Preparation method, product and application of carbamido-containing macrocyclic peptide compound

Abstract

The invention provides a preparation method, a product and application of a carbamido-containing macrocyclic peptide compound, and belongs to the technical field of organic synthesis of peptide macrocyclization. The preparation method comprises the following steps: dissolving polypeptide containing two exposed amino groups in an organic solution, and then adding diisocyanate for reaction; then dropwise adding alkali into a reaction system for reaction to obtain a carbamido-containing macrocyclic peptide compound; the polypeptide containing the two exposed amino groups is a polypeptide of N-terminal amino group-side chain amino group or a polypeptide of side chain amino group-side chain amino group. The preparation method provided by the invention has the advantages of high reaction economy, mild conditions, wide substrate applicability, simplicity and convenience in operation and the like. The method can be used for diversity modification of polypeptide / protein, provides a theoretical basis for research on structure functions of polypeptide drugs and compounds with specific functions, and has a wide application prospect in the fields of pharmaceutical chemistry and polypeptide drug innovation.

Description

Technical Field

[0001] The present invention relates to the technical field of organic synthesis of peptide macrocyclization, and in particular to a preparation method, product and application of a urea-containing macrocyclic peptide compound. Background Art

[0002] In recent years, with the increasing maturity of biotechnology and peptide synthesis technology, peptide drugs have received extensive attention from drug developers at home and abroad. Moreover, compared with general small molecule chemical drugs, peptide drugs have higher activity and stronger selectivity, and have obvious advantages in treating complex diseases. Moreover, since peptides themselves are compounds composed of amino acids, their metabolites are amino acids, which generally have no side effects or very little side effects on the human body; compared with protein drugs, peptides have relatively good stability, high purity, low production cost, low immunogenicity or no immunogenicity, and the quality control level can also be close to traditional small molecule chemical drugs; in the drug development stage, chemical modification can also be used to improve the affinity, solubility, pharmacokinetic properties, toxicity, etc. of drug candidates. In short, peptide drugs have a good combination of the advantages of small molecule chemical drugs and protein drugs.

[0003] However, due to the limitation of cell permeability, most peptides are prevented from further development as therapeutic agents, and studies have shown that the macrocyclization of peptides and peptides is a feasible strategy to overcome this problem. Just as in order to improve the cell permeability of peptides, Lin and colleagues previously reported a strategy for conjugating modified peptides with tetrazoles and alkenes through light-mediated 1,3-dipolar cycloaddition. Compared with previous studies that required double side chain modification to semi-prepare the peptides, it was fully demonstrated that the cyclization between tetrazoles and natural Lys side chains in peptides is favorable (Chem. Commun, 2009, 5588-5590).

[0004] In addition, cyclized peptides can increase the in vivo stability of peptide drugs. Studies have shown that due to the proteolytic stability and conformational rigidity of cyclized peptides, they exhibit good antibacterial activity and strong cell selectivity, and can also reduce host cell toxicity (Chinese, 2022 Dec 25; 39 (6): 1247-1253). Peptide drugs generally present four common types of cyclization, including: head-tail cyclization, head-side chain cyclization, side chain-tail cyclization, and side chain-side chain cyclization (Chemistry, 2021, 27 (5): 1487-1513). At present, stapled peptides obtained by cyclizing the peptide chain by covalently bonding two amino acids on the same helical surface (such as positions i and i+4, i and i+7) are often used to stabilize the α-helical conformation, thereby maintaining its binding ability to the target, such as ring-closing metathesis (RCM), click chemistry between alkynes and azides, disulfide bonds and thioethers, etc. (Journal of Peptide Science. 2022, 28(6): e3387). Moreover, compared with linear peptides, stapled peptides have outstanding advantages such as higher binding affinity, target selectivity, cell permeability, proteolytic stability and the ability to regulate protein-protein interactions (PPIs).

[0005] However, current stapling strategies often need to be performed on "treated" peptide substrates, such as introducing non-natural amino acids as specific reaction sites or protecting highly active side chain groups. However, if the pretreatment of the substrate can be avoided and the natural peptide can be selectively stapled directly, the applicability of stapled peptides will be greatly broadened, providing a powerful modification tool for drug development.

[0006] Therefore, it is of great significance to develop green and mild methods to achieve peptide macrocyclization and apply them to the development of peptide drugs with specific functions and the study of the structural functions of compounds. Summary of the invention

[0007] Based on the above, the present invention aims to provide a preparation method, product and application of a urea-containing macrocyclic peptide compound. The preparation method provided by the present invention has the advantages of high reaction economy, mild conditions, wide substrate applicability, and simple operation. The present invention can be used for the diverse modification of polypeptides / proteins, providing a theoretical basis for the study of polypeptide drugs with specific functions and the structural functions of compounds, and has broad application prospects in the fields of medicinal chemistry and polypeptide drug innovation.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] One of the technical solutions of the present invention is a method for preparing a urea-containing macrocyclic peptide compound, characterized in that it comprises the following steps:

[0010] A polypeptide containing two exposed amino groups is dissolved in an organic solution, and then a diisocyanate is added to react, and then a base is added dropwise to the reaction system to react, thereby obtaining a urea-containing macrocyclic peptide compound;

[0011] The polypeptide containing two exposed amino groups is a polypeptide having an N-terminal amino group-side chain amino group or a side chain amino group-side chain amino group.

