A bicyclic peptide and its application

Abstract

The present invention discloses a bicyclic peptide and its application. Specifically, the present invention provides a bicyclic peptide or a pharmaceutically acceptable salt thereof. The bicyclic peptide provided by the present invention has good serum stability, good in vitro cell activity and binding, high synthesis efficiency, good purity and yield, simple preparation, and good drug development prospects. #imgabs0#

Description

Technical Field

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

[0002] EphA2 is a key member of the Eph receptor family, and its canonical signaling relies 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 that bind to EphA2 with high affinity can be used to stimulate the tumor-suppressing activity of the EphA2 receptor in tumor cells. Furthermore, because ephrins and artificial ligands can activate EphA2 and promote receptor internalization from the cell surface into lysosomes, these peptides can also be used for the 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 the development of more druggable compounds is needed. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the shortcomings of the existing EphA2-targeting bicyclic peptide compounds, which are limited in variety and have poor biostability, 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 one 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 one of the following:

[0010] 、 or .

[0011] The present invention also provides a chelate or a pharmaceutically acceptable salt thereof, comprising any of the aforementioned compounds or pharmaceutically acceptable salts thereof, and a radioactive isotope; the radioactive isotope is a diagnostic radioisotope or a therapeutic radioisotope, and the radioisotope is chelated with a chelating group. The chelating group is the DOTA chelating group of any 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 Second, 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 applications, 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 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 "chelate group" refers to a group having two or more coordinating atoms that can combine with the same central atom to form a ring structure.

[0019] The term "treat" refers to any of the following: (1) alleviating 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 "prevent" refers to reducing the risk of developing a disease.

[0021] The positive progress of the present invention is that the compound provided by the present invention has good serum stability, good in vitro cell activity and binding effect, high synthesis efficiency and 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; Column: Shim-pack VP-ODS (4.6 × 150 mm, 5 μm); Mobile phase A: H2O (0.1% TFA); Mobile phase B: acetonitrile (0.1% TFA). Flow rate: 1.0 mL / min.

[0025] Column temperature: 30°C;

[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 as follows:

[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( A mixture of Fmoc(tBu)-(Fmoc-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 was condensed using a PyBOP / DIEA (3 eq.) system. The Fmoc protecting group was removed using 20% Pip / DMF. After condensation, the resin was washed with MeOH and dried by vacuum to yield 1.8 g. The resin was then cleaved with 20 mL of a cleavage solution (87.5% TFA / 7.5% DTT / 2.5% TIS / 2.5% H2O) for 4 h. Isopropyl ether was added to the filtered cleavage 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). Saturated NH4HCO3 was then added to adjust the pH to 8. The reaction was allowed to proceed for 1 h. LCMS analysis showed that JH200023-A1 was completely consumed and the product was generated. 280 mg of the product was lyophilized and flash purified. 100 mg of the product was used to prepare 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] JH200023 (5 mg; 1.7 μmol) and LuCl3*6H2O (4.5 mg; 27 μmol) were dissolved in 0.1 M, pH 4.7 buffer (5 mL) at room temperature and heated to 90°C for 20 min. LCMS analysis showed complete consumption of the starting material and the formation of the product, which was then prepared and purified. Lyophilization afforded 2.27 mg of the final product with a purity of 99.40%. 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, 254nm).

[0048] Preparation method:

[0049] .

[0050] The remaining compounds were synthesized using the solid phase Fmoc synthesis 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 test compound and dissolve it in DMSO (Biyuntian, ST038) to a 1 mM stock solution for later use. Store the dissolved solution at -20°C.

[0055] 1.2 Take HELA epha2 11# (Crystal Biosciences) 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 were inoculated into U-bottom 96-well plates (Corning, 3799), i.e., 1×10 5 cells / well.

[0058] 1.5 Prepare 3x the test compound in FACS buffer and set up control wells without sample. Add 50 μL of the corresponding compound to each well. Mix thoroughly and incubate on ice for 15 min.

[0059] 1.6 Prepare 3× the amount of the competing fluorescent molecule JH200009-FITC in FACS buffer. Set up blank wells without fluorescent molecule and add 50 μL of the corresponding compound to each well. Mix thoroughly 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 (GenWeizhi). The pLent-EF1a-Epha2-puro lentiviral expression vector was constructed and packaged into lentivirus. HELA (Cell Bank, Chinese Academy of Sciences) cells were revived and cultured. HT1080 WT cells were plated in six-well plates and allowed to adhere overnight. Polybrene (Biyuntian) was added at a final concentration of 10 μg / mL, and the packaged lentivirus was used to infect HELA cells at an MOI of 40. 72 hours after viral infection, 5 μg / mL puromycin (Biyuntian) was added for resistance screening. After 3 days of screening, a positive cell pool was obtained. The high-positive region of the positive cell pool was sorted by flow cytometry (BD, FACS AriaIII). The collected cells were isolated and expanded for monoclonal cell line selection. After FACS identification of human Epha2 expression on the surface of the monoclonal cells, a HELA cell line overexpressing hEphA2 was obtained.

[0068] 1.2 JH200023 177 Lu Mark

[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 by radio-high performance liquid chromatography to determine the radiochemical purity of the radioligand (>90% was qualified).

[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. and housed in individually ventilated cages with 5 animals per cage. HELA hEpha2 11# cells were gently suspended in equal volumes of PBS and matrigel matrix (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 administered intravenously to each mouse at the desired dose concentration of 3.7 MBq 177 Lu-JH200023 was injected in a 200 μL volume. Six hours after injection, mice were sacrificed by CO₂ inhalation. Blood and specific organs (heart, liver, spleen, lungs, kidneys, muscles, femurs, esophagus, and tumors) were collected, weighed, and measured in a gamma counter (2470 Wizard, PerkinElmer, Inc.). Accumulated radiotracer levels were expressed as the percentage of the injected dose per gram (%ID / g) of the corresponding organ.

[0072] 2. Experimental Data

[0073] ;

[0074] The compound is taken up by tumors in mice to a certain extent 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 experiment

[0076] 1. Experimental Procedure

[0077] 1.1 Experimental Preparation

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

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

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

[0081] 1.2 Experimental operation

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

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

[0084] 1.2.3 Prepare serum samples with a concentration of 2 μM for each species by mixing 4 μL of the 200 μM test sample working solution with 396 μL of serum from each species. Mix thoroughly.

[0085] 1.2.4 Take 20 μL of the test 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 sedimentation tube containing the internal standard;

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

[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 It is 15.5h.

[0094] Although specific embodiments of the present invention have been described above, those skilled in the art will appreciate that these are merely illustrative and that the 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, and such changes and modifications are intended to fall within the scope of the present invention.

Claims

1. A bicyclic peptide or a pharmaceutically acceptable salt thereof, wherein the structure is as shown below: or 。 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 according to claim 2 or a pharmaceutically acceptable salt thereof, and a radioactive isotope; the radioactive isotope is a diagnostic radioisotope or a therapeutic radioisotope, and the radioisotope is chelated with a chelating group.

4. The chelate or pharmaceutically acceptable salt thereof according to claim 3, wherein: 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.

5. The chelate or pharmaceutically acceptable salt thereof according to claim 3, wherein: The therapeutic radioisotope is 177 Lu, 90 Y. 89 Sr. 188 Re、 225 Ac, 212 Bi, 213 Bi, 203 Pb, 212 Pb, 165 Second, 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.

6. The chelate or pharmaceutically acceptable salt thereof according to claim 3, wherein: The diagnostic radioisotope is 68 Ga.

7. The chelate or pharmaceutically acceptable salt thereof according to claim 3, wherein: The therapeutic radioisotope is 177 Lu.

8. Use of the chelate compound or a pharmaceutically acceptable salt thereof according to any one of claims 3 to 7 in the preparation of an imaging agent for tumor imaging.

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