Bicyclic peptide and application thereof

Abstract

The invention discloses a bicyclic peptide and application thereof, and particularly provides a bicyclic peptide or pharmaceutically acceptable salt thereof, the bicyclic peptide provided by the invention has good serum stability, good in-vitro cell viability and binding effect, high synthesis efficiency, good purity and yield, simple and convenient preparation, and good patent medicine prospect. # imgabs0 #

Description

Technical Field

[0001] The present application relates to a bicyclic peptide and its application. Background Art

[0002] EphA2 is an important member of the Eph receptor family, and its canonical signaling depends on the binding of ephrin-A1 and its tyrosine kinase activity to maintain the normal epithelial cell state. In cancer, EphA2 is often overexpressed and accompanied by the loss of ephrin ligands, promoting malignancy and cancer progression in a ligand-independent manner in the absence of ligand binding.

[0003] Agonist peptides targeting EphA2 with high affinity can be used to stimulate the tumor-suppressing activity of EphA2 receptors in tumor cells. In addition, since ephrin and artificial ligands can cause EphA2 activation and promote the internalization of receptors from the cell surface to lysosomes, these peptides can also be used for targeted delivery of chemotherapeutic drugs or toxins to tumor cells. Finally, molecules that bind to EphA2 with high affinity can be used for tumor imaging. However, the affinity and stability of existing EphA2-targeting compounds are not high enough, and more druggable compounds need to be developed. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the defects of the single type and poor biological stability of EphA2-targeted bicyclic peptide compounds in the prior art, and to provide a bicyclic peptide compound with good stability and in vitro cell activity. The compound labeled with Lu-177 has a certain uptake in tumors.

[0005] The present invention solves the above technical problems through the following technical solutions:

[0006] The present invention provides a bicyclic peptide or a pharmaceutically acceptable salt thereof, the structure of which is shown in any of the following:

[0007] ,

[0008] , , , .

[0009] The present invention also provides a compound or a pharmaceutically acceptable salt thereof, the structure of which is shown in any of the following:

[0010] , or .

[0011] The present invention also provides a chelate or a pharmaceutically acceptable salt thereof, which comprises any one of the aforementioned compounds or a pharmaceutically acceptable salt thereof, and a radioactive isotope; the radioactive isotope is a diagnostic radioactive isotope or a therapeutic radioactive isotope, and the radioactive isotope is chelated with a chelating group. The chelating group refers to the DOTA chelating group of any one of the aforementioned compounds.

[0012] In some embodiments, the diagnostic radioisotope may be, but is not limited to, 68 Ga, 177 Lu, 67 Ga, 61 Cu, 64 Cu, 99m Tc, 114m In, 111 In, 44 Sc, 86 Y. 89 Zr or 90 Nb, preferably 68 Ga.

[0013] In some embodiments, the therapeutic radioisotope may be, but is not limited to, 177 Lu, 90 Y. 89 Sr. 188 Re, 225 Ac, 212 Bi, 213 Bi, 203 Pb, 212 Pb, 165 2. 211 At 166 Ho, 149 Pm, 159 Gd, 105 Rh, 109 Pd, 198 Au, 199 Au, 175 Yb, 142 Pr, 111 In, 47 Sc, 117m Sn, 153 Sm, 149 Tb, 161 Tb, 223 Ra, 224 Ra, 227 Th or 67 Cu; e.g. 177 Lu.

[0014] The present invention also provides use of any of the aforementioned bicyclic peptides, any of the aforementioned compounds and any of the aforementioned chelates in the preparation of EphA2 agonists.

[0015] In some embodiments, in the application, the EphA2 agonist can be used in mammals in vivo; it can also be used in vitro, mainly for experimental purposes, for example: as a standard sample or control sample for comparison, or prepared into a kit according to conventional methods in the art to provide rapid detection of EphA2 agonist effects.

[0016] The present invention also provides the use of any of the aforementioned bicyclic peptides or pharmaceutically acceptable salts thereof, any of the aforementioned compounds and any of the aforementioned chelates in the preparation of drugs for treating or preventing EphA2-related diseases; the EphA2-related diseases are, for example, cancer, preferably solid tumors.

[0017] The present invention also provides the use of any of the aforementioned bicyclic peptides or pharmaceutically acceptable salts thereof, any of the aforementioned compounds or pharmaceutically acceptable salts thereof, and any of the aforementioned chelates or pharmaceutically acceptable salts thereof in the preparation of tumor imaging agents.

[0018] The term "chelating group" refers to a group having two or more coordinating atoms and capable of combining with the same central atom to form a ring structure.