[0012] The second technical solution of the present invention is a urea-containing macrocyclic peptide compound prepared by the above preparation method.

[0013] The third technical solution of the present invention is the use of the above-mentioned urea-containing macrocyclic peptide compound in the preparation of a drug for treating human non-small cell lung cancer (A549-Luc).

[0014] A fourth technical solution of the present invention is the use of the above-mentioned urea-containing macrocyclic peptide compound in the preparation of a drug for treating human colorectal adenocarcinoma (Caco-2).

[0015] The present invention discloses the following technical effects:

[0016] (1) The method provided by the present invention uses diisocyanate as a symmetrical cross-linking agent to react with the same two Lys residue side chains or N-terminal amino-Lys residue side chains on a natural polypeptide to form a novel urea-containing macrocyclic peptide compound, which has the advantages of being convenient, simple to operate, and mild conditions.

[0017] (2) The method of the present invention is green and environmentally friendly, easy to operate, does not require heating, does not require directing groups, does not involve metals, and can be exposed to the air for reaction without the need for inert gas protection.

[0018] (3) The synthetic strategy of the stapled peptides in the present invention has very high chemical selectivity and specificity, high raw material conversion rate, high yield, and wide substrate adaptability.

[0019] (4) The present invention can reduce the deformability of the molecule or increase the hydrophobicity through cyclization to enhance the drug-like properties and diversify the application of cyclic peptides.

[0020] (5) The present invention can be extended to selectively modify more complex peptides or even proteins, and can also be applied to the design of stapled peptide molecular libraries to achieve the construction of stapled peptide drug screening platforms.

[0021] In summary, the method of the present invention can greatly enrich the library of cyclic peptide synthesis methods and provide a theoretical basis for the development of specific functional polypeptide drugs and the study of compound structure and function. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the LC tracer of the reaction solution of the target product in Example 1;

[0023] Figure 2 This is the pure LC tracer of the target product in Example 1;

[0024] Figure 3 This is the LC tracer of the reaction solution of the target product in Example 2;

[0025] Figure 4 This is the pure LC tracer of the target product in Example 2;

[0026] Figure 5 This is the LC tracer of the reaction solution of the target product in Example 3;

[0027] Figure 6 This is the pure LC tracer of the target product in Example 3;

[0028] Figure 7 This is the LC tracer of the reaction solution of the target product in Example 4;

[0029] Figure 8 This is the pure LC tracer of the target product in Example 4;

[0030] Fig. 9 This is the LC tracer of the reaction solution of the target product in Example 5;

[0031] Fig.10 This is the pure LC tracer of the target product in Example 5;

[0032] Fig.11 This is the LC tracer of the reaction solution of the target product in Example 6;

[0033] Fig.12 This is the pure LC tracer of the target product in Example 6;

[0034] Fig.13 This is the LC tracer of the reaction solution of the target product in Example 7;

[0035] Fig.14 This is the pure LC tracer of the target product in Example 7;

[0036] Fig.15 This is the LC tracer of the reaction solution of the target product in Example 8;

[0037] Fig.16 This is the pure LC tracer of the target product in Example 8;

[0038] Fig.17 This is the LC tracer of the reaction solution of the target product in Example 9;

[0039] Fig.18 This is the pure LC tracer of the target product in Example 9;

[0040] Fig.19 This is the LC tracer of the reaction solution of the target product in Example 10;

[0041] Fig. 20 This is the LC tracer of the pure target product in Example 10;

[0042] Fig.21 This is the LC tracer of the reaction solution of the target product in Example 11;

[0043] Fig. 22 This is the pure LC tracer of the target product in Example 11;

[0044] Fig.23 This is the LC tracer of the reaction solution of the target product in Example 12;

[0045] Fig.24 This is the pure LC tracer of the target product in Example 12;

[0046] Fig.25 This is the LC tracer of the reaction solution of the target product in Example 13;

[0047] Fig.26 This is the pure LC tracer of the target product in Example 13;

[0048] Fig. 27 This is the LC tracer of the reaction solution of the target product in Example 14;

[0049] Fig.28 This is the pure LC tracer of the target product in Example 14;

[0050] Fig.29 This is the LC tracer of the reaction solution of the target product in Example 15;

[0051] Fig.30 This is the pure LC tracer of the target product in Example 15;

