Cyclic peptide ligand and nuclide labeled compound targeting Nectin-4 and application of cyclic peptide ligand and nuclide labeled compound

Abstract

The invention belongs to the field of biological medicine, and relates to a Nectin-4-targeted cyclic peptide ligand, a nuclide labeled compound and application. The ligand has a structure as shown in a formula I. The nuclide labeled compound provided by the invention has high tumor uptake, high tumor-muscle uptake ratio and good in-vivo metabolism performance, and has great clinical application prospects. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of nuclear medicine, and in particular, relates to a cyclic peptide ligand targeting nectin-4, a nuclide-labeled compound based on the ligand, and applications thereof. Background Art

[0002] Nectin-4 (gene name PVRL4, poliovirus receptor 4) is a type I transmembrane immunoglobulin-like cell-to-cell adhesion molecule that belongs to the Nectin family of Ig superfamily proteins. It plays a role in the formation of cell-to-cell adhesion junctions, mediation of cell-to-cell junctions, and regulation of cell proliferation, differentiation, migration and other physiological processes. Nectin-4 is limitedly expressed in normal adult tissues, but abnormally high expression has been detected in a variety of cancer samples, especially in bladder cancer, breast cancer and lung cancer. More than half of the patient specimens showed moderate to strong staining of Nectin-4, making it a reliable marker for a variety of cancers.

[0003] Antibody-drug conjugates (ADCs) targeting nectin-4 can effectively treat malignant tumors such as breast cancer and urothelial carcinoma. For example, Enfortumab Vedotin is a new, fully humanized ADC, which is composed of an anti-nectin-4 antibody coupled to a highly efficient microtubule inhibitor, MMAE. It can bind to nectin-4 expressed on the cell surface with high affinity, and then be endocytosed and hydrolyzed by the cell to release MMAE to destroy tubulin polymerization, leading to mitotic arrest and inducing cell death. It was approved for marketing by the FDA in 2019 and is mainly used to treat locally advanced or metastatic urothelial carcinoma.

[0004] In the field of nuclear medicine, the development of nuclear medicine probes targeting Nectin-4 has important clinical significance for the diagnosis, precise positioning and efficacy evaluation of Nectin-4 positive solid tumors. Currently reported Nectin-4 targeted nuclear medicine diagnostic and therapeutic agents include radionuclide labeled Nectin-4 monoclonal antibodies, but these imaging agents have defects such as large molecular weight, long in vivo circulation time, high radiation dose, poor cell membrane penetration, and low target / background ratio. Therefore, the development of new Nectin-4 targeted probes is of great significance for improving the diagnosis and treatment effects. Summary of the invention

[0005] The purpose of the present invention is to provide a novel nectin-4 targeting ligand and radionuclide labeled compound and application thereof.

[0006] In order to achieve the above object, the first aspect of the present invention provides a cyclic peptide ligand targeting Nectin-4, wherein the ligand has a structure shown in Formula I:

[0007]

[0008] Wherein, R is selected from the group shown in formula II, formula III or formula IV:

[0009]

[0010] In formula III, m is an integer of 1-5, preferably 1, 2 or 3; in formula IV, n is an integer of 3-7, preferably 4, 5 or 6;

[0011] R1 is selected from the group represented by formula V or the group represented by formula VI:

[0012]

[0013] According to a preferred embodiment of the present invention, R is a group represented by formula II.

[0014] Specifically preferably, the ligand is compound 1, compound 2 or compound 3 (corresponding to compounds N230A, N231A, and N232A herein, respectively):

[0015] Compound 1: R is a group represented by formula II, and R1 is a group represented by formula V;

[0016] Compound 2: R is a group represented by formula III, and R1 is a group represented by formula V;

[0017] Compound 3: R is a group represented by formula IV, and R1 is a group represented by formula V;

[0018] The above compounds of the present invention can be prepared by various methods known in the art.

[0019] The second aspect of the present invention provides a radionuclide-labeled nectin-4 targeting compound, which is the above-mentioned cyclic peptide ligand labeled with a radionuclide.

