WO2023098072A1 - Nectin-4-targeted bicyclic peptide nuclide ligand and probe - Google Patents

Abstract

Provided are a Nectin-4-targeted bicyclic peptide nuclide ligand and probe. The ligand has a structure represented by formula I, where X is Lys or Arg; and R is a group represented by formula II, formula III, formula IV or formula V. The probe has high tumor uptake and a high uptake ratio of tumor to muscle, and has good clinical application prospects.

Description

Bicyclic peptide nuclide ligands and probes targeting Nectin-4 technical field

The invention belongs to the field of nuclear medicine, and in particular relates to a bicyclic peptide nuclide ligand targeting Nectin-4 and a bicyclic peptide nuclide probe targeting Nectin-4 formed by the ligand.

Background technique

Cell Adhesion Molecular (CAM) is a class of cell membrane surface glycoprotein molecules that mediate cell-cell and cell-extracellular matrix interactions, and are involved in cell polarization, differentiation, adhesion, proliferation, migration, etc. It plays an important role in physiological processes and is also involved in cell signal transduction processes. Therefore, the abnormal expression of CAM can reflect the pathological changes of the body to a certain extent. CAM can be roughly divided into selectin (Selectin) family, integrin family (Integrin), cadherin family (Cadherin) and immunoglobulin superfamily (Immunoglobulin superfamily, IgSF). The expression of most CAMs in the body is not specific, and there is no significant difference in the expression of cells in various tissues. However, the Nectin-4 protein of the IgSF family is only expressed in embryos, placenta, and mature epithelial tissues in normal humans, but it is specifically overexpressed on the surface of various cancer cells such as breast cancer and bladder cancer. Studies have confirmed that Nectin-4 protein is involved in It mediates cancer cell apoptosis resistance, new blood vessels, lymphatic vessels and other cancer progression and metastasis processes, and its expression level in malignant tumors is closely related to the malignancy of the disease and the survival of patients. Therefore, Nectin-4 protein is an ideal biomarker for high-sensitivity and high-specificity cancer lesion localization imaging and targeted therapy.

In the field of cancer drug development, Enfortumab Vedotin, a humanized monoclonal antibody targeting Nectin-4 and a microtubule-disrupting agent monomethyl auristatin E (MMAE) antibody conjugate drug, was launched in December 2019. Approved by the FDA as a drug for the treatment of advanced metastatic bladder cancer. However, in the fields of early diagnosis of cancer and precise positioning of metastases in recurrent patients, the development of imaging reagents targeting Nectin-4 is still in the exploratory stage. Compared with traditional imaging modalities, nuclear medicine imaging methods with extremely high sensitivity can reflect the function and metabolism of tissues and organs in a specific, accurate and non-invasive way by cooperating with highly specific molecular probes. Therefore, the development of Nectin-targeted The nuclear medicine probe of 4 has important clinical significance for the diagnosis and precise localization of bladder cancer.

The currently reported Nectin-4-targeted nuclear medicine diagnosis and treatment reagents are radionuclide ⁸⁹ Zr and ¹⁸ F-labeled Nectin-4 monoclonal antibody (AGS-22M6), but the monoclonal antibody is limited by its large molecular weight and metabolism in vivo. Due to defects such as long time, high blood background, and poor cell membrane penetration, the direct application of monoclonal antibodies to clinical routine molecular imaging reagents has certain limitations. Transformation has a natural advantage.

Contents of the invention

The purpose of the present invention is to provide bicyclic peptide nuclide ligands and probes targeting Nectin-4. The inventors of the present invention relied on the phage display technology to obtain the bicyclic peptide sequence targeting Nectin-4, and carried out structural optimization, functional labeling, A series of evaluations, such as imaging, obtained a small molecule diagnostic reagent for nuclear medicine that can be used for Nectin-4 targeting.

In order to achieve the above object, the first aspect of the present invention provides a bicyclic peptide nuclide ligand targeting Nectin-4, which has the structure shown in formula I:

in,

X is Lys or Arg;

R is a group shown in formula II, formula III, formula IV or formula V,

Wherein, a is an integer of 1-5, preferably an integer of 1-3, b is an integer of 3-8, preferably an integer of 4-6, c is an integer of 1-5, preferably an integer of 1-3.