[0052] Fig.31 The in vitro anti-tumor test results of the target product C10 in Example 16;

[0053] Fig.32 These are the in vitro anti-tumor experimental results of the target product C11 in Example 17. DETAILED DESCRIPTION

[0054] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0055] The first aspect of the present invention provides a method for preparing a urea-containing macrocyclic peptide compound, comprising the following steps:

[0056] A polypeptide containing two exposed amino groups is dissolved in an organic solvent, and then a diisocyanate is added to react, and then a base is added dropwise to the reaction system to react to obtain a urea-containing macrocyclic peptide compound;

[0057] The polypeptide containing two exposed amino groups is a polypeptide with an N-terminal amino group and a side chain amino group or a polypeptide with a side chain amino group and a side chain amino group.

[0058] In some embodiments of the present invention, the polypeptide containing two naked amino groups is an oligopeptide containing two naked amino groups, a bioactive peptide containing two naked amino groups, or a polypeptide drug containing two naked amino groups.

[0059] In some embodiments of the present invention, the polypeptide containing two naked amino groups includes an acetyl, 9-fluorenylmethoxycarbonyl or methyl ester protected polypeptide.

[0060] In some embodiments of the present invention, the polypeptide containing two naked amino groups includes a polypeptide having a naked guanidine group, an amide group, an imidazole group, a hydroxyl group, a phenolic hydroxyl group, an indole group, a thiol group or a carboxyl group on the side chain.

[0061] In some embodiments of the present invention, the structural formula of the polypeptide is as shown in Formula A:

[0062]

[0063] In formula A, R 1 is hydrogen or acetyl; R 2 is hydroxy, amino, benzyl or methoxy.

[0064] In some embodiments of the present invention, the structural formula of the diisocyanate is: Wherein, R is xylene, cyclohexane dimethyl, tetramethylxylene or dicyclohexylmethane.

[0065] In some embodiments of the present invention, the molar ratio of the diisocyanate to the polypeptide containing two exposed amino groups is 1:1-1:2.

[0066] In some embodiments of the present invention, the organic solvent is one or a mixed solvent of two of acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, methanol, tetrahydrofuran, and acetone.

[0067] In the present invention, when adding diisocyanate, the diisocyanate is first dissolved in an organic solvent; the organic solvent for dissolving the diisocyanate is consistent with the organic solvent for dissolving the polypeptide containing two exposed amino groups.

[0068] The present invention does not impose any particular limitation on the amount of organic solvent used, as long as the amount of the organic solvent can fully dissolve the polypeptide and the diisocyanate and ensure the amount of solvent required for the reaction.

[0069] In some embodiments of the present invention, the reaction temperature is room temperature and the reaction time is 1 h-5 h.

[0070] In some embodiments of the present invention, the base is triethylenediamine (DABCO), triethylamine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1,5-diazabicyclo[4.3.0]non-5-ene (DBN), 4-dimethylaminopyridine (DMAP), tetramethylethylenediamine (TMEDA) or tetramethylguanidine (TMG); the amount of the base used is 1 to 5 eq.

[0071] In some embodiments of the present invention, the reaction is an air atmosphere, non-direct exposure reaction.

[0072] The reaction formula of the preparation method of the above-mentioned urea-containing macrocyclic peptide compound is as follows:

[0073]

[0074] In the present invention, the separation and purification is preferably carried out by semi-preparative HPLC. The present invention does not specifically limit the methods and parameters of concentration, drying and freeze-drying, and conventional technical means of those skilled in the art can be used.

[0075] The second aspect of the present invention provides a urea-containing macrocyclic peptide compound prepared by the above preparation method.

[0076] The third aspect of the present invention provides a use of the above-mentioned urea-containing macrocyclic peptide compound in the preparation of a drug for treating human non-small cell lung cancer (A549-Luc).

[0077] The fourth aspect of the present invention provides a use of the above-mentioned urea-containing macrocyclic peptide compound in the preparation of a drug for treating human colorectal adenocarcinoma (Caco-2).

[0078] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0079] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with the embodiments, but the content of the present invention is not limited to the following embodiments.

[0080] Example 1

[0081] Peptide A1 (1.0 eq, 0.01 mmol) with two exposed amino groups was added to a 25 ml glass flask equipped with a magnetic stirrer. After dissolving it with 10 ml of DMSO:DMF mixed solvent (volume ratio 1:1), m-phenylenediisocyanate B1 (1.0 eq, 0.01 mmol) with the same volume of solvent was slowly added dropwise to the flask. After the reaction was allowed to proceed for 1 hour, triethylamine (1.6 eq, 0.016 mmol) was added and the mixture was stirred at room temperature for 4 hours. Finally, the reaction solution was concentrated and dried, dissolved with a small amount of methanol, and then directly injected into a semi-preparative HPLC for separation and purification. After freeze-drying, the cyclization product C1 was obtained as a white solid. The product molecular formula is C 36 H 48 N 8 O 7 , yield 84%.

[0082] The structural formula and product characterization data of the obtained product are shown below:

[0083]

[0084] HRMS-ESI(m / z):calcd for C 36 H 47 N 8 O 7 - [MH] - :703.3573; found:703.3575.

[0085] Example 2

[0086] Peptide A2 (1.0 eq, 0.01 mmol) with two exposed amino groups was added to a 25 ml glass flask equipped with a magnetic stirrer. After dissolving it with 10 ml of DMSO:DMF mixed solvent (volume ratio 1:1), m-phenylenediisocyanate B1 (1.0 eq, 0.01 mmol) with the same volume of solvent was slowly added dropwise to the flask. After the reaction was allowed to proceed for 1 hour, triethylamine (1.6 eq, 0.016 mmol) was added and the mixture was stirred at room temperature for 4 hours. Finally, the reaction solution was concentrated and dried, dissolved with a small amount of methanol, and then directly injected into a semi-preparative HPLC for separation and purification. After freeze-drying, the cyclization product C2 was obtained as a white solid. The product molecular formula is C 29 H 37 N 7 O 5 , yield 54%.

[0087] The structural formula and product characterization data of the obtained product are shown below:

[0088]

[0089] HRMS-ESI(m / z):calcd forC 29 H 37 N 7 O 5 [M]:563.2783; found:563.2779.

[0090] Example 3

[0091] Peptide A3 (1.0 eq, 0.01 mmol) with two exposed amino groups was added to a 25 ml glass flask equipped with a magnetic stirrer. After dissolving it with 10 ml of DMSO:DMF mixed solvent (volume ratio 1:1), m-phenylenediisocyanate B1 (1.0 eq, 0.01 mmol) with the same volume of solvent was slowly added dropwise to the flask. After the reaction was allowed to proceed for 1 hour, triethylamine (1.6 eq, 0.016 mmol) was added and the mixture was stirred at room temperature for 4 hours. Finally, the reaction solution was concentrated and dried, dissolved with a small amount of methanol, and then directly injected into a semi-preparative HPLC for separation and purification. After freeze-drying, the cyclization product C3 was obtained as a white solid. The product molecular formula is C 32 H 47 N 11 O 7 , yield 44%.

[0092] The structural formula and product characterization data of the obtained product are shown below:

[0093]

[0094] HRMS-ESI(m / z):calcd for C 32 H 46 N 11 O 7 - [MH] - :696.3587; found:696.3584.

[0095] Example 4

[0096] Peptide A4 (1.0 eq, 0.01 mmol) with two exposed amino groups was added to a 25 ml glass flask equipped with a magnetic stirrer. After dissolving it with 10 ml of DMSO:DMF mixed solvent (volume ratio 1:1), m-phenylenediisocyanate B1 (1.0 eq, 0.01 mmol) with the same volume of solvent was slowly added dropwise to the flask. After the reaction was allowed to proceed for 1 hour, triethylamine (1.6 eq, 0.016 mmol) was added and the mixture was stirred at room temperature for 4 hours. Finally, the reaction solution was concentrated and dried, dissolved with a small amount of methanol, and then directly injected into a semi-preparative HPLC for separation and purification. After freeze-drying, the cyclization product C4 was obtained as a white solid. The product molecular formula is C 20 H 29 N 5 O 5 , yield 47%.

[0097] The structural formula and product characterization data of the obtained product are shown below:

[0098]

[0099] HRMS-ESI(m / z):calcd for C 20 H 29 N 5 O 5 [M]:419.2129; found:419.2130.

[0100] Example 5

[0101] Peptide A5 (1.0 eq, 0.01 mmol) with two exposed amino groups was added to a 25 ml glass flask equipped with a magnetic stirrer. After dissolving it with 10 ml of DMSO:DMF mixed solvent (volume ratio 1:1), m-phenylenediisocyanate B1 (1.0 eq, 0.01 mmol) with the same volume of solvent was slowly added dropwise to the flask. After the reaction was allowed to proceed for 1 hour, triethylamine (1.6 eq, 0.016 mmol) was added and the mixture was stirred at room temperature for 4 hours. Finally, the reaction solution was concentrated and dried, dissolved with a small amount of methanol, and then directly injected into a semi-preparative HPLC for separation and purification. After freeze-drying, the cyclization product C5 was obtained as a white solid. The product molecular formula is C 21 H 32 N 6 O 4 , yield 44%.

[0102] The structural formula and product characterization data of the obtained product are shown below:

[0103]

[0104] HRMS-ESI(m / z):calcd forC21 H 31 N 6 O 4 - [MH] - :431.2412; found:431.2408.

