, 7, 8, 9, and 19. FGFRs are a type of single-chain glycoprotein, 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. The intracellular region is the tyrosine kinase domain.

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

[0008] As a therapeutic target across tumor types, FGFR2 protein is overexpressed in approximately 3% of breast cancers, including triple-negative breast cancer, and approximately 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 CH02 derivative containing an amino acid chain and a hydrazinonicotinamide group and its application. The radioactive preparation obtained by labeling it with a radionuclide has high tumor uptake and a high tumor-to-muscle uptake ratio in tumors with overexpressed FGFR2, and it is a novel tumor radioactive drug with promotional significance.

[0010] There remains a significant unmet medical need for diagnostic, tracer, and / or therapeutic reagents / drugs that are effective in cancers expressing FGFR2, especially anti-cancer drugs specifically targeting the FGFR2-IIIc protein. Summary of the Invention

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

[0012] The technical solution of the present invention is as follows:

[0013] In the first aspect of the present invention, the present invention provides a polypeptide radionuclide ligand targeting FGFR2, which has a structure as shown in general formula (1) or (2):

[0014] X-L1-SEQ-R1

[0015] Formula (1),

[0016] R2-SEQ-L1-L2-X

[0017] Formula (2)

[0018] Wherein,

[0019] SEQ is a polypeptide shown by the amino acid sequence DAMMFIYQYLNQLKH (SEQ ID No 1);

[0020] X is a radionuclide complexing group, and the X is one of the following radionuclide complexing groups:

[0021] 、 、 、 、 、 、 、 、 ;

[0022] L1 is a linking bond or

[0023] m is an integer from 1 to 12;

[0024] L2 is a linking bond, lysine or arginine;

[0025] R1 is one of the following groups: -OH, -NH2, -NHMe, -NMe2, -NHEt;

[0026] R2 is one of the following groups: -H, formyl, acetyl, propionyl.

[0027] In a preferred embodiment of the present invention, the polypeptide radionuclide ligand has a structure as shown in general formula (3) or (4):

[0028]

[0029] Formula (3),

[0030]

[0031] Formula (4)

[0032] Wherein,

[0033] X is a radioactive nuclide complexing group;

[0034] L1 is a linking bond or ;

[0035] m is an integer from 1 to 12;

[0036] L2 is a linking bond, lysine or arginine;

[0037] R1 is one of the following groups: -OH, -NH2, -NHMe, -NMe2, -NHEt;

[0038] R2 is one of the following groups: -H, formyl, acetyl, propionyl.

[0039] In a second aspect of the present invention, the present invention provides a polypeptide radionuclide probe targeting FGFR2, and the polypeptide radionuclide probe comprises the above-mentioned polypeptide radionuclide ligand labeled with a radioactive nuclide.

[0040] In a third aspect of the present invention, the present invention provides a pharmaceutical composition, and the pharmaceutical composition comprises the above-mentioned polypeptide radionuclide ligand or polypeptide radionuclide probe and a pharmaceutically acceptable carrier or excipient.

[0041] In a fourth aspect of the present invention, the present invention provides the use of the above-mentioned polypeptide radionuclide ligand, polypeptide radionuclide probe or pharmaceutical composition in the preparation of reagents for the diagnosis, tracing or treatment of tumors expressing FGFR2.

[0042] The present invention provides a polypeptide radionuclide ligand and a polypeptide radionuclide probe targeting FGFR2. Through cell uptake studies and receptor binding studies, it is found that the polypeptide radionuclide ligand and polypeptide radionuclide probe of the present invention have good binding affinity with FGFR2IIIc. By investigating the tumor imaging effect of the polypeptide radionuclide probe of the present invention with a small animal PET / CT, the probe of the present invention has high sensitivity, clear imaging, low non-specific tissue uptake, high tumor uptake and a high tumor-to-muscle uptake ratio, and good in vivo metabolic performance; compared with existing pathological detections, this technology has the characteristics of non-invasiveness, quantification, real-time dynamics, high specificity and high sensitivity. It has good clinical application prospects.

[0043] As used herein, "polypeptide" or "peptide" refers to a compound composed of a series of amino acid residues connected to each other by amide (or peptide) bonds.