Further preferably, R is a group represented by formula II, formula VI, formula VII or formula VIII,

More preferably, the bicyclic peptide nuclide ligand is a compound represented by formula (1), formula (2), formula (3), formula (4) or formula (5), that is, the application number DXJ187, DXJ188, DXJ194 , DXJ196, DXJ204 compounds:

The bicyclic peptide nuclide ligand targeting Nectin-4 of the present invention can be synthesized through the synthetic route shown in FIG. 2 or FIG. 4 .

Specifically, the reaction conditions of each step of the synthetic route in Figure 2 are as follows: (a) 20% piperidine, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid tri-tert DMF solution of butyl ester; (b) trifluoroacetic acid, water and triisopropylsilane; (c) 20% acetonitrile buffer (20mM NH ₄ HCO ₃ , 5mM EDTA) at pH=8, 30°C, after 1h Add 10 times TATA equivalent of cysteine.

The reaction conditions of each step of the synthetic route of Fig. 4 are as follows: Reaction conditions: (a) the DMF solution of 20% piperidine, the DMF solution of Fmoc-amino acid, HBTU, HOBt and EIPEA; (b) 20% piperidine, 1,4, 7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid tri-tert-butyl ester (DOTA) in DMF; (c) trifluoroacetic acid, water and triisopropylsilane; (d ) pH=8 containing 20% acetonitrile buffer solution (20mM NH ₄ HCO ₃ , 5mM EDTA), at 30°C, after 2h, 10 times of TATA equivalent of Cys was added.

The raw materials used in each step are commercially available or prepared by conventional organic synthesis methods.

The second aspect of the present invention provides a Nectin-4-targeting bicyclic peptide nuclide probe, which is the above-mentioned Nectin-4-targeting bicyclic peptide nuclide ligand labeled with a radionuclide.

The bicyclic peptide nuclide probe of the present invention can be prepared by labeling the bicyclic peptide nuclide ligand with a radionuclide, specifically, dissolving the ligand in a radioactive labeling buffer, and then adding different radionuclides to carry out reaction to obtain the corresponding molecular probes.

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

According to some preferred embodiments of the present invention, the diagnostic radionuclides are ⁶⁸ Ga, ⁶⁴ Cu, ¹⁸ F, ⁸⁶ Y, 90 Y, ⁸⁹ Zr, ¹¹¹ In, ^99m Tc, ¹¹ C, ^{123 I} , ¹²⁵ I and ¹²⁴ At least one of I.

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

The probe of the present invention has higher tumor uptake and higher ratio of tumor to muscle uptake, good metabolic performance in vivo, and good clinical application prospect.

Other features and advantages of the present invention will be described in detail in the detailed description that follows.

Description of drawings

The above and other objects, features and advantages of the present invention will become more apparent by describing the exemplary embodiments of the present invention in more detail with reference to the accompanying drawings.

Figure 1 shows the structural formula of DXJ187.

Figure 2 shows the general route for the preparation of DXJ187.

Figure 3 shows the general structural formula (A) and specific structural formula (B) of DXJ188, DXJ194, DXJ196, DXJ204.

Figure 4 shows the general preparation route of DXJ188, DXJ194, DXJ196, DXJ204.

Figures 5-1 to 5-5 show the mass spectra of DXJ187, DXJ188, DXJ194, DXJ196, and DXJ204, respectively.

Figure 6 shows the PET images of ⁶⁸ Ga-labeled DXJ187, DXJ188, DXJ194, DXJ196, and DXJ204 tumor-bearing mice at 1 h and 2 h after injection.

Figure 7 shows the SPECT imaging and biodistribution of ¹⁷⁷ Lu-DXJ188 in tumor-bearing nude mice. The left picture is the SPECT/CT image of the tumor-bearing mice in the experimental group injected with ¹⁷⁷ Lu-DXJ188 for 2 hours, and the right picture is the SPECT/CT image of the tumor-bearing mice in the inhibition group co-injected with cold ligand and ¹⁷⁷ Lu-DXJ188 for 2 hours.

Detailed ways

Preferred embodiments of the present invention will be described in more detail below. Although preferred embodiments of the present invention are described below, it should be understood that the present invention can be embodied in various forms and should not be limited by the embodiments set forth herein.

