Preparation method, product and application of novel ureido cyclopeptide compound

Abstract

The invention provides a preparation method, a product and application of a novel ureido cyclopeptide compound, and belongs to the technical field of polypeptide chemical cyclization synthesis. The preparation method comprises the following steps: dissolving polypeptide containing amino or lysine residues in an organic solvent A to obtain a reaction solution 1, and dissolving carbonyl diimidazole in an organic solvent B to obtain a reaction solution 2; and sequentially adding the reaction liquid 2 and alkali into the reaction liquid 1, and reacting to obtain the novel ureido cyclopeptide compound. The preparation method provided by the invention can be carried out at room temperature, does not need an energy source, and has the advantages of convenience, simplicity in operation, mild conditions, environment friendliness and the like; the method has the advantages of high chemical reaction selectivity and specificity, high yield and wide substrate adaptability, can be used for polypeptide diversity modification, and can provide a theoretical basis for development of polypeptide drugs with specific functions and research of compound structure functions.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide chemical cyclization synthesis, and in particular to a preparation method, product and application of a novel urea-based cyclic peptide compound. Background Art

[0002] In recent years, cyclic peptides have been widely used in the fields of chemistry, life sciences and medicine. As a polypeptide chain with a cyclic structure, cyclic peptides have many significant advantages over linear peptides and other traditional drugs. Peptide cyclization has now become an important research direction in the fields of protein chemistry and medicinal chemistry, aiming to convert linear polypeptide chains into cyclic structures through specific chemical reactions. The formation of cyclic peptides by cyclization technology reduces the conformational flexibility of polypeptides, improves the stability of polypeptide drugs and prolongs their half-life in the body; at the same time, the cyclic structure of cyclic peptides is conducive to reducing entropy loss, improving its binding force with the target, better simulating the structure of natural proteins, and enhancing the efficacy of drugs; some polypeptides can enter cells by direct translocation due to their small molecular weight after cyclization, showing good cell membrane permeability, regulating protein-protein interactions (PPIs) and oral bioavailability; cyclic peptides provide more structural diversity, and by introducing different chemical modifications, the drug properties of polypeptides can be optimized and new polypeptide drugs can be discovered. Therefore, as therapeutic drug molecules, cyclic peptides help improve patients' medication convenience and compliance, and can show great potential in multiple therapeutic fields such as anti-infection, endocrine, and anti-cancer.

[0003] According to the type of bond between the two amino acids that make up the cyclic peptide, cyclic peptides can be divided into two categories: complete (containing only peptide bonds) and heterogeneous (different functional groups are also used to connect amino acids). The cyclization methods of polypeptides are: head-to-tail cyclization, that is, an amide bond is formed between the N-terminal amino group and the C-terminal carboxyl group; terminal-side chain cyclization (head to side chain or side chain to tail), that is, a covalent bond is formed between the N or C-terminus and the side chain functional group of the amino acid; side chain-side chain cyclization, that is, a covalent bond is formed between the two side chains of the amino acid. Common cyclopeptide synthesis strategies mainly include (a) ring condensation reactions based on carbonyl or carboxyl groups, including the most common amide condensation to construct amide bridges ("head-to-tail" ring formation, side chain-side chain ring formation, head-side chain ring formation, tail side chain ring formation), lactonization condensation reaction to construct lactone bond bridges, and ammaldehyde condensation reaction to construct carbon-nitrogen bridging groups (J.Am.Chem.Soc., 2023, 145, 27218–27224; J.Am.Chem. Soc.2024,146,20868-20877); (b) Construction of SS bond bridge based on cysteine ​​side chain sulfhydryl oxidation reaction (J.Org.Chem.2020,85,1495-1503; Chem.Pharm.Bull.71,435–440(2023)); (c) Construction of nitrogen heterocyclic bridging group based on copper-catalyzed alkyne-azide click reaction (CuAAC) (Nature Communications|(2023)14:3935); (d) Based on CC bonding reactions, including the development of relatively mature olefin metathesis reactions to construct olefin bridges, Suzuki-Miyaura cross-coupling reactions to construct biphenyl bridge groups, and the rise of CH activation under mild conditions in recent years, the synthesis of cyclopeptides with CH bonds has been realized (Chem. Soc. Rev., 2024, 53, 11725; Angew. Chem. Int. Ed. 10.1002 / anie.202007226). It is worth noting that in these cyclization reaction methods, both amide condensation reactions and cyclic lactone condensation reactions are highly dependent on the amino acid composition of the chain polypeptide substrate and the size of the final ring. Olefin metathesis cyclization and alkyne-azide click reactions require the introduction of non-natural amino acids in advance. Therefore, in order to provide the active sites or specific chemical properties required for the reaction, most methods still have certain limitations. They usually require the protection of amino acids in advance, or the activation of amino acid functional groups or the pre-synthesis of peptides containing special functional groups. They rely on non-natural amino acids in the peptide chain, which not only increases the complexity of the synthesis steps, but also limits the efficiency and product diversity of cyclopeptide synthesis. There are still many challenges in developing efficient and universal cyclopeptide synthesis technology and constructing cyclopeptide molecules with novel and diverse bridging groups, especially the direct cyclization based on natural amino acids is still difficult. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a preparation method, product and application of a novel urea-based cyclic peptide compound. The 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 polypeptide proteins, providing a theoretical basis for the research on 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.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] One of the technical solutions of the present invention is a method for preparing a novel urea-based cyclic peptide compound, comprising the following steps:

[0007] The polypeptide containing an amino group or a lysine residue is dissolved in an organic solvent A to obtain a reaction solution 1, and carbonyl diimidazole is dissolved in an organic solvent B to obtain a reaction solution 2;

[0008] The reaction solution 2 and a base are sequentially added to the reaction solution 1 to carry out a reaction to obtain a novel urea-based cyclic peptide compound.

[0009] The polypeptide containing an amino group or a lysine residue is a polypeptide having an N-terminal amino group and a side chain amino group or a polypeptide having a side chain amino group and a side chain amino group.

[0010] The second technical solution of the present invention is a novel urea cyclic peptide compound prepared by the above preparation method.

[0011] The third technical solution of the present invention is the use of the above-mentioned novel urea cyclic peptide compound in the preparation of a drug for treating human non-small cell lung cancer (A549-Luc).

[0012] The fourth technical solution of the present invention is a human non-small cell lung cancer (A549-Luc) drug, the raw materials of which include the above-mentioned novel urea cyclic peptide compound and pharmaceutically acceptable excipients.

[0013] The present invention discloses the following technical effects:

[0014] The preparation method provided by the present invention can be carried out at room temperature, does not require an energy source, and has the advantages of being convenient, simple to operate, mild conditions, and being green and environmentally friendly.

[0015] The method provided by the invention has high chemical reaction selectivity and specificity, high yield and wide substrate adaptability.

[0016] The method of the present invention can be used for polypeptide diversity modification, and can provide a theoretical basis for the development of specific functional polypeptide drugs and the study of compound structure and function.

[0017] The invention can be used for modifying lysine in natural active peptides, and the modified natural active polypeptide analogs have certain anti-cancer activity.

[0018] In summary, the method of the present invention can be used for the cyclization modification of polypeptide molecules containing amino or lysine residues, and can 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

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 This is the LC tracer of the reaction solution before purification of the target product obtained in Example 1;

[0021] Figure 2 This is the LC tracer diagram of the target product obtained in Example 1 after purification;

[0022] Figure 3 This is the LC tracer of the reaction solution before purification of the target product obtained in Example 2;

[0023] Figure 4 This is the LC tracer diagram of the target product obtained in Example 2 after purification;

[0024] Figure 5 This is the LC tracer of the reaction solution before purification of the target product obtained in Example 3;

[0025] Figure 6 This is the LC tracer diagram of the target product obtained in Example 3 after purification;

[0026] Figure 7 This is the LