A cyclic peptide ligand and radionuclide-labeled compound targeting nectin-4 and its application

Abstract

The present invention belongs to the field of biomedicine and relates to a cyclic peptide ligand targeting nectin-4, a radionuclide-labeled compound, and their applications. The ligand has the structure shown in Formula I. The radionuclide-labeled compound of the present invention has high tumor uptake and a high tumor-to-muscle uptake ratio, as well as good in vivo metabolic properties, 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 (PVRL4, poliovirus receptor 4) is a type I transmembrane immunoglobulin-like intercellular adhesion molecule that belongs to the nectin family of Ig superfamily proteins. It plays a role in the formation of intercellular adhesion junctions, mediating intercellular connections, and regulating physiological processes such as cell proliferation, differentiation, and migration. Nectin-4 expression is limited in normal adult tissues, but abnormally high expression has been detected in various cancer samples, particularly in bladder cancer, breast cancer, and lung cancer. Over half of patient specimens show moderate to strong staining of nectin-4, making it a reliable marker for various cancers.

[0003] Nectin-4-targeting antibody-drug conjugates (ADCs) are effective in treating malignancies such as breast cancer and urothelial carcinoma. For example, Enfortumab Vedotin is a novel, fully humanized ADC consisting of an anti-nectin-4 antibody conjugated to the highly potent microtubule inhibitor MMAE. It binds to nectin-4 expressed on the cell surface with high affinity, is subsequently internalized and hydrolyzed to release MMAE, disrupting microtubule polymerization, leading to mitotic arrest and inducing cell death. It was approved for marketing by the FDA in 2019 and is primarily 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 implications for the diagnosis, precise localization, and therapeutic efficacy assessment of nectin-4-positive solid tumors. Currently reported nectin-4-targeted nuclear medicine diagnostic and therapeutic agents include radionuclide-labeled nectin-4 monoclonal antibodies. However, these imaging agents suffer from limitations such as large molecular weight, long circulation time, high radiation dose, poor cell membrane penetration, and low target / background ratios. Therefore, the development of novel nectin-4-targeted probes is crucial for improving diagnostic and therapeutic outcomes. Summary of the Invention

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

[0006] In order to achieve the above objectives, 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 represented by 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-mentioned 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 I at least one, 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℃ 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 auxiliary.

[0030] A 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, and Al 18 The intratumoral uptake of F-N230A is 68 The uptake of Ga-N188 is 1.58 times 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 compensate 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 These are the mass spectra of ligands N230, N231, N232, N230A, N231A, and N232A, respectively.

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

[0038] Figure 4 Al 18 F-N230A and 68 MicroPET / CT imaging 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 Shows Al 18 The overall synthetic route of the F-labeled necitn-4 targeting probe. Reaction conditions: (a) 20% piperidine in DMF, Fmoc-R-OH, HBTU, HOBt, and DIPEA in DMF; (b) 20% piperidine in DMF, DOTA-TBu, HBTU, HOBt, and DIPEA in DMF; (c) trifluoroacetic acid, water, and triisopropylsilane; (d) pH = 8 containing 20% ​​acetonitrile buffer (20mM NH4HCO3, 5mM EDTA), react at 30℃ for 1h, and then add 10 times the equivalent of TATA to terminate the reaction; (e) Na 18 F, potassium hydrogen phthalate, AlCl3, 100℃. 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 and add 2 mL of dichloromethane (DCM) to swell for 5 minutes each time, repeat three times. Then wash with 2 mL of DMF three times, 5 minutes each time. Use 2 mL of DMF solution containing 20% ​​piperidine (v / v) 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 the linker R (N230 / N230A: no linker structure, N231 / N231A: 4-aminomethylbenzoic acid, N232 / N232A: sarcosine) to the peptide was performed according to the standard Fmoc solid-phase synthesis method: 3 times the stoichiometric amount of Fmoc-R-OH relative to the resin (8.4 μmol) was dissolved in 3 mL of DMF containing 20 μL of DIPEA and 11.5 mg of HBTU, added to a solid-phase synthesis tube, and reacted under electromagnetic stirring for 1 hour to obtain compound 7. Subsequently, 43 mg of NOTA-TBu was dissolved in 3 mL of DMF containing 20 μL of DIPEA and 3.2 mg of HBTU, added to a solid-phase synthesis tube, and reacted at room temperature under electromagnetic stirring for 1 hour to obtain compound 8. A solid-phase synthesis tube was filled with 5 mL of a TFA / TIPS / water (95:2.5:2.5, v / v / v) solution. After stirring for 2 hours, the filtrate was collected to cleave compound 8 from the resin and remove the tert-butyl ester and trityl protecting groups. The resin was washed with 2 mL of TFA, and the filtrate was collected. After removing the TFA under reduced pressure, compound 9 or 9A was prepared by reverse HPLC. 4.5 mg of compound 9 or 9A was dissolved in 5 mL of cyclization buffer (pH 8.0, 20 mM NH4HCO3, 5 mM EDTA, 20% acetonitrile, v / v), and 1.3 mg of TATA (1,3,5-triacryloylhexa-1,3,5-triazine) was added. The reaction was allowed to react at 30°C for 1 hour. Finally, 100 mg of L-Cys was added, and the reaction was allowed to terminate at 30°C for 10 minutes. The product was isolated by reverse HPLC and lyophilized to yield a yellow powder (compound 10 or 10A). 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+ , found value 1222.48; N231A, molecular formula C 125 H 161 N 29 O 30 S4, theoretical value 1355.54 [M+2H] 2+ , found 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 labeling 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 normal saline and activated Sep-pak QMA cartridge (eluted and activated with 10 mL of normal 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: 10mL of anhydrous ethanol and 10mL of pure water were used to activate the Sep-Pak C18 column, 5-6mL of pure water was added to the labeled reaction solution to dilute it, and then the column was loaded, and the C18 column was rinsed 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 to dilute it for later use.

[0047] The radiochemical purity of the labeled product was determined by high-performance liquid chromatography (HPLC). The HPLC analyzer was a Shimadzu company in Japan, equipped with an LC-20AT pump, a Phenomenex Gemini 5μ100AC-18 analytical column (4.6×150 mm), and a FLOW-COUNT flow radioactivity detector. Quality control conditions were as follows: Mobile phase: Phase A: H2O (0.1% TFA), Phase B: acetonitrile (0.1% TFA), flow rate: 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 The F probe (5.6-7.4 MBq, 100-200 μL) was injected into female BALB / c nude mice bearing SW780 tumors (Nectin-4 positive) via the tail vein (n=4 per group). Static PET / CT images were acquired 1 and 2 hours after injection (Figure 2). ROIs (tumor, muscle, liver, and kidney) were circled based on the anatomical information provided by CT, and the uptake values ​​(SUVmax) were calculated. The results are shown in Table 1. 18 Among the F-labeled probes, 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 is in 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 that in 68 Ga-N188, 60 minutes and 120 minutes after injection, Al 18 The intake of F-N230A is 68 The uptake of Ga-N188 is 1.58 times 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 compensate for 68 Disadvantages of Ga-N188 delayed imaging.

[0054] While various embodiments of the present invention have been described above, the above description is intended to be illustrative, not exhaustive, and not limited to the disclosed embodiments. Many modifications and variations 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: Formula I Wherein, R is selected from the group represented by Formula II, Formula III or Formula IV: Formula II Formula III Formula IV In formula III, m is 2, and in formula IV, n is 5; R1 is a group represented by formula V: Formula V.

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

3. A radionuclide-labeled nectin-4 targeting compound, wherein the compound is a cyclic peptide ligand according to any one of claims 1 to 2 labeled with a radionuclide; the radionuclide is a diagnostic radionuclide or a therapeutic radionuclide; The diagnostic radionuclide is 18 F. 68 Ga, 64 Cu, 86 Y. 90 Y. 89 Zr, 111 In and 99m At least one of Tc; The therapeutic radionuclide is 177 Lu, 211 At 111 In, 153 Sm, 186 Re、 188 Re、 67 Cu, 225 Ac, 213 Bi, 212 Bihe 212 At least one of Pb.

4. The nectin-4 targeting compound according to claim 3, wherein The diagnostic radionuclide is Al 18 F.

5. A pharmaceutical composition comprising the compound according to any one of claims 3 to 4, and pharmaceutically acceptable excipients.

6. Use of the compound according to any one of claims 3 to 4 in the preparation of a diagnostic drug or a therapeutic drug for a disease mediated by Nectin-4; the disease mediated by Nectin-4 is bladder cancer.

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