[0105] Example 6

[0106] Peptide A6 (1.0 eq, 0.01 mmol) with two exposed amino groups was added to a 25 ml glass flask equipped with a magnetic stirrer. After dissolving it with 10 ml of DMSO:DMF mixed solvent (volume ratio 1:1), m-phenylenediisocyanate B1 (1.0 eq, 0.01 mmol) with the same volume of solvent was slowly added dropwise to the flask. After the reaction was allowed to proceed for 1 hour, triethylamine (1.6 eq, 0.016 mmol) was added and the mixture was stirred at room temperature for 4 hours. Finally, the reaction solution was concentrated and dried, dissolved with a small amount of methanol, and then directly injected into a semi-preparative HPLC for separation and purification. After freeze-drying, the cyclization product C6 was obtained as a white solid. The product molecular formula is C 48 H 69 N 11 O 10 , yield 87%.

[0107] The structural formula and product characterization data of the obtained product are shown below:

[0108]

[0109] HRMS-ESI(m / z):calcd for C 48 H 68 N 11 O 10 - [MH] - :958.5156; found:958.5155.

[0110] Example 7

[0111] A polypeptide A7 (1.0 eq, 0.01 mmol) with two exposed amino groups was added to a 25 ml glass flask equipped with a magnetic stirrer. After dissolving it with 10 ml of a DMSO:DMF mixed solvent (volume ratio 1:1), m-phenylenediisocyanate B1 (1.0 eq, 0.01 mmol) with the same volume of solvent was slowly added dropwise to the flask. After the reaction was allowed to proceed for 1 hour, triethylamine (1.6 eq, 0.016 mmol) was added and the mixture was stirred at room temperature for 4 hours. Finally, the reaction solution was concentrated and dried, dissolved with a small amount of methanol, and then directly injected into a semi-preparative HPLC for separation and purification. After freeze-drying, the cyclized product C7 was obtained as a white solid. The product molecular formula is C48 H 57 N 11 O 7 , yield 64%.

[0112] The structural formula and product characterization data of the obtained product are shown below:

[0113]

[0114] HRMS-ESI(m / z):calcd for C 49 H 56 N 11 O 7 - [MH] - :898.4370; found:898.4371.

[0115] Example 8

[0116] Peptide A8 (1.0 eq, 0.01 mmol) with two exposed amino groups was added to a 25 ml glass flask equipped with a magnetic stirrer. After dissolving it with 10 ml of DMSO:DMF mixed solvent (volume ratio 1:1), m-phenylenediisocyanate B1 (1.0 eq, 0.01 mmol) with the same volume of solvent was slowly added dropwise to the flask. After the reaction was allowed to proceed for 1 hour, triethylamine (1.6 eq, 0.016 mmol) was added and the mixture was stirred at room temperature for 4 hours. Finally, the reaction solution was concentrated and dried, dissolved with a small amount of methanol, and then directly injected into a semi-preparative HPLC for separation and purification. After freeze-drying, the cyclized product C8 was obtained as a white solid. The product molecular formula is C 40 H 57 N 11 O 11 , yield 68%.

[0117] The structural formula and product characterization data of the obtained product are shown below:

[0118]

[0119] HRMS-ESI(m / z):calcd for C 40 H 56 N 11 O 11 - [MH] - :866.4166; found:866.4163.

[0120] Example 9

[0121] Peptide A9 (1.0 eq, 0.01 mmol) with two exposed amino groups was added to a 25 ml glass flask equipped with a magnetic stirrer. After dissolving it with 10 ml of DMSO:DMF mixed solvent (volume ratio 1:1), m-phenylenediisocyanate B1 (1.0 eq, 0.01 mmol) with the same volume of solvent was slowly added dropwise to the flask. After the reaction was allowed to proceed for 1 hour, triethylamine (1.6 eq, 0.016 mmol) was added and the mixture was stirred at room temperature for 4 hours. Finally, the reaction solution was concentrated and dried, dissolved with a small amount of methanol, and then directly injected into a semi-preparative HPLC for separation and purification. After freeze-drying, the cyclized product C9 was obtained as a white solid. The product molecular formula is C 75 H 127 N 25 O 21 , yield 45%.

[0122] The structural formula and product characterization data of the obtained product are shown below:

[0123]

[0124] HRMS-ESI(m / z):calcd for C 75 H 129 N 25 O 21 2+ [(M+2H + ) / 2]:857.9892; found:857.9901.