[0020] According to the present invention, the radionuclide may be a diagnostic radionuclide or a therapeutic radionuclide.

[0021] According to some preferred embodiments of the present invention, the diagnostic radionuclide is 18 F. 68 Ga, 64 Cu, 86 Y. 90 Y. 89 Zr, 111 In, 99m Tc, 11 C. 123 I. 125 I and 124 At least one of I, preferably 18F mark, the specific mark form is Al 18 F.

[0022] According to some preferred embodiments of the present invention, the therapeutic radionuclide is 177 Lu, 125 I. 131 I. 211 At 111 In, 153 Sm, 186 Re, 188 Re, 67 Cu, 212 Pb, 225 Ac, 213 Bi, 212 Bihe 212 At least one of Pb.

[0023] The compound of the present invention can be prepared by labeling the ligand with a radioactive nuclide. Specifically, the ligand is dissolved in a radioactive labeling buffer, and then different radionuclides are added to react to obtain the corresponding compound.

[0024] The raw materials used in each step of the method for preparing the ligand and nuclide-labeled compound of the present invention can be commercially available or prepared by conventional organic synthesis methods.

[0025] According to a preferred embodiment of the present invention, the structure of the radionuclide-labeled nectin-4 targeting compound is shown in Formula A:

[0026]

[0027] The compound of formula A can be Figure 1-1 and Figure 1-2 Prepared by the synthetic route shown. Reaction conditions: (a) 20% piperidine in DMF solution, Fmoc-R-OH, HBTU, HOBt and DIPEA in DMF solution; (b) 20% piperidine in DMF solution, DOTA-TBu, HBTU, HOBt and DIPEA in DMF solution; (c) trifluoroacetic acid, water and triisopropylsilane; (d) pH = 8 containing 20% ​​acetonitrile buffer (20mM NH4HCO3, 5mM EDTA), react at 30°C for 1h, then add 10 times TATA equivalent of cysteine ​​to terminate the reaction. Figure 1-2 The reaction conditions are as follows: (e) Na 18 F, potassium hydrogen phthalate, AlCl3, 100℃.

[0028] The third aspect of the present invention provides a pharmaceutical composition comprising the above-mentioned cyclic peptide ligand or the above-mentioned compound, and pharmaceutically acceptable excipients.

[0029] The pharmaceutically acceptable excipients refer to non-toxic solid, semi-solid or liquid fillers, diluents, capsule materials or any type of formulation aids.

[0030] The fourth aspect of the present invention provides the use of the above-mentioned cyclic peptide ligand and / or compound in the preparation of diagnostic drugs or therapeutic drugs for diseases mediated by Nectin-4.

[0031] The nectin-4 mediated diseases include but are not limited to at least one of bladder cancer, lung cancer, breast cancer, prostate cancer, urothelial cancer, gastric cancer, cervical cancer, ovarian cancer, pancreatic cancer, hepatocellular carcinoma, thyroid cancer, melanoma and skin cancer.

[0032] The radionuclide labeled compound of the present invention has high tumor uptake and high tumor to muscle uptake ratio, and good in vivo metabolic performance. 18 F-N230A had the highest tumor uptake value and target to non-target ratio. At 60 minutes after injection, Al 18 The SUVmax of F-N230A in the tumor was 0.60±0.07, and the ratios of tumor to muscle and liver were 10.94±0.92 and 3.25±0.24, respectively. At 120 minutes, the SUVmax in the tumor was still 0.52±0.04, the ratio of tumor to muscle reached 11.80±2.14, and the ratio of tumor to liver was 3.40±0.31. In particular, Al 18 F-N230A is a better probe than the one previously developed by the applicant 68 Ga-N188 has a greater advantage. At 60 minutes and 120 minutes after injection, Al 18 The intratumor uptake of F-N230A was 68 The uptake of Ga-N188 is 1.58 and 1.73 times that of Al 18 The tumor-to-muscle and tumor-to-liver ratios of F-N230A were also significantly higher than those of 68 Ga-N188. At 180 minutes of injection, Al 18 F-N230A can still obtain clear PET / CT images, which can effectively make up for 68 Ga-N188 has better application prospects because it overcomes the shortcomings of delayed imaging.