[0044] As used herein, "amino acid" includes canonical amino acids (genetically encoded) and non-natural amino acids.

[0045] As used herein, "the twenty α-amino acids that form naturally encoded polypeptides" is understood in the art and refers 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).

[0046] As used herein, "pharmaceutically acceptable carrier or excipient" refers to any type of non-toxic, inert solid, semi-solid 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 carboxymethyl cellulose, ethyl cellulose and cellulose acetate; yellow flower tea powder; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; diols 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; depending on the judgment of the formulator, the composition may also contain ethanol and phosphate buffer solution, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, and coloring agents, release agents, coating agents, sweeteners, flavoring agents and fragrances, preservatives and antioxidants. Description of the Drawings

[0047] Figure 1 For the HPLC detection result of Compound 1;

[0048] Figure 2 For the LC-MS detection result of Compound 1, wherein Figure A is the liquid phase detection result and Figure B is the mass spectrometry detection result;

[0049] Figure 3 For the HPLC detection result of Compound 2;

[0050] Figure 4For the LC-MS detection results of Compound 2, where Figure A is the liquid-phase detection result and Figure B is the mass-spectrometry detection result;

[0051] Figure 5 For the Micro-PET / CT imaging and analysis after injecting Compound I into mice;

[0052] Figure 6 For the Micro-PET / CT imaging and analysis after injecting Compound II into mice;

[0053] Figure 7 For the uptake analysis of Compound II in tumor cell line H716;

[0054] Figure 8 For the uptake analysis of Compound II in tumor cell line KATO III;

[0055] Figure 9 For the uptake analysis of Compound II in tumor cell line SNU-16;

[0056] Figure 10 For the structural schematic diagrams of Compound I and Compound II. Detailed implementation manners

[0057] To make the technical solutions of the present invention clearer and more understandable to those skilled in the art, the following examples are provided for illustration. It should be noted that the following examples do not limit the scope of protection required by the present invention.

[0058] As described above, the first aspect of the present invention provides a polypeptide-radionuclide ligand targeting FGFR2, which has a structure as shown in General Formula (1) or (2):

[0059] X-L1-SEQ-R1

[0060] Formula (1),

[0061] R2-SEQ-L1-L2-X

[0062] Formula (2)

[0063] Wherein,

[0064] SEQ is a polypeptide shown by the amino acid sequence DAMMFIYQYLNQLKH (SEQ ID No 1);

[0065] X is a radionuclide complexing group, and the X is one of the following radionuclide complexing groups:

[0066] , , , , , , , , ;

[0067] L1 is a linking group or ;

[0068] m is an integer from 1 to 12;

[0069] L2 is a linking group, lysine or arginine;

[0070] R1 is one of the following groups: -OH, -NH2, -NHMe, -NMe2, -NHEt;

[0071] R2 is one of the following groups: -H, formyl, acetyl, propionyl.

[0072] In a preferred embodiment of the present invention, the polypeptide-radionuclide ligand has the structure of general formula (3) or (4):

[0073]

[0074] Formula (3),

[0075]

[0076] Formula (4)

[0077] In a preferred embodiment of the present invention, the X is one of the following radionuclide complexing groups:

[0078] , , , , ;

[0079] In a preferred embodiment of the present invention, the X is one of the following radionuclide complexing groups:

[0080] , , , 。

[0081] In a preferred embodiment of the present invention, the X is one of the following radionuclide complexing groups:

[0082] , 。

[0083] In a preferred embodiment of the present invention, in the general formula, L1 is , m is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12.

[0084] In a preferred embodiment of the present invention, in the general formula, L1 is , and m is 4, 6 or 8.

[0085] In a preferred embodiment of the present invention, in general formulas (1) and (3), L1 is , and m is an integer from 1 to 12;

[0086] In a preferred embodiment of the present invention, in general formulas (1) and (3), L1 is , and m is 4, 6 or 8.

[0087] In a preferred embodiment of the present invention, in general formulas (1) and (3), L1 is , and m is 6.

[0088] In a preferred embodiment of the present invention, in general formulas (2) and (4), L1 is , m is an integer from 1 to 12; L2 is lysine or arginine; X is linked to the side chain of L2.

[0089] In a preferred embodiment of the present invention, in general formulas (2) and (4), L1 is , m is 4, 6 or 8; L2 is lysine; X is linked to the side chain of L2.

[0090] In a preferred embodiment of the present invention, in general formulas (2) and (4), L1 is , m is 6; L2 is lysine; X is linked to the side chain of L2.

[0091] In a preferred embodiment of the present invention, the polypeptide radionuclide ligand is one of the following structures:

[0092]

[0093] Compound 1,

[0094]

[0095] Compound 2,

[0096]

[0097] Compound 3,

[0098]

[0099] Compound 4,

[0100]

[0101] Compound 5,

[0102]

[0103] Compound 6,

[0104]

[0105] Compound 7.

[0106] In a preferred embodiment of the present invention, the polypeptide-radionuclide ligand is Compound 1 or Compound 2.

[0107] The second aspect of the present invention provides a polypeptide-radionuclide probe targeting FGFR2, which comprises the above-mentioned polypeptide-radionuclide ligand labeled with a radionuclide.

[0108] The polypeptide-radionuclide probe targeting FGFR2 of the present invention can be prepared by radiolabeling the polypeptide-radionuclide ligand targeting FGFR2. Specifically, the ligand is dissolved in a radiolabeling buffer, and then different radionuclides are added for reaction to obtain the corresponding molecular probe.

[0109] In a preferred embodiment of the present invention, the radionuclide is selected from diagnostic radionuclides or therapeutic radionuclides, wherein:

[0110] The diagnostic radionuclide is selected from 68 Ga, 64 Cu, 18 F, 99m Tc, 123 I, 125 I, 124 I;

[0111] The therapeutic radionuclide is selected from 177 Lu, 125 I, 131 I, 67 Cu, 225 Ac.

[0112] In a preferred embodiment of the present invention, the diagnostic radionuclide is selected from 68 Ga, 64 Cu, 18 F, 99m Tc.

[0113] In a preferred embodiment of the present invention, the diagnostic radionuclide is selected from 68 Ga or 99m Tc.

[0114] In a preferred embodiment of the present invention, the therapeutic radionuclide is 177Lu.

[0115] In a preferred embodiment of the present invention, the polypeptide-radionuclide probe targeting FGFR2 is obtained by radiolabeling one of Compounds 1 to 7.

[0116] In a preferred embodiment of the present invention, the polypeptide-radionuclide probe targeting FGFR2 is obtained by radiolabeling Compound 1 or Compound 2.

[0117] In some specific embodiments, the polypeptide-radionuclide probe targeting FGFR2 is one of the following structures:

[0118]

[0119] Compound I,

[0120]

[0121] Compound II.

[0122] The third aspect of the present invention provides a pharmaceutical composition, which comprises the above-mentioned polypeptide-radionuclide ligand or polypeptide-radionuclide probe and a pharmaceutically acceptable carrier or excipient.

[0123] The fourth aspect of the present invention provides the use of the above-mentioned polypeptide-radionuclide ligand, polypeptide-radionuclide probe or pharmaceutical composition in the preparation of reagents for the diagnosis, tracing or treatment of FGFR2-expressing tumors.

[0124] In a preferred embodiment of the present invention, the reagent for tumor diagnosis is at least one of SPECT imaging agent and PET imaging agent.

[0125] In a preferred embodiment of the present invention, the FGFR2-expressing tumors are selected from any one or more of rectal cancer, gastric cancer, endometrial cancer, breast cancer, and ovarian tumors.

[0126] The present invention will be described in detail below by way of examples.

[0127] The polypeptide compound and its derivatives provided by the present invention are synthesized by solid-phase synthesis to obtain their linear precursors, and the target compound is directly purified from the crude peptide after cleavage. The synthesis carrier is Rink Amide-AM Resin or Wang resin. During the synthesis process, first, the resin is fully swollen in N,N-dimethylformamide (DMF). The solid-phase carrier and the activated amino acid derivatives are repeatedly subjected to the operations of condensation → washing → deprotection of Fmoc → washing → the next round of amino acid condensation to reach the desired polypeptide chain length. Then, DOTA is coupled to the lysine side chain at the N-terminus or C-terminus of the solid-phase carrier. 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 the solid crude product of the linear precursor is obtained after precipitation with cold methyl tert-butyl ether. The crude linear precursor after cleavage is oxidized to form a disulfide bond in a neutral solution to obtain the crude target polypeptide. The solid crude product or the oxidized polypeptide crude product is purified and separated by a C18 reversed-phase preparative chromatography column in an acetonitrile / water system containing 0.1% trifluoroacetic acid to obtain the pure product of the polypeptide and its derivatives.

[0128] The experimental reagents used in the present invention are shown in Table 1.

[0129]

[0130] Example 1 Preparation of Compound 1

[0131]

[0132] Compound 1

[0133] Step 1: Synthesis of the linear precursor peptide chain

[0134] The linear precursor peptide chain of Compound 1: {PEG6}-D-A-M-M-F-I-Y-Q-Y-L-N-Q-L-K-H.

[0135] 294 mg (0.2 mmol) of Rink Amide-AM Resin was fully swollen in DMF for 1 h. Then, the synthesis was carried out in the order from the carboxyl terminus to the amino terminus according to the linear precursor sequence. Each coupling cycle was carried out as follows:

[0136] 20% piperidine / DMF (20% v / v, 10 mL) was used for Fmoc-deprotection twice, 8 min each time;

[0137] The resin was rinsed with DMF 6 - 8 times until the pH was neutral;

[0138] Dissolve 1.0 mmol of Fmoc-AA, 1.0 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 2 mmol of 4-methylmorpholine (NMM) in DMF, add the resin and react at room temperature for 1 h;

[0139] Before coupling the next amino acid, rinse the resin with DMF 4 - 6 times.

[0140] After the synthesis of the linear polypeptide, rinse the resin with DMF 5 times.

[0141] Step 2: Coupling of DOTA at the N-terminus

[0142] Weigh 1.0 mmol of tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid and 1.0 mmol of ethyl 2-oximinocyanoacetate, dissolve them in 8 mL of DMF, then add 160 μL of DIC for pre-activation for 3 min, and then add the mixed solution to the resin obtained in the previous step and react with shaking for 16 h. After the reaction, drain the reaction solution, wash with DMF 4 - 5 times and wash with DCM 5 times.

[0143] Step 3: Cleavage of the linear precursor peptide chain

[0144] 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 react with shaking at room temperature for 2 h. After the reaction, filter the reaction solution, wash the resin with trifluoroacetic acid, combine with the reaction solution, and precipitate with 4 volumes of cold MTBE to obtain the crude product. Wash the crude product with MTBE 3 times and dry it in vacuo.

[0145] Step 4: Purification and preparation of the polypeptide radionuclide ligand

[0146] Dissolve the crude polypeptide radionuclide ligand obtained in Step 3 in 20% aqueous acetonitrile solution, filter through a 0.45 μm membrane, and separate it using a reverse-phase high-performance liquid chromatography system. The buffer solutions are A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). Among them, the chromatographic column is a BR-C18 (Sepax) reverse-phase chromatographic column. During the purification process, the detection wavelength of the chromatograph is set at 230 nm, the flow rate is 15 mL / min, and the gradient is 30 - 50% acetonitrile in 40 min. Collect the relevant fractions of the product, combine the fractions with a purity > 95% after HPLC identification, and lyophilize to obtain the pure polypeptide radionuclide ligand.

[0147] Step 5: Detection and characterization methods

[0148] The pure polypeptide-radionuclide ligand from Step 4 is analyzed by analytical high-performance liquid chromatography and liquid chromatography / mass spectrometry to determine the purity and to complete the N-terminal conjugation of DOTA. The results of the liquid phase and liquid chromatography / mass spectrometry detections of the polypeptide-radionuclide ligand are as Figure 1 , Figure 2 shown.