[0125] Example 10

[0126] Peptide A10 (1.0 eq, 0.01 mmol) with two exposed amino groups was added to a 25 ml glass flask equipped with a magnetic stirrer. After dissolving it with 10 ml of DMSO:DMF mixed solvent (volume ratio 1:1), m-phenylenediisocyanate B1 (1.0 eq, 0.01 mmol) with the same volume of solvent was slowly added dropwise to the flask. After the reaction was allowed to proceed for 1 hour, triethylamine (1.6 eq, 0.016 mmol) was added and the mixture was stirred at room temperature for 4 hours. Finally, the reaction solution was concentrated and dried, dissolved with a small amount of methanol, and then directly injected into a semi-preparative HPLC for separation and purification. After freeze-drying, the cyclization product C10 was obtained as a white solid. The product molecular formula is C 84 H 110 N 26 O 12 , yield 76%.

[0127] The structural formula and product characterization data of the obtained product are shown below:

[0128]

[0129] HRMS-ESI(m / z):calcd for C 84 H 112 N 26 O 12 2+ [(M+2H + ) / 2]:838.4471; found:838.4481

[0130] Embodiment 11

[0131] Peptide A11 (1.0 eq, 0.01 mmol) with two exposed amino groups was added to a 25 ml glass flask equipped with a magnetic stirrer. After dissolving it with 10 ml of DMSO:DMF mixed solvent (volume ratio 1:1), m-phenylenediisocyanate B1 (1.0 eq, 0.01 mmol) with the same volume of solvent was slowly added dropwise to the flask. After the reaction was allowed to proceed for 1 hour, triethylamine (1.6 eq, 0.016 mmol) was added and the mixture was stirred at room temperature for 4 hours. Finally, the reaction solution was concentrated and dried, dissolved with a small amount of methanol, and then directly injected into a semi-preparative HPLC for separation and purification. After freeze-drying, the cyclized product C11 was obtained as a white solid. The product molecular formula is C 65 H 114 N 18 O 12 , yield 19%.

[0132] The structural formula and product characterization data of the obtained product are shown below:

[0133]

[0134] HRMS-ESI(m / z):calcd for C 65 H 116 N 18 O 12 2+ [(M+2H + ) / 2]:670.4505; found:670.4520

[0135] Example 12

[0136] Peptide A12 (1.0 eq, 0.01 mmol) with two exposed amino groups was added to a 25 ml glass flask equipped with a magnetic stirrer. After dissolving it with 10 ml of DMSO:DMF mixed solvent (volume ratio 1:1), m-phenylenediisocyanate B1 (1.0 eq, 0.01 mmol) with the same volume of solvent was slowly added dropwise to the flask. After the reaction was allowed to proceed for 1 hour, triethylamine (1.6 eq, 0.016 mmol) was added and the mixture was stirred at room temperature for 4 hours. Finally, the reaction solution was concentrated and dried, dissolved with a small amount of methanol, and then directly injected into a semi-preparative HPLC for separation and purification. After freeze-drying, the cyclized product C12 was obtained as a white solid. The product molecular formula is C 78 H 112 N 22 O 16 , yield 53%.

[0137] The structural formula and product characterization data of the obtained product are shown below:

[0138]

[0139] HRMS-ESI(m / z):calcd for C 78 H 114 N 22 O 16 2+ [(M+2H + ) / 2]:807.4386; found:807.4384

[0140] Embodiment 13

[0141] Peptide A13 (1.0 eq, 0.01 mmol) with two exposed amino groups was added to a 25 ml glass flask equipped with a magnetic stirrer. After dissolving it with 10 ml of DMSO:DMF mixed solvent (volume ratio 1:1), m-phenylenediisocyanate B1 (1.0 eq, 0.01 mmol) with the same volume of solvent was slowly added dropwise to the flask. After the addition was completed, the reaction was continued for 1 hour. Triethylamine (1.6 eq, 0.016 mmol) was then added and stirred at room temperature for 4 hours. Finally, the reaction solution was concentrated and dried, and then dissolved with a small amount of methanol and directly injected into semi-preparative HPLC for separation and purification. After freeze-drying, the cyclization product C13, a white solid, was obtained. The product molecular formula is C 87 H 137 N 27 O 17 , yield 23%.

[0142] The structural formula and product characterization data of the obtained product are shown below:

[0143]

[0144] HRMS-ESI(m / z):calcd for C 87 H 140 N 27 O 17 3+ [(M+3H + ) / 2]:917.5432; found:917.5419

[0145] Embodiment 14

[0146] Peptide A14 (1.0 eq, 0.01 mmol) with two exposed amino groups was added to a 25 ml glass flask equipped with a magnetic stirrer. After dissolving it with 10 ml of DMSO:DMF mixed solvent (volume ratio 1:1), m-phenylenediisocyanate B1 (1.0 eq, 0.01 mmol) with the same volume of solvent was slowly added dropwise to the flask. After the reaction was allowed to proceed for 1 hour, triethylamine (1.6 eq, 0.016 mmol) was added and the mixture was stirred at room temperature for 4 hours. Finally, the reaction solution was concentrated and dried, dissolved with a small amount of methanol, and then directly injected into a semi-preparative HPLC for separation and purification. After freeze-drying, the cyclized product C14 was obtained as a white solid. The product molecular formula is C 92 H 127 N 23 O 23 S, yield 81%.

