A cyclic peptide and its application

Abstract

The present invention discloses a cyclic peptide and its application. Specifically, the present invention provides a cyclic peptide as shown in formula I. The cyclic peptide provided by the present invention has good biological stability and good cell activity, and is suitable for clinical promotion and application.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, in particular to the field of cancer, and specifically, to a cyclic peptide and application thereof. Background Art

[0002] EpCAM (Epithelial cell adhesion molecule) is a transmembrane glycoprotein with a molecular weight of 40 kDa encoded by the GA-733-2 gene. Immunohistochemical studies have shown that EpCAM is overexpressed in various cancer cells isolated from patients with breast, prostate, ovarian, lung, colon, kidney and gastric cancer, which means the importance of EpCAM as a diagnostic biomarker for various cancers. In addition, EpCAM has recently been recognized as an important biomarker for cancer stem cells, which makes the development of specific probes for in vitro / in vivo imaging and drug-conjugated carriers even more exciting.

[0003] However, the existing compounds targeting EpCAM have poor biological stability and are easily decomposed in animal serum, making them difficult to use in drug development and clinical application. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the defect of low stability of cyclic peptides in the prior art and to provide a cyclic peptide and its application. The cyclic polypeptide compound has good biological stability and good cell activity, and is suitable for clinical promotion and application.

[0005] The present invention provides a cyclic peptide as shown in formula I:

[0006] ;

[0007] R 1 for or ;

[0008] When R 1 for When R 2 is amino or halogen;

[0009] When R 1 for When R 2 is hydroxy, amino or halogen.

[0010] In some embodiments, when R 1 for When R 2 It is hydroxyl.

[0011] In some embodiments, R 2wherein the halogen is fluorine.

[0012] In some embodiments, the structure of the cyclic peptide as shown in Formula I is any of the following:

[0013] , , .

[0014] The present invention also provides a cyclic peptide as shown in formula II:

[0015] ;

[0016] R 1 for or ;

[0017] When R 1 for When R 2 is amino or halogen;

[0018] When R 1 for When R 2 is hydroxy, amino or halogen;

[0019] A is ; * end and R 3 connected; R is independently H or -CH2OH; n is 4 to 8;

[0020] R 3 for ; M is a chelating group.

[0021] In some embodiments, when R 1 for When R 2 It is hydroxyl.

[0022] In some embodiments, R 2 wherein the halogen is fluorine.

[0023] In some embodiments, the chelating group is DOTA or NOTA; preferably DOTA.

[0024] In some embodiments, n is 6.

[0025] In some embodiments, for , * end and R 3 connected.

[0026] In some embodiments, the structure of the cyclic peptide as shown in Formula II is any of the following:

[0027] , , .

[0028] The present invention also provides a chelate, which comprises any one of the aforementioned cyclic peptides as shown in formula II 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.

[0029] 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, 111 In, 44 Sc, 86 Y. 89 Zr or 90 Nb, preferably 68 Ga.

[0030] 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, preferably 177 Lu.

[0031] The present invention also provides the use of the cyclic peptide as shown in formula I as described in any of the preceding items, the cyclic peptide as shown in formula II as described in any of the preceding items, and the chelate as described in any of the preceding items in the preparation of specific probes or drugs targeting EpCAM.

[0032] In some embodiments, the medicament is for diagnosing or treating EpCAM-positive tumors.

[0033] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0034] The "-" at the end of a group means that the group is attached to another fragment in the molecule through this site. For example, -CH2OH refers to a hydroxymethyl group.

[0035] The positive and progressive effect of the present invention is that the cyclic polypeptide compounds provided by the present invention have improved stability and good cell activity, and are suitable for clinical promotion and application. DETAILED DESCRIPTION

[0036] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0037] This patent uses the following compounds as Yangshen (DOI 10.1007 / s00239-015-9710-z)

[0038] .

[0039] The present invention adopts the liquid phase analysis method conditions as follows:

[0040] ;

[0041] ;

[0042] ;

[0043] The abbreviations and corresponding structures used in the present invention are shown in the following table:

[0044] ;

[0045] The compounds synthesized in this application are as follows:

[0046] , , , , , , , .

[0047] Example 1 Synthesis of Compound JH220017

[0048] Step 1: Synthesis of JH220017-A1

[0049] ;

[0050] Using 0.6 mmol CTC resin (1 g), 3 eq. of amino acids Fmoc-Lys(Boc)-OH, Fmoc-Ser(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Ser(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Ser(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ile-OH, Fmoc-Trp(Boc)-OH were added in sequence according to the peptide sequence. H, Fmoc-Trp(Boc)-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-HoArg(Pbf)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Pro-OH, Fmoc-Arg(Pbf)-OH, Fmoc-D-Trp-OH and bromoacetic acid. Condensation was performed using HATU / DIEA (3eq. / 6eq.) system, and Fmoc protecting group was removed using 20% ​​Pip / DMF. After the condensation is completed, wash with MeOH and drain to obtain 3 g of peptide resin; then use 30 mL of cutting solution (87.5% TFA / 7.5% DTT / 2.5% TIS / 2.5% H2O) for 4 hours, and add isopropyl ether to the filtered cutting solution to precipitate 1.6 g of solid.

[0051] Step 2: Synthesis of JH220017-A2

[0052] ;

[0053] JH220017-A1 (1.6 g) was dissolved in H2O (300 mL) and ACN (300 mL) solution, and then saturated NH4HCO3 was added to adjust the pH to 8. The reaction was continued for 1 h. After LCMS detection, the raw material was completely consumed and the product was generated. The product was freeze-dried and flash purified to obtain 378 mg.

[0054] Step 3: Synthesis of JH220017

[0055] ;

[0056] JH220017-A2 (50 mg) and DOTA-NHS (1.5 eq.) were dissolved in DMF (0.5 mL), DIEA (3 eq.) was added, and the reaction was continued for 1 h. After LCMS detection, the reaction was completed and the product was prepared and purified to obtain 14.3 mg. HPLC: 95.02%, RT: 12.617 min; MS: [M / 3+H] + =983.74.

[0057] Step 4: Compositing 175 Lu-JH220017

[0058] ;

[0059] JH220017 (8 mg; 2.7 μmol) and LuCl3·6H2O (7 mg; 27 μmol) were dissolved in 0.1M, pH=4.7 sodium acetate buffer solution (8 mL) at room temperature, 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. The final product was lyophilized to obtain 3.15 mg; the purity was 97.46%, t R =11.189min, [M / 3+H] + =1040.98.

[0060] other 175 Lu-labeled compounds were also prepared using the above synthesis method, and the analysis methods and results are summarized as follows.

[0061] ;

[0062] ;

[0063] During the compound preparation process, the preparative HPLC was:

[0064] Preparative HPLC: 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 (214.4 nm, 254 nm)

[0065] The analysis equipment involved in the embodiment is:

[0066] HPLC-MS Waters 2696-Micromass system: 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: 1.0 mL / min.

[0067] Column temperature: 30 ºC

[0068] Detection: UV (214.4 nm) and MS (ESI, Positive mode, 110 to 1000 amu).

[0069] Preparation method:

[0070] ;

[0071] The remaining compounds were synthesized using the solid phase synthesis Fmoc strategy according to the above method, see the table below.

[0072] ;

[0073] Effect Example 1

[0074] Cell competition binding (IC 50 )experiment

[0075] 1. Experimental Procedure

[0076] 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℃.

[0077] 1.2 Take CT26-human-epcam (Kangyuan Bochuang) 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.

[0078] 1.3 Resuspend each cell line in FACS buffer and count the cells.

[0079] 1.4 Adjust the cell density to 2×10 6 The cells were seeded into U-bottom 96-well plates (Corning, 3799) at a density of 50 μL / well, i.e., 1×105 cells / well.

[0080] 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.

[0081] 1.6 Use FACS buffer to prepare 3× competitive fluorescent molecules JH220023 or JH220004, 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.

[0082] 1.7 Wash away unbound compounds with ice-cold FACS buffer, centrifuge at 400 g for 5 min, discard the supernatant, and repeat once.

[0083] 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).

[0084] 2. Experimental Data

[0085] The results are shown in the following table, and the compounds of the present invention have good cell competitive binding ability with EpCAM positive cells.