[0019] The term "treat" refers to any of the following: (1) ameliorating one or more biological manifestations of a disease; (2) interfering with one or more points in the biological cascade that leads to a disease; or (3) slowing the progression of one or more biological manifestations of a disease.

[0020] The term "prevention" refers to reducing the risk of developing a disease.

[0021] The positive and progressive effects of the present invention are that the compounds provided by the present invention have good serum stability, good in vitro cell activity and binding effect, high synthesis efficiency, good purity and yield, simple preparation, and good drug development prospects. DETAILED DESCRIPTION

[0022] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples.

[0023] The present invention adopts the liquid phase analysis method conditions as follows:

[0024] Analytical equipment: HPLC-MS Waters 2696-Micromass system: Chromatographic column: Shim-pack VP-ODS, (4.6 × 150mm, 5μm), Mobile phase A: H2O (0.1% TFA), Mobile phase B: acetonitrile (0.1% TFA). Flow rate: 1.0mL / min.

[0025] Column temperature: 30 ºC;

[0026] Detection: UV (214.4 nm) and MS (ESI, Positive mode, 110 to 1000 amu).

[0027] .

[0028] .

[0029] ;

[0030] .

[0031] The abbreviations and corresponding structures used in the present invention are shown in the following table:

[0032] .

[0033] The following compounds were synthesized in this application:

[0034] , , , , , , , , , , , .

[0035] Example 1 Preparation of Compounds

[0036] Take the synthesis steps of compound JH200023 as an example.

[0037] ;

[0038] Step 1: Synthesis of JH200023-A1

[0039] ;

[0040] Using 0.6 mmol Rink amino resin (1 g), 3 eq. of amino acids (Fmoc-Cys(Trt)-OH, Fmoc-HoArg(Pbf)-OH, Fmoc-Trp(Boc)-OH, Fmoc-D-Cysacid-OH, Fmoc-Pro-OH, Fmoc-SeMet-OH, Fmoc-Leu-OH, Fmoc-Cys(Trt)-OH, Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Asn( Trt)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Hyp(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-HoArg(Pbf)-OH, Fmoc-Ala-OH and DOTA(tBu)3 were condensed using PyBOP / DIEA (3eq.) system, and the Fmoc protecting group was removed using 20% ​​Pip / DMF. After the condensation was completed, it was washed with MeOH and dried to obtain 1.8 g of resin; it was then cleaved with 20 mL of cutting solution (87.5% TFA / 7.5% DTT / 2.5% TIS / 2.5% H2O) for 4 hours, and isopropyl ether was added to the filtered cutting solution to precipitate 550 mg of solid.

[0041] Step 2: Synthesis of JH200023

[0042] ;

[0043] JH200023-A1 (550 mg) and TATA (280 mg, 1.5 eq.) were dissolved in H2O (300 mL) and ACN (300 mL) solution, and then saturated NH4HCO3 was added to adjust pH=8. The reaction was continued for 1 h. LCMS showed that JH200023-A1 was completely consumed and the product was generated. The product was freeze-dried and flash-purified to obtain 280 mg of the product. 100 mg was taken to obtain 41.15 mg of the product. HPLC: 95.53%, RT: 10.578 min; m / z=1433.66[1 / 2M+H] + 。

[0044] Step 3: Synthesis 175 Lu-JH200023;

[0045] ;

[0046] At room temperature, JH200023 (5 mg; 1.7 μmol) and LuCl3*6H2O (4.5 mg; 27 μmol) were dissolved in 0.1M, pH=4.7 buffer solution (5 mL), heated to 90°C, and reacted for 20 min. LCMS detection showed that the raw material was completely consumed and the product was generated. The final product was prepared and purified. Lyophilization gave 2.27 mg of the final product; the purity was 99.40%, t R =8.463 min, [M / 3+H] + = 1012.99.

[0047] Preparation equipment: Shimadzu LC-20AP-SPD40 system, column: Bonnasil-BS C 18 (30 x 250mm, 5μm), mobile phase A: H2O (0.1% TFA), mobile phase B: acetonitrile (0.1% TFA), flow rate: 20 mL / min, detection: UV (214.4nm, 254 nm).

[0048] Preparation method:

[0049] .

[0050] The remaining compounds were synthesized using the solid phase synthesis Fmoc strategy according to the above method, see the table below:

[0051] .

[0052] Effect Example 1 Cell competition binding (IC 50 )experiment

[0053] 1. Experimental Procedure

[0054] 1.1 Accurately weigh the compound to be tested, add DMSO (Biyuntian, ST038) to dissolve it into 1 mM stock solution for later use, and store the dissolved solution at -20℃.