[0033] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The above and other objects, features and advantages of the present invention will become more apparent through a more detailed description of exemplary embodiments of the present invention with reference to the accompanying drawings.

[0035] Figure 1-1 and 1-2 Shows Al 18 The overall synthetic route of the F-labeled necitn-4 targeting probe.

[0036] Figures 2-1 to 2-6 They are the mass spectra of ligands N230, N231, N232, N230A, N231A, and N232A, respectively.

[0037] Figure 3 For Al 18 MicroPET / CT images of F-labeled probe in SW780 tumor-bearing mice (MIP images, n=4).

[0038] Figure 4 For Al 18 F-N230A and 68 MicroPET / CT images of Ga-N188 in SW780 tumor-bearing mice (MIP images, n=4). DETAILED DESCRIPTION

[0039] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0040] Example

[0041] Figure 1-1 and 1-2 It shows that Al 18 The overall synthetic route of the F-labeled necitn-4 targeting probe. Reaction conditions: (a) 20% piperidine in DMF solution, Fmoc-R-OH, HBTU, HOBt and DIPEA in DMF solution; (b) 20% piperidine in DMF solution, DOTA-TBu, HBTU, HOBt and DIPEA in DMF solution; (c) trifluoroacetic acid, water and triisopropylsilane; (d) pH = 8 containing 20% ​​acetonitrile buffer (20mM NH4HCO3, 5mM EDTA), react at 30℃ for 1h, then add 10 times TATA equivalent of cysteine ​​to terminate the reaction; (e) Na 18 F, potassium hydrogen phthalate, AlCl3, 100°C. The specific steps are as follows:

[0042] Ligand synthesis

[0043] Linear peptide resins Fmoc-Cys-Pro-1Nal-(D)Asp-Cys-Met-Arg-Asp-Trp-Ser-Thr-Pro-HyP-Trp-Cys-Resin (resin) and Fmoc-Cys-Pro-1Nal-(D)Asp-Cys-Met-Arg-Asp-Trp-Ser-Thr-Pro-HyP-Trp-Cys-AM-Resin (amino resin) were commissioned to Shanghai Chupeptide Biotechnology Co., Ltd. for synthesis.

[0044] Take 70 mg of linear peptide resin or linear peptide amino resin in a 10 mL solid phase synthesis tube, add 2 mL of dichloromethane (DCM) to swell, 5 minutes each time, repeat three times. Then wash three times with 2 mL of DMF, 5 minutes each time. Use 2 mL of DMF solution (v / v) containing 20% ​​piperidine to react with the resin for 2 minutes, 10 minutes, and 10 minutes respectively to remove the amino protecting group Fmoc. Then use 2 mL of DMF to wash 3-5 times, 2 minutes each time. The coupling of linker R (N230 / N230A without linker structure, N231 / N231A: 4-aminomethylbenzoic acid, N232 / N232A: sarcosine) and the polypeptide was carried out according to the standard Fmoc solid phase synthesis method: 3 times the chemical amount of Fmoc-R-OH relative to the resin (8.4 μmol) was dissolved in 3 mL DMF solution containing 20 μL DIPEA and 11.5 mg HBTU, added to the solid phase synthesis tube, and reacted for 1 hour under electromagnetic stirring to obtain compound 7. Subsequently, 43 mg NOTA-TBu was dissolved in 3 mL DMF solution containing 20 μL DIPEA and 3.2 mg HBTU, added to the solid phase synthesis tube, and reacted at room temperature for 1 hour under electromagnetic stirring to obtain compound 8. Add 5 mL of TFA / TIPS / water (95:2.5:2.5, v / v / v) solution to the solid phase synthesis tube, stir for 2 hours and collect the filtrate, dissociate compound 8 from the resin, and remove the tert-butyl ester, trityl and other protecting groups. Wash the resin with 2 mL of TFA, collect all the filtrate, remove TFA under reduced pressure, and prepare compound 9 or 9A by reverse HPLC. Take 4.5 mg of compound 9 or 9A and dissolve it in 5 mL of cyclization reaction buffer (pH=8.0, 20 mM NH4HCO3, 5 mM EDTA, 20% acetonitrile, v / v), add 1.3 mg of TATA (1,3,5-triacryloyl hexa-1,3,5-triazine), and react at 30°C for 1 hour. Finally, add 100 mg of L-Cys and react at 30°C for 10 minutes to terminate the cyclization reaction. Separate the product by reverse HPLC and obtain a yellow powder (compound 10 or 10A) after freeze-drying. The probe structure was identified by mass spectrometry. Figures 2-1 to 2-6The mass spectra of N230, N231, N232, N230A, N231A, and N232A are shown respectively. N230, molecular formula C 109 H 146 N 26 O 31 S4, theoretical value 1222.99 [M+2H] 2+ , measured value 1222.88; N231, molecular formula C 125 H 160 N 28 O 33 S4, theoretical value 1356.04 [M+2H] 2+ , measured value 1356.02; N232, molecular formula C 124 H 171 N 31 O 36 S4, theoretical value 1400.58 [M+2H] 2+ , measured value 1400.07; N230A, molecular formula C 109 H 147 N 27 O 30 S4, theoretical value 1222.49 [M+2H] 2+ , measured value 1222.48; N231A, molecular formula C 125 H 161 N 29 O 30 S4, theoretical value 1355.54 [M+2H] 2+ , measured value 1355.52; N232A, molecular formula C 124 H 171 N 31 O 36 S4, theoretical value 1400.58 [M+2H] 2+ , the measured value is 1400.56.