[0147] The structural formula and product characterization data of the obtained product are shown below:

[0148]

[0149] HRMS-ESI(m / z):calcd for C 92 H 128 N 23 O 23 S + [M+H] + :1954.9269; found:1954.9274

[0150] Embodiment 15

[0151] Peptide A15 (1.0 eq, 0.01 mmol) with two exposed amino groups was added to a 25 ml glass flask equipped with a magnetic stirrer. After dissolving it with 10 ml of DMSO:DMF mixed solvent (volume ratio 1:1), m-phenylenediisocyanate B1 (1.0 eq, 0.01 mmol) with the same volume of solvent was slowly added dropwise to the flask. After the reaction was allowed to proceed for 1 hour, triethylamine (1.6 eq, 0.016 mmol) was added and the mixture was stirred at room temperature for 4 hours. Finally, the reaction solution was concentrated and dried, dissolved with a small amount of methanol, and then directly injected into a semi-preparative HPLC for separation and purification. After freeze-drying, the cyclized product C15 was obtained as a white solid. The product molecular formula is C 114 H 144 N 24 O 25 S, yield 84%.

[0152] The structural formula and product characterization data of the obtained product are shown below:

[0153]

[0154] HRMS-ESI(m / z):calcd for C 114 H 146 N 24 O 25 S 2+ [(M+2H + ) / 2]:1141.5300; found:1141.5294

[0155] Example 16

[0156] This example is an in vitro antitumor experiment of the urea-containing macrocyclic peptide compound C10.

[0157] 1 Experimental methods:

[0158] 1.1 Cell culture

[0159] Human non-small cell lung cancer (A549-Luc) cells were cultured in 10% FBS (Fetal bovine serum) and 1% Penicillin-Streptomycin and 89% RPMI Medium 1640 basic (Roswell Park Memorial Institute) medium (containing (L)-glutamine) at 37°C and 5% CO 2 , cultured under saturated humidity, and cells in the logarithmic growth phase were used for the experiment.

[0160] 1.2 CCK8 colorimetric assay to detect the effect of drugs on cell viability

[0161] (1) Collect cells in the logarithmic growth phase, centrifuge at 1000r for 3 minutes, discard the old culture medium, and add an appropriate amount of fresh complete culture medium for cell counting.

[0162] (2) Adjust the cell concentration to 5x10 4 / mL was inoculated into a 96-well plate, 100 μl per well, which means 5000 cells per well.

[0163] (3) Place the 96-well plate in a 37°C, 5% CO 2 , cultured under saturated humidity for 24 hours.

[0164] (4) Prepare the drug into four concentrations of 400 μM, 200 μM, 100 μM, 50 μM, 25 μM, and 12.5 μM, and add 100 μl of each to the experimental well.

[0165] The concentrations in the wells were adjusted to 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, and 6.25 μM; 100 μl of culture medium was added to the control wells and blank wells.

[0166] (5) Cultivate cells at 37°C and 5% CO 2 , cultured for 24 hours under saturated humidity; after the culture was completed, 10 μl CCK-8 (Cell Counting Kit-8) was added to each well, and after culture for 2-4 hours, the light absorption value at 450 nm was detected using an enzyme reader.

[0167] (6) Calculate the inhibition rate of the drug according to the following formula: Inhibition rate = (OD 实验 -OD 空白 ) / OD 对照 *100%.

[0168] As: OD experiment: experimental group (culture medium containing cells, CCK-8, test substance), OD blank: blank well (culture medium without cells and test substance, CCK-8), OD control: control well (culture medium containing cells, CCK-8, no test substance).

[0169] 2 The results of in vitro antitumor experiments of urea-containing macrocyclic peptide compound C10 are shown in Fig.31 As shown, the inhibitory activity against A549-Luc cells was measured to be 20.94 μM. It can be seen that the compound C10 containing the urea macrocyclic peptide of the present invention has inhibitory activity against A549-Luc cells.

[0170] 3. The present invention also refers to the experimental process of Example 16 to verify the inhibitory activity of the compounds containing urea macrocyclic peptides in Examples 1-9 and Examples 11-15 (i.e., the target products) on human non-small cell lung cancer (A549-Luc) cells. The experimental results show that the compounds containing urea macrocyclic peptides of the present invention have inhibitory activity on human non-small cell lung cancer (A549-Luc) cells.

[0171] Embodiment 17

[0172] This example is an in vitro antitumor experiment of the urea-containing macrocyclic peptide compound C11.