Nectin-4 targeting ligand synthesis

Preparation of Ligand DXJ187

The specific structure of DXJ187 is shown in Figure 1. The synthesis steps of compound DXJ187 are shown in Figure 2. The precursor polypeptide resin 187resin was purchased from Shanghai Chupeptide Biotechnology Company, and other reagents were purchased from Reagent Company without purification. The purchased resin was deprotected after coupling with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid tri-tert-butyl ester, followed by the cyclization linker TATA The reaction affords compound 187. The synthetic route of the above-mentioned DOTA-Nectin 4 ligand (DXJ187) is shown in FIG. 2 . Reaction conditions: (a) 20% piperidine, DMF solution of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid tri-tert-butyl ester; (b) trifluoro Acetic acid, water and triisopropylsilane; (c) buffer solution containing 20% acetonitrile (20mM NH ₄ HCO ₃ , 5mM EDTA) at pH=8, 30°C, add 10 times TATA equivalent of cysteine after 1h.

Take a certain amount of resin (70 mg) in a 10 mL solid-phase synthesis tube, add 2 mL of dichloromethane (DCM) to swell, repeat three times, each time for 5 minutes, and then wash with N,N-dimethylformamide (DMF) three times, 5 minutes each time. Use 20% piperidine in DMF solution (v/v) to remove the amino protecting group Fmoc, the specific operation is 2 mL of 20% piperidine in DMF solution for 2 minutes, 10 minutes, 10 minutes, and then use 2 mL of DMF to wash 3-5 times, 2 minutes each time. 6 times the stoichiometric amount of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid tri-tert-butyl ester relative to the resin (70mg) was activated by 7.2 times the stoichiometric amount of HBTU Add it into a synthesis tube, and react for 1 hour under electromagnetic stirring. The ligand is dissociated from the resin and the t-butyl ester and trityl group are removed using 5mL trifluoroacetic acid/triisopropylsilane/water (95:2.5:2.5, v/v/v), after stirring for 2 hours The filtrate was collected, and the resin was washed with 2 mL of trifluoroacetic acid, all the filtrate was collected, and the trifluoroacetic acid was removed under reduced pressure, and compound 2 was prepared by reverse HPLC. 4.5 mg of compound 2 was dissolved in 5 mL of pH=8 containing 20% acetonitrile buffer (20 mM NH ₄ HCO ₃ , 5 mM EDTA), and 1.3 mg of TATA (1,3,5-triacryloylhexa-1,3, 5-triazine), after reacting at 30° C. for 1 hour, add cysteine 10 times the equivalent of TATA, and react at 30° C. for 10 minutes to terminate the cyclization reaction. The target ligand DXJ187 was obtained after reverse preparation by HPLC and freeze-drying. The ligand structure was identified by mass spectrometry, as shown in Figure 3-1.

Preparation of Ligands DXJ188, DXJ194, DXJ196, DXJ204

The specific structure of DXJ188, 194, 196, 204 is shown in Figure 3, and the synthesis steps are shown in Figure 4. The precursor polypeptide resin 188resin was purchased from Shanghai Tripeptide Biotechnology Company, and other reagents were purchased from reagent companies without further purification. The purchased resin was directly coupled with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid tri-tert-butyl ester (DOTA) after removing the methoxycarbonyl group (Fmoc). (DXJ188); or coupled with Wat methoxycarbonyl (Fmoc) protected 8-aminocaprylic acid (DXJ194), sarcosine (DXJ196), 4-aminomethylbenzoic acid (DXJ204), followed by 1,4, 7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid tri-tert-butyl ester (DOTA) coupling; finally remove the protecting group, and react with the cyclization linker TATA to obtain compounds DXJ188, DXJ194 , DXJ196, DXJ204.

Figure 4 shows the general preparation route of DXJ188, DXJ194, DXJ196, DXJ204. Reaction conditions: (a) 20% piperidine in DMF, Fmoc-amino acid, HBTU, HOBt and EIPEA in DMF; (b) 20% piperidine, 1,4,7,10-tetraazacyclododecane - DMF solution of tri-tert- butyl 1,4,7,10-tetraacetate (DOTA); (c) trifluoroacetic acid, water and triisopropylsilane; (d) pH=8 buffer containing 20% acetonitrile (20mM NH ₄ HCO ₃ , 5mM EDTA), at 30°C, after 2h, 10 times the equivalent of TATA of Cys was added.