[0045] Probe preparation: Al 18 F Marking and Quality Control

[0046] A certain volume of DMSO was added to dissolve the ligand to 40 μg / μL. The ligand was prepared by displacement accelerator using 0.5 mL of saline and activated Sep-pak QMA column (eluted and activated by 10 mL of saline and 10 mL of pure water, and air-dried for later use). 18 F - ions, discard the first 0.1 mL of eluent, and use the remaining 0.4 mL of Na 18The F solution (activity of 3.7-5.6GBq) was collected into a vial with 10μL (400μg) of ligand added, and 7μL of 20mmol / L aluminum chloride solution was added for complexation, 30μL of 0.5mol / L potassium hydrogen phthalate buffer was used to adjust the pH of the reaction system, and 200μL of ethanol was used to dissolve the ligand. The solution in the vial was shaken and sealed, and reacted at 100°C for 10 minutes. After the reaction solution was cooled to room temperature, it was purified by Sep-Pak C18 column. The specific steps are as follows: Use 10mL of anhydrous ethanol and 10mL of pure water to activate the Sep-Pak C18 column, add 5-6mL of pure water to the labeled reaction solution to dilute it and load it on the column, and rinse the C18 column with 10mL of pure water to remove free 18 F - After ionization, the purified labeled product was eluted with 0.5 mL of 80% ethanol solution (v / v), and an appropriate amount of physiological saline was added to the eluate for dilution and use.

[0047] The radiochemical purity of the labeled product was determined by high performance liquid chromatography (HPLC). HPLC analyzer, Shimadzu Corporation, Japan, equipped with LC-20AT pump, analytical column Phenomenex Gemini 5μ100AC-18 (4.6×150 mm), FLOW-COUNT flow radioactivity detector. Quality control conditions: mobile phase: phase A: H2O (0.1% TFA), phase B: acetonitrile (0.1% TFA), flow rate of 1.0 mL / min, gradient: 0-5 min, 10%-90% phase B (0.1% TFA); 5-20 min, 90% phase B (0.1% TFA). 18 The radiochemical purity of the FA1 labeled products was no less than 95%.

[0048] Al 18 MicroPET / CT imaging with F probe

[0049] According to the conventional microPET / CT imaging method, radiolabeled Al 18 F probe (5.6-7.4MBq, 100-200μL) was injected into female BALB / c nude mice bearing SW780 tumors (Nectin-4 positive) via the tail vein (n=4 per group), and static PET / CT images were collected at 1h and 2h after injection (Figure 2). ROI (tumor, muscle, liver and kidney) were circled and the uptake value (SUVmax) was calculated based on the anatomical information provided by CT. The results are shown in Table 1. 18 Among the probes labeled with F, the main metabolic pathways were kidneys, among which Al 18 F-N230A had the highest tumor uptake value and target to non-target ratio. At 60 minutes after injection, Al 18The SUVmax of F-N230A in the tumor was 0.60±0.07, and the ratios of tumor to muscle and liver were 10.94±0.92 and 3.25±0.24, respectively. At 120 minutes, the SUVmax in the tumor was still 0.52±0.04, the ratio of tumor to muscle reached 11.80±2.14, and the ratio of tumor to liver was 3.40±0.31, making it the preferred probe.

[0050]

[0051] 68 Ga-N188 is a necitn-4 targeting probe previously developed by the applicant, and its structure is as follows:

[0052]

[0053] Al 18 F-N230A 68 Ga-N188 has obvious advantages in comparison, such as Figure 3 As shown in Table 1, Al 18 The uptake of F-N230A in tumors was significantly higher than 68 Ga-N188, Al 18 The intake of F-N230A is 68 The uptake of Ga-N188 is 1.58 and 1.73 times that of Al 18 The tumor-to-muscle and tumor-to-liver ratios of F-N230A were also significantly higher than those of 68 Ga-N188. At 180 minutes of injection, Al 18 F-N230A can still obtain clear PET / CT images, which can effectively make up for 68 Disadvantages of Ga-N188 delayed imaging.

[0054] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and changes will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A cyclic peptide ligand targeting nectin-4, characterized in that: The ligand has a structure shown in Formula I: Wherein, R is selected from the group shown in formula II, formula III or formula IV: In formula III, m is an integer of 1-5, and in formula IV, n is an integer of 3-7; R1 is selected from the group represented by formula V or the group represented by formula VI:

2. The cyclic peptide ligand targeting Nectin-4 according to claim 1, wherein m is 1, 2 or 3, and n is 4, 5 or 6.

3. The cyclic peptide ligand targeting Nectin-4 according to claim 1, wherein R is a group represented by formula II.

4. The cyclic peptide ligand targeting Nectin-4 according to claim 1, wherein The ligand is compound 1, compound 2 or compound 3: Compound 1: R is a group represented by formula II, and R1 is a group represented by formula V; Compound 2: R is a group represented by formula III, and R1 is a group represented by formula V; Compound 3: R is a group represented by formula IV, and R1 is a group represented by formula V.

5. A radionuclide-labeled nectin-4 targeting compound, which is a cyclic peptide ligand according to any one of claims 1 to 4 labeled with a radionuclide.

6. The Nectin-4 targeting compound according to claim 5, wherein The radionuclide is a diagnostic radionuclide or a therapeutic radionuclide.

7. The Nectin-4 targeting compound according to claim 6, wherein The diagnostic radionuclide is 18 F. 68 Ga, 64 Cu, 86 Y. 90 Y. 89 Zr, 111 In, 99m Tc, 11 C. 123 I. 125 I and 124 At least one of I, preferably Al 18 F; The therapeutic radionuclide is 177 Lu, 125 I. 131 I. 211 At 111 In, 153 Sm, 186 Re, 188 Re, 67 Cu, 212 Pb, 225 Ac, 213 Bi, 212 Bihe 212 At least one of Pb.

8. A pharmaceutical composition comprising the cyclic peptide ligand according to any one of claims 1 to 4 or the compound according to any one of claims 5 to 7, and a pharmaceutically acceptable excipient.

9. Use of the cyclic peptide ligand according to any one of claims 1 to 4 and / or the compound according to any one of claims 5 to 7 in the preparation of diagnostic drugs or therapeutic drugs for diseases mediated by nectin-4.

10. The use according to claim 9, wherein: The nectin-4 mediated disease is at least one of bladder cancer, lung cancer, breast cancer, prostate cancer, urothelial cancer, gastric cancer, cervical cancer, ovarian cancer, pancreatic cancer, hepatocellular carcinoma, thyroid cancer, melanoma and skin cancer.

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