[0173] 1 Experimental methods:

[0174] 1.1 Cell culture

[0175] Human colorectal adenocarcinoma (Caco-2) cells were cultured in 10% FBS (fetal bovine serum) and 1% Penicillin-Streptomycin and 89% RPMI Medium 1640 basic (Roswell Park Memorial Institute) medium (containing (L)-glutamine) at 37°C and 5% CO. 2 , cultured under saturated humidity, and cells in the logarithmic growth phase were used for the experiment.

[0176] 1.2 CCK-8 colorimetric assay to detect the effect of drugs on cell viability

[0177] (1) Collect cells in the logarithmic growth phase, centrifuge at 1000r for 3 minutes, discard the old culture medium, and add an appropriate amount of fresh complete culture medium for cell counting.

[0178] (2) Adjust the cell concentration to 5x10 4 / mL was inoculated into a 96-well plate, 100 μl per well, which means 5000 cells per well.

[0179] (3) Place the 96-well plate in a 37°C, 5% CO 2 , cultured under saturated humidity for 24 hours.

[0180] (4) Prepare the drug into four concentrations of 400 μM, 200 μM, 100 μM, 50 μM, 25 μM, and 12.5 μM, and add 100 μl of each into the experimental wells to make the concentrations in the wells 200 μM, 100 μM, 50 μM, 25 μM, 12.5 μM, and 6.25 μM; add 100 μl of culture medium into the control wells and blank wells.

[0181] (5) Cultivate cells at 37°C and 5% CO 2, cultured for 24 hours under saturated humidity; after the culture was completed, 10 μl CCK-8 (Cell Counting Kit-8) was added to each well, and after culture for 2-4 hours, the light absorption value at 450 nm was detected using an enzyme reader.

[0182] (6) Calculate the inhibition rate of the drug according to the following formula: Inhibition rate = (OD 实验 -OD 空白 ) / OD 对照 *100%.

[0183] As: OD experiment: experimental group (culture medium containing cells, CCK-8, test substance), OD blank: blank well (culture medium without cells and test substance, CCK-8), OD control: control well (culture medium containing cells, CCK-8, no test substance).

[0184] 2 The results of in vitro antitumor experiments of urea-containing macrocyclic peptide compound C11 are shown in Fig.32 As shown, the inhibitory activity against human colorectal adenocarcinoma (Caco-2) cells was measured to be 16.39 μM. It can be seen that the compound C11 containing the urea macrocyclic peptide of the present invention has inhibitory activity against human colorectal adenocarcinoma (Caco-2) cells.

[0185] 3. The present invention also refers to the experimental process of Example 17 to verify the inhibitory activity of the compounds containing urea macrocyclic peptides in Examples 1-10 and Examples 12-15 (i.e., the target products) on human colorectal adenocarcinoma (Caco-2) cells. The experimental results show that the compounds containing urea macrocyclic peptides of the present invention have inhibitory activity on human colorectal adenocarcinoma (Caco-2) cells.

[0186] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a urea-containing macrocyclic peptide compound, characterized in that: The following steps are involved: A polypeptide containing two exposed amino groups is dissolved in an organic solvent, a diisocyanate is added to react, and then a base is added dropwise to the reaction system to react, thereby obtaining a urea-containing macrocyclic peptide compound; The polypeptide containing two exposed amino groups is a polypeptide having an N-terminal amino group and a side chain amino group or a side chain amino group and a side chain amino group.

2. The method for preparing a urea-containing macrocyclic peptide compound according to claim 1, characterized in that: The structural formula of the diisocyanate is: Wherein, R is xylene, cyclohexane dimethyl, tetramethylxylene or dicyclohexylmethane.

3. The method for preparing a urea-containing macrocyclic peptide compound according to claim 1 or 2, characterized in that: The molar ratio of the diisocyanate to the polypeptide containing two exposed amino groups is 1:1-1:

2.

4. The method for preparing a urea-containing macrocyclic peptide compound according to claim 1, characterized in that: The organic solvent is one or a mixed solvent of two of acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, methanol, tetrahydrofuran and acetone.

5. The method for preparing a urea-containing macrocyclic peptide compound according to claim 1, characterized in that: The reaction temperature is room temperature, the reaction time is 1 h-5 h, and the reaction is carried out in air atmosphere and is not a direct open reaction.

6. The method for preparing a urea-containing macrocyclic peptide compound according to claim 1, characterized in that: The base is triethylenediamine, triethylamine, 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5-diazabicyclo[4.3.0]non-5-ene, 4-dimethylaminopyridine, tetramethylethylenediamine or tetramethylguanidine; the amount of the base is 1-5eq.

7. A urea-containing macrocyclic peptide compound prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the urea-containing macrocyclic peptide compound according to claim 7 in the preparation of a drug for treating human non-small cell lung cancer A549-Luc.

9. Use of the urea-containing macrocyclic peptide compound according to claim 7 in the preparation of a drug for treating human colorectal adenocarcinoma Caco-2.

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