[0086] ;

[0087] Compared with the positive reference compound JH220001, several compounds covered by the present invention showed better competitive binding ability in the EpCAM positive cell test, which is of great significance for the development and application of the compounds in the field of biomedicine.

[0088] Effect Example 2

[0089] Stability test

[0090] 1. Experimental Procedure

[0091] 1.1 Preparation of dilution solution

[0092] Dilution: 100 mL methanol (Honeywell) added to 100 mL acetonitrile (Honeywell) added to 200 mL water, mixed and stored at 4°C;

[0093] Internal standard precipitant: methanol containing 5ng / ml verapamil, 50ng / ml glibenclamide, and 500ng / ml tolbutamide (internal standard and positive compounds were from China Food and Drug Inspection Institutes);

[0094] 1.2 Compound preparation

[0095] Accurately weigh 175 Lu-JH22 and the positive control benfluorex hydrochloride were dissolved in DMSO (VWR) to form a 1 mM stock solution for later use. 175Lu-JH22 compounds and positive controls were prepared into 200 μM working solutions.

[0096] 1.3 Experimental procedures

[0097] 1.3.1 Mouse serum (CD-1 mice were purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd.) and 1 concentration (2 μM) of 175 Lu-JH22 compound was used for serum stability test, and the samples were incubated in parallel twice.

[0098] 1.3.2 Take 4 μL of 200 μM test substance working solution or positive control and mix with 396 μL human serum to prepare a 2 μM human serum sample.

[0099] 1.3.3 Incubate in a 37°C reciprocating shaking water bath at a shaking speed of 80 times / min. The equilibrium incubation time for the test samples is 0h, 0.5h, 1h, 4h, 8h, and 24h, and the incubation time for the positive reference is 0h, 1h, and 2h.

[0100] 1.3.4 Transfer 20 μL of serum sample, add 200 μL of precipitant acetonitrile (containing 50 ng / mL internal standard), and mix thoroughly to precipitate protein.

[0101] 1.3.5 After protein precipitation, all samples were centrifuged at 3700 r / min for 10 min, the supernatant was taken, diluted 1:1 with water and analyzed using LC-MS / MS.

[0102] 1.4 Biological analysis

[0103] The LC-MS / MS method was used to analyze the signal peak areas of the test compound and the positive control in the test sample and the positive control sample, and then the corresponding parameters were calculated based on the peak area ratio of the analyte to the internal standard. There was no standard curve or QC sample in the analysis batch of the positive control group. For detailed analysis conditions, see "Analysis Method".

[0104] Data analysis

[0105] The LC-MS / MS method was used to measure the 175 The Lu-JH22 series of compounds (positive control for determination of benfluorex) were measured by the instrument to obtain the analyte / internal standard peak area ratio (Analyte / AIS). The remaining percentage (%Control) was calculated by the ratio of Analyte / AIS in the non-zero time point sample to the zero time point sample. Ln (%Control) was plotted against the incubation time and a linear fit was performed. The clearance constant (k, min) of the test compound was calculated by the following equation: -1 ) and elimination half-life (T 1 / 2 , min).

[0106] k = - slope

[0107] T 1 / 2 = 0.693 / k

[0108] Analytical methods:

[0109] ;

[0110] ;

[0111] ;

[0112] 2. Experimental Data

[0113] The results are shown in the following table

[0114] ;

[0115] The experimental results show that the cyclic peptide molecules obtained by modifying and replacing specific amino acid sequences on the ring have good biological stability, and their biological stability is improved to varying degrees compared with the positive reference compound JH220001. Combined with their good targeting affinity, these compounds have great prospects for drug development and its application.

[0116] 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 cyclic peptide having a structure of any of the following: 、 、 。 2. A cyclic peptide having a structure of any of the following: 、 、 。 3. A chelate, characterized in that: It comprises the cyclic peptide 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 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, 111 In, 44 Sc, 86 Y. 89 Zr or 90 Nb; (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 according to claim 3, characterized in that It meets one or both of the following conditions: (1) The diagnostic radioisotope is 68 Ga; (2) The therapeutic radioisotope is 177 Lu.

6. Use of the cyclic peptide according to claim 1, the cyclic peptide according to claim 2 or the chelate according to any one of claims 3 to 5 in the preparation of a specific probe targeting EpCAM.

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