[0149] Example 2 Preparation of Compound I

[0150]

[0151] Compound I

[0152] The germanium-gallium generator is eluted stepwise with hydrochloric acid solution, and the eluate with the highest activity is taken for ligand compound 1 68 Ga labeling. The amount of compound 1 to be added is calculated according to the activity. The solution of compound 1 is mixed with the eluate, and the reaction system is adjusted to pH 3-4 using a pH = 7 metal-free 1 M sodium acetate buffer. The reaction temperature is set at 95 °C and the reaction time is 10 min. After the reaction is completed, the reaction solution is purified by a pre-activated C18 column. First, unlabeled 68 Ga ions are removed with pure water, and then the compound I is eluted with an ethanol solution. The eluate is diluted with physiological saline and sterile filtered to obtain an injectable solution of compound I for experimental use.

[0153] Example 3 Preparation of Compound 2

[0154]

[0155] Compound 2

[0156] Step 1: Coupling of the first amino acid Fmoc-Lys(Mtt)-OH

[0157] 177 mg (0.2 mmol) of Wang resin is fully swollen in DCM for 1 h. An amino acid solution is prepared by weighing 0.16 mmol of Fmoc-Lys(Mtt)-OH and 0.16 mmol of HOBt and dissolving them in 10-15 mL of DCM, and then adding 0.32 mmol of DIC. After the resin is swollen, the DCM is drained, the prepared amino acid solution is added, and 0.02 mmol of DMAP is added. The reaction is carried out at room temperature for 15 h. After the reaction is completed, a blocking solution (10 mL) of DCM:methanol:DIEA (85:10:5, v:v:v) is added at room temperature for 10 min for blocking. The blocked resin is washed 5 times with DCM and 5 times with DMF.

[0158] Step 2: Synthesis of the linear precursor peptide chain

[0159] Linear precursor peptide chain of Compound 2: D-A-M-M-F-I-Y-Q-Y-L-N-Q-L-K-H-{PEG6}-{K(Mtt)}.

[0160] The resin obtained in Step 1 was fully swollen in DMF for 1 h, and then synthesized in the order from the second position {PEG6} at the carboxyl terminus to the amino terminus according to the linear precursor sequence. Each coupling cycle was carried out as follows:

[0161] 20% piperidine / DMF (20% v / v, 10 mL) was used for Fmoc-deprotection twice, 8 min each time;

[0162] The resin was rinsed with DMF 6 - 8 times until neutral pH;

[0163] 1 mmol of Fmoc-AA, 1 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 2 mmol of 4-methylmorpholine (NMM) were dissolved in DMF, and added to the resin and reacted at room temperature for 1 h;

[0164] Before coupling one amino acid, the resin was rinsed with DMF 4 - 6 times.

[0165] After the synthesis of the linear polypeptide, the resin was rinsed with DMF 5 times and with DCM 5 times. The resin was dried in vacuo.

[0166] Step 3: N-terminal acetylation

[0167] Prepare 10 mL of acetylation reagent: 500 µL of acetic anhydride and 500 µL of DIEA were dissolved in 9 mL of DMF. 10 mL of the prepared acetylation reagent was added to the resin obtained in Step 2, shaken well, and oscillated for 10 min. After the reaction, the reaction solution was drained, and the resin was rinsed with DMF 6 - 8 times and with DCM 5 times.

[0168] Step 4: Coupling of DOTA to the C-terminal lysine side chain

[0169] Remove the Mtt protecting group on the lysine side chain: After swelling the resin in DCM for 1 h, a mixed solution of hexafluoroisopropanol / dichloromethane (30% v / v, 10 mL) was added to the resin, and the reaction was oscillated at room temperature for 45 minutes and then removed. The operation was repeated once. After the reaction, the resin was rinsed with DCM 5 times and washed with DMF 6 times.

[0170] Lysine side chain coupling with DOTA: Dissolve 1.0 mmol of tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid and 1.0 mmol of ethyl 2-oximecyanoacetate in 8 mL of DMF. Preactivate with 160 µL of DIC for 3 minutes. Add the resulting mixture to the resin obtained in the previous step and shake for 16 hours. Drain the reaction mixture and wash with DMF 4-5 times and then DCM 5 times.