[0055] 1.2 Take HELA epha2 11# (Crystal Nucleus Biotechnology) in the logarithmic growth phase, wash the cells once with PBS, add trypsin containing 0.05% EDTA (Gibco, 25300062) for digestion, centrifuge at 400 g for 5 min, and discard the supernatant.

[0056] 1.3 Resuspend each cell line in FACS buffer and count the cells.

[0057] 1.4 Adjust the cell density to 2×10 6cells / mL, and 50 μL / well was inoculated into a U-bottom 96-well plate (Corning, 3799), that is, 1×10 5 cells / well.

[0058] 1.5 Prepare 3× test compounds using FACS buffer, set up control wells without samples, add 50 μL of the corresponding compound to each well, mix well, and incubate on ice for 15 min.

[0059] 1.6 Use FACS buffer to prepare 3× competitive fluorescent molecules JH200009-FITC, set up blank wells without fluorescent molecules, add 50 μL of the corresponding compound to each well, mix well, and incubate on ice for 60 min.

[0060] 1.7 Wash away unbound compounds with ice-cold FACS buffer, centrifuge at 400 g for 5 min, discard the supernatant, and repeat once.

[0061] 1.8 Resuspend the cells in each well with 50 μL FACS buffer, mix well, and detect the fluorescence signal using a flow cytometer (ACEA biocsience, Novocyte 3130).

[0062] 2. Experimental Data

[0063] ;

[0064] Effect Example 2 In vivo distribution experiment

[0065] 1. Experimental Procedure

[0066] 1.1 Construction of overexpression cell lines

[0067] The human EphA2 gene was synthesized and cloned into the lentiviral vector pLent-EF1a-puro (Jinweizhi), and the pLent-EF1a-Epha2-puro lentiviral expression vector was constructed and packaged into lentivirus; HELA (Cell Bank of Chinese Academy of Sciences) cells were revived and cultured; HT1080 WT were plated in six-well plates and adhered overnight, and polybrene (Biyuntian) was added at a final concentration of 10μg / mL, and the HELA cells were infected with the packaged lentivirus at an MOI of 40; 72h after virus infection, 5μg / mL puromycin (Biyuntian) was added for resistance screening, and the positive cell pool was obtained after 3 days of screening; the high positive area of ​​the positive cell pool was sorted by flow cytometry (BD, FACS AriaIII), and the sorted and collected cells were sorted and expanded for monoclonal cell lines. After FACS identification of human Epha2 expression on the surface of monoclonal cells, HELA cell lines overexpressing hEphA2 were obtained;

[0068] 1.2 JH200023 177 Lu Marking

[0069] Dissolve JH200023 in 0.45M ascorbic acid / sodium ascorbate buffer at pH 4.5, and add the corresponding volume of 177 LuCl3 (ITM Medical Isotopes GmbH), the mixture was heated to 95 °C for 20 min, and after the reaction, quality control was performed using radio-HPLC to determine the radiochemical purity of the radioligand (>90% was acceptable).

[0070] 1.3 177 Lu-JH200023 distribution experiment

[0071] BALB / c nude female mice (18-20 g) were provided by Zhejiang Weitong Lihua Experimental Animal Technology Co., Ltd. The animals were housed in individually ventilated cages, with 5 animals per cage. HELA hEpha2 11# cells were lightly suspended in equal volumes of PBS and matrigelmatrix (Corning) to a final density of 8×10 7 8×10 / mL were injected subcutaneously on the right side of the mouse 6 When the tumor volume reaches 100-300 mm 3 (about 1 week after cell inoculation), biodistribution studies were performed. 177 Lu-JH200023 was injected intravenously at a concentration of 3.7 MBq per mouse. 177 Lu-JH200023, injection volume was 200 μL. Mice were killed by CO2 inhalation 6 h after injection. Blood and characteristic organs (heart, liver, spleen, lung, kidney, muscle, femur, esophagus and tumor) were collected, weighed, and measured in a gamma counter (2470 Wizard, PerkinElmer, Inc.). The accumulated amount of radioactive tracer was expressed as the percentage of the injected dose per gram of the corresponding organ (%ID / g).

[0072] 2. Experimental Data

[0073] ;

[0074] The compound has a certain uptake in the tumors of mice and is distributed relatively cleanly in the body, which has broad prospects for the further development of the compound and its clinical application.

[0075] Effect Test Example 3 In vitro stability test

[0076] 1. Experimental Procedure

[0077] 1.1 Experimental Preparation

[0078] 1.1.1 Intermediate dilution: Add 100 ml methanol, 100 ml acetonitrile and 200 ml water, mix well and store at 4°C.

[0079] 1.1.2 Internal standard precipitant: 5ng / ml Verapamil, 50ng / ml Glibenclamide, 500ng / ml Tolbutamide in MeOH (all obtained from China Food and Drug Inspection Institute);

[0080] 1.1.3 Test compound: accurately weigh a certain mass 175 Lu-JH200023 was dissolved in DMSO to prepare a 2 mM stock solution. The stock solution was diluted with an intermediate diluent to prepare a 200 μM working solution.

[0081] 1.2 Experimental procedures

[0082] 1.2.1 Prepare eight tubes required for incubation and sampling, and mark the species and time points;

[0083] 1.2.2 Add 200 μL of internal standard precipitant to the EP tube or 96-well plate and store at 4°C for later use;

[0084] 1.2.3 Take 4 μL of 200 μM test sample working solution and mix with 396 μL of serum of each species to prepare serum samples of each species with a concentration of 2 μM, and mix them evenly;

[0085] 1.2.4 Take 20 μL of the test incubation tube at 0, 0.5, 1, 4, 8, and 24 h, and the positive control group at 0, 30, and 60 min and add it to the precipitation tube containing the internal standard;

[0086] 1.2.5 Centrifuge at 3700 rpm for 10 min, take the supernatant and dilute it with water in a 1:1 ratio before injection.

[0087] 1.3 Analysis methods

[0088] ;

[0089] .

[0090] 2. Experimental Data

[0091] The results are shown in the following table

[0092] ;

[0093] 175Lu-JH200023 has good in vitro stability and mouse serum stability. 1 / 2 is 15.5h.

[0094] Although the specific embodiments of the present invention are described above, it should be understood by those skilled in the art that this is only for illustration and the protection scope of the present invention is defined by the appended claims. Those skilled in the art may make various changes or modifications to these embodiments without departing from the principles and essence of the present invention, but these changes and modifications all fall within the protection scope of the present invention.

Claims

1. A bicyclic peptide or a pharmaceutically acceptable salt thereof, wherein the structure is as shown in any of the following: 、 、 、 、 。 2. A compound or a pharmaceutically acceptable salt thereof, the structure of which is shown in any of the following: 、 or 。 3. A chelate or a pharmaceutically acceptable salt thereof, comprising the compound or a pharmaceutically acceptable salt thereof as claimed in claim 2, and a radioactive isotope; the radioactive isotope is a diagnostic radioactive isotope or a therapeutic radioactive isotope, and the radioactive isotope is chelated with a chelating group.

4. The chelate or pharmaceutically acceptable salt thereof according to claim 3, characterized in that: It meets one or both of the following conditions: (1) The diagnostic radioisotope is 68 Ga, 177 Lu, 67 Ga, 61 Cu, 64 Cu, 99m Tc, 114m In, 111 In, 44 Sc, 86 Y. 89 Zr or 90 Nb; and (2) The therapeutic radioisotope is 177 Lu, 90 Y. 89 Sr. 188 Re, 225 Ac, 212 Bi, 213 Bi, 203 Pb, 212 Pb, 165 2. 211 At 166 Ho, 149 Pm, 159 Gd, 105 Rh, 109 Pd, 198 Au, 199 Au, 175 Yb, 142 Pr, 111 In, 47 Sc, 117m Sn, 153 Sm, 149 Tb, 161 Tb, 223 Ra, 224 Ra, 227 Th or 67 Cu.

5. The chelate or pharmaceutically acceptable salt thereof according to claim 3, characterized in that: It meets one or both of the following conditions: (1) The diagnostic radioisotope is 68 Ga; and, (2) The therapeutic radioisotope is 177 Lu.

6. Use of the bicyclic peptide or a pharmaceutically acceptable salt thereof according to claim 1, the compound or a pharmaceutically acceptable salt thereof according to claim 2, or the chelate or a pharmaceutically acceptable salt thereof according to any one of claims 3 to 5 in the preparation of an EphA2 agonist.

7. The use according to claim 6, characterized in that The EphA2 agonist is used in vivo and / or in vitro in mammals.

8. Use of the bicyclic peptide or a pharmaceutically acceptable salt thereof according to claim 1, the compound or a pharmaceutically acceptable salt thereof according to claim 2, or the chelate or a pharmaceutically acceptable salt thereof according to any one of claims 3 to 5 in the preparation of a medicament for treating or preventing EphA2-related diseases.

9. The use according to claim 8, characterized in that The EphA2-related disease is cancer.

10. The use according to claim 8, characterized in that The EphA2-related disease is a solid tumor.

11. Use of the bicyclic peptide or a pharmaceutically acceptable salt thereof according to claim 1, the compound or a pharmaceutically acceptable salt thereof according to claim 2, or the chelate or a pharmaceutically acceptable salt thereof according to any one of claims 3 to 5 in the preparation of an imaging agent for tumor imaging.

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