Synthesis of DXJ188/194/196/204:

Take a certain amount of resin (70 mg) in a 10 mL solid-phase synthesis tube, add 2 mL of dichloromethane (DCM) to swell, repeat three times, each time for 5 minutes, and then wash with N,N-dimethylformamide (DMF) three times, 5 minutes each time. Use 20% piperidine in DMF solution (v/v) to remove the amino protecting group Fmoc, then the coupling of amino acid Linker is carried out according to the standard Fmoc solid-phase synthesis method, HBTU-activated DOTA tri-tert-butyl ester coupling, and continue The amino protecting group Fmoc was removed using 20% piperidine in DMF (v/v). The specific operation is to react with 2 mL of 20% piperidine in DMF for 2 minutes, 10 minutes, and 10 minutes, and then wash with 2 mL of DMF for 3-5 times, each time for 2 minutes. 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid tri-tert-butyl ester (DOTA) by 7.2 times the stoichiometric amount relative to the resin (70mg) 6 times the stoichiometric amount After activation, HBTU was added into the synthesis tube and reacted for 1 hour under electromagnetic stirring. Dissociation of the ligand from the resin and removal of the tert-butyl ester, trityl and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl groups was performed using 5 mL of trifluoroacetic acid/tris Isopropylsilane/water (95:2.5:2.5, v/v/v) was stirred for 2 hours and the filtrate was collected, and the resin was washed with 2 mL of trifluoroacetic acid, all the filtrate was collected, and the trifluoroacetic acid was removed under reduced pressure to obtain the compound 11/12/13/14. Dissolve 4.5 mg of compound 11/12/13/14 in 5 mL of pH=8 containing 20% acetonitrile buffer (20 mM NH ₄ HCO ₃ , 5 mM EDTA), add 1.3 mg of TATA, react at 30°C for 1 hour, add Cysteine with 10 times the equivalent of TATA was reacted at 30°C for 10 minutes to terminate the cyclization reaction. The target ligand DXJ188/194/196/204 was obtained after reverse preparation by HPLC and lyophilization. The ligand structure was identified by mass spectrometry.

The mass spectrometric identification results are summarized in Table 1, and the mass spectrograms of DXJ188, DXJ194, DXJ196, and DXJ204 are shown in Figures 5-2 to 5-5, respectively.

Table 1 Summary of mass spectrometry results of compounds DXJ188, DXJ194, DXJ196, and DXJ204

Labeling and Quality Control

mark:

⁶⁸ Ga: Accurately weigh a certain mass of ligand, add a certain volume of DMSO (dimethyl sulfoxide) to dissolve to a solution with a concentration of 20 μg/μL. Draw 4 μL of ligand solution and 130 μL of NaOAc solution (1mol/L) into a vial, add 2mL of ⁶⁸ Ga ³⁺ ion solution (solvent: 0.05mol/L hydrochloric acid solution, radioactivity 7-10mCi ), shake well, seal and react at 95°C for 10 minutes. After the reaction solution was cooled to room temperature, it was analyzed by radio-TLC for quality control.

¹⁷⁷ Lu: Accurately weigh a certain mass of ligand, add a certain volume of DMSO (dimethyl sulfoxide) to dissolve to a solution with a concentration of 20 μg/μL. Dissolve 5 μL of DXJ188 solution in 20 μL of 1mol/L sodium acetate solution; dilute ¹⁷⁷ LuCl ₃ solution to 300 μL with 0.05 M hydrochloric acid solution and place in a reaction flask; mix the two solutions and react at 95°C for 10 minutes. After the reaction solution was cooled to room temperature, it was analyzed by radio-TLC for quality control.

Quality control:

The radiochemical purity of ^{the 68} Ga and ¹⁷⁷ Lu labeled complexes was determined using radio-TLC (radioactive thin layer chromatography). First, mix 10 μL saturated EDTA (ethylenediaminetetraacetic acid) solution with 5 μL reaction solution, take the mixed solution and put it on the iTLC-SG chromatography plate, the developing agent is physiological saline (0.9% NaCl), and the radiochemical purity of all complexes is If it is greater than 95%, the next step is carried out without purification.