[0171] Step 5: Cleavage of the linear precursor peptide chain

[0172] 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, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate with 4 volumes of cold MTBE to obtain the crude product. Wash the crude product three times with MTBE and dry it in a vacuum.

[0173] Step 6: Purification and preparation of peptide nuclide ligand

[0174] The crude peptide nuclide ligand obtained in step 5 was dissolved in 20% acetonitrile-water solution, filtered through a 0.45 μm membrane, and separated using a reversed-phase high-performance liquid chromatography system with buffers A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column. During purification, the detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 30-50% acetonitrile in 40 minutes. Product-related fractions were collected and, after HPLC analysis of purity, fractions exceeding 95% were pooled and lyophilized to obtain the pure peptide nuclide ligand.

[0175] Step 6: Detection and Characterization Methods

[0176] The purity of the peptide nuclide ligand in step 5 was confirmed by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry to confirm the compound's C-terminal lysine side chain coupling with DOTA. Figure 3 、 Figure 4 shown.

[0177] Example 4 Preparation of Compound II

[0178]

[0179] Compound II

[0180] 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. 68Ga labeling. Calculate the amount of Compound 2 to be added according to the activity, mix the Compound 2 solution with the eluent, use a pH = 7 metal-free 1M sodium acetate buffer to adjust the reaction system to pH 3 - 4. Set the reaction temperature at 95 °C and the reaction time at 10 min. After the reaction, purify the reaction solution with a pre-activated C18 column to obtain the final product Compound II. First, remove the unlabeled 68 Ga ions with pure water, and then elute with an ethanol solution to obtain Compound II. The eluent is diluted with physiological saline and sterile filtered to obtain an injection of Compound II for experimental use.

[0181] Other polypeptide radionuclide ligands and polypeptide radionuclide probes of the present invention can be synthesized by referring to the synthesis methods of Examples 1 - 4.

[0182] Test Example 1 SPR test for the affinity (KD) of the polypeptide radionuclide ligand and FGFR2IIIC

[0183] 1. Experimental materials and methods

[0184] (1) Experimental materials

[0185] The experimental materials are shown in Table 2.

[0186] Table 2 Experimental materials

[0187]

[0188] (2) Experimental methods

[0189] Use Biacore T200 to test the affinity of the polypeptide with FGFR2IIIb and FGFR2IIIc. Use a ProteinA chip to capture about 1800 RU and 1300 RU of FGFR2IIIb and FGFR2IIIc proteins at 25 °C. Use 1×HBS-EP+, pH 7.4 as the running buffer, and perform the binding experiment at 25 °C. The flow rate of the polypeptide analysis sample is 30 μL / min, Association 120 s, dissociation 600 s. Select single-cycle multi-concentration to detect the binding of the polypeptide sample to FGFR2IIIb and FGFR2IIIc proteins. Use Gly-HCl pH = 1.5, 30 μL / min, 30 s for chip regeneration, and fit the data with a 1:1 binding model.

[0190] 2. Experimental results

[0191] By SPR single-cycle kinetic analysis, the polypeptide radionuclide ligand of the present invention has a good binding affinity with FGFR2IIIc. The binding affinities of Compound 1 and Compound 2 with FGFR2IIIc are shown in Table 3.

[0192] Table 3 Affinity of Compounds for Binding to FGFR2IIIc

[0193]

[0194] Test Example 2 PET-CT Images of Tumor Animal Models

[0195] Experimental procedure: Operate according to the operating procedures of Micro-PET / CT. The probe obtained after drug labeling in the steps of the foregoing examples was diluted with physiological saline to 1 mCi / mL, and an appropriate amount was drawn for injection. For the randomly grouped mice, the tumor-bearing mice were anesthetized with isoflurane. After the righting reflex of the tumor-bearing mice disappeared, the probe was injected into the tail vein. PET scanning and whole-body CT scanning were performed 30 min after injection. The uptake in the tumor region was quantified, and the uptake in the contralateral muscle, heart and its contents, and liver were used as controls. <{

[0196] The experimental results are shown in Figure 5 、 Figure 6 , Figure 5 and Figure 6 are the maximum density projection images of Micro PET of H716 tumor-bearing mice, KATO III tumor-bearing mice, and SNU-16 tumor-bearing mice 30 min after intravenous injection of Compound I and Compound II into mice, respectively. As can be seen from Figure 5 and Figure 6 , at the time point of image acquisition, the tumors were clearly visible with good tissue contrast. Moreover, the tumor uptake of Compound II was higher than that of Compound I. Both Compound II and Compound I had obvious tumor targeting and could be used as potential molecules for the diagnosis and treatment of tumors with high expression of FGFR2IIIC. Compound II may have better effects.