Imaging of ⁶⁸ Ga/ ¹⁷⁷ Lu Labeled Products

Take 0.1 mL of newly prepared ⁶⁸ Ga-labeled complexes (5.6MBq-7.4MBq) and inject them into female Balb/c nude mice bearing SW780 tumors (the tumor diameter is about 1.0cm) through the tail vein, and use them after 1h and 2h, respectively. Under isoflurane anesthesia, small animal PET/CT (SUPER-NOVA, Bingsheng Technology, China) imaging was performed, and the standard uptake value SUV was delineated for the region of interest.

In the experimental group, ^0.1 mL of freshly prepared ¹⁷⁷ Lu-DXJ188 (18.5 MBq) was injected via tail vein into female Balb/c nude mice bearing SW780 tumors; ^177Lu -DXJ188 with an activity of 18.5MBq was co-injected through the tail vein of the mice. The radioactive complex was injected together with the experimental group under isoflurane anesthesia, and SPECT/CT static imaging was performed 2 hours after injection.

As shown in Figure 6 and Table 2, ^{the 68} Ga complexes ⁶⁸ Ga-DXJ187, ⁶⁸ Ga-DXJ188, ⁶⁸ Ga-DXJ194, ⁶⁸ Ga-DXJ196 and ⁶⁸ Ga-DXJ204 can all be concentrated in the tumor area and excreted mainly through the kidney In vitro, non-target organ clearance is rapid. Over time, the uptake ratio of the complex in tumor and muscle increased. Among them, ⁶⁸ Ga-DXJ204 had the highest SUVmax value in the tumor, which were 1.00±0.25 and 1.01±0.26 at 1h and 2h of injection, respectively. ⁶⁸ Ga-DXJ188 has higher uptake in tumor, the lowest uptake in muscle, and the highest uptake ratio in tumor and muscle. At 1h and 2h after injection, the ratio of tumor to muscle is 10.21±1.15 and 10.64, respectively. ±1.35. These two ligands have better prospects for clinical application.

Figure 7 shows the SPECT/CT imaging results of ^177Lu -DXJ188 in the tumor-bearing mice of the experimental group and the inhibition group. According to the quantitative analysis of the tumor and normal muscle area, ^177Lu -DXJ188 has a higher The uptake in muscle is the lowest, and the uptake ratio between tumor and muscle reaches 6.92 at 2 hours after injection.

Having described various embodiments of the present invention, the foregoing description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and alterations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.

Claims (8)

1. A bicyclic peptide nuclide ligand targeting Nectin-4, having the structure shown in formula I:

   

   in,

   X is Lys or Arg;

   R is a group shown in formula II, formula III, formula IV or formula V,

   

   Wherein, a is an integer of 1-5, b is an integer of 3-8, and c is an integer of 1-5.
2. The bicyclic peptide nuclide ligand targeting Nectin-4 according to claim 1, wherein a is an integer of 1-3, b is an integer of 4-6, and c is an integer of 1-3.
3. The bicyclic peptide nuclide ligand targeting Nectin-4 according to claim 2, wherein R is a group represented by formula II, formula VI, formula VII or formula VIII,

   

   
4. The bicyclic peptide nuclide ligand targeting Nectin-4 according to claim 3, wherein the bicyclic peptide nuclide ligand is formula (1), formula (2), formula (3), formula (4) Or the compound shown in formula (5):

   

   
5. A bicyclic peptide nuclide probe targeting Nectin-4, the probe is a radionuclide-labeled bicyclic peptide nuclide ligand targeting Nectin-4 according to any one of claims 1-4.
6. The bicyclic peptide nuclide probe targeting Nectin-4 according to claim 5, wherein the radionuclide is a diagnostic radionuclide or a therapeutic radionuclide.
7. The bicyclic peptide nuclide probe targeting Nectin-4 according to claim 6, wherein the diagnostic radionuclide is ⁶⁸ Ga, ⁶⁴ Cu, ¹⁸ F, ⁸⁶ Y, ⁹⁰ Y, ⁸⁹ Zr, ¹¹¹ In , ^99m Tc, ¹¹ C, ¹²³ I, ¹²⁵ I and ¹²⁴ I at least one.
8. The bicyclic peptide nuclide probe targeting Nectin-4 according to claim 6, wherein 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 Bi and ²¹² Pb.

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