[0197] Test Example 3 Cell Uptake and Inhibition Experiments

[0198] Experimental procedure: H716 cells, KATO III cells, and SNU-16 cells were respectively plated in 6-well plates and cultured in an incubator. After overnight culture, the culture medium was aspirated and washed one or two times. 68 The probe labeled with 68After incubation with the Ga-labeled probe at 37 °C for 15 min, 30 min, 60 min, and 120 min, the cell uptake was blocked with the unlabeled ligand compound for 60 min. After the incubation, the incubation solution was aspirated, and the cells were washed three times with pre-cooled phosphate buffer at 4 °C to terminate the cell uptake. All cells were lysed with the lysis solution, and the lysate was collected for radioactivity counting. After decay correction of the radioactivity counting and conversion with reference to the standard, the probe cell uptake rate was calculated, and the uptake of the probe by the cells and the change of the uptake rate over time were analyzed to judge the binding specificity of the probe and the probe distribution ability. The experimental results are as Figures 7 - 9 shown.

[0199] According to the results of the cell uptake experiment, it can be seen that Compound II has a high uptake in H716 cells, KATO III cells, and SNU-16 cells in vitro, and shows an upward trend with the increase of the incubation time. The significant difference in cell uptake between the group without the unlabeled ligand compound and the group with the unlabeled ligand compound indicates that Compound II specifically binds to FGFR2, suggesting that the novel FGFR2-targeted probe maintains comparable specificity with the ligand compound while accommodating more types of radionuclide labeling.

[0200] As mentioned above, it is only a preferred embodiment of the present invention, and thus cannot limit the scope of implementation of the present invention accordingly. That is, equivalent changes and modifications made according to the scope of the present invention patent and the content of the specification should still fall within the scope covered by the present invention.

Claims

1. A polypeptide-radionuclide ligand targeting FGFR2, characterized in that, The polypeptide radionuclide ligand has the following structure: Compound 2.

2. A polypeptide-radionuclide probe targeting FGFR2, characterized in that, The polypeptide radionuclide probe comprises the polypeptide radionuclide ligand according to claim 1 labeled with a radionuclide.

3. The polypeptide radionuclide probe according to claim 2, wherein The radionuclide is selected from 68 Ga, 64 Cu, 18 F, 99m Tc, 123 I, 125 I, 124 I, 177 Lu, 131 I, 67 Cu, 225 one of Ac.

4. A pharmaceutical composition, characterized in that, Comprising the polypeptide radionuclide ligand according to claim 1 or the polypeptide radionuclide probe according to claim 2 or 3, and a pharmaceutically acceptable carrier or excipient.

5. Use of the polypeptide radionuclide probe according to claim 2 or 3, or a pharmaceutical composition comprising the polypeptide radionuclide probe according to claim 2 or 3 and a pharmaceutically acceptable carrier or excipient, in the preparation of a diagnostic reagent for FGFR2-expressing tumors; the FGFR2-expressing tumors are selected from any one or more of colorectal adenocarcinoma, gastric cancer, endometrial cancer, breast cancer, and ovarian cancer.

6. Use of the polypeptide radionuclide probe according to claim 2 or 3, or a pharmaceutical composition comprising the polypeptide radionuclide probe according to claim 2 or 3 and a pharmaceutically acceptable carrier or excipient, in the preparation of a tracer reagent for FGFR2-expressing tumors; the FGFR2-expressing tumors are selected from any one or more of colorectal adenocarcinoma, gastric cancer, endometrial cancer, breast cancer, and ovarian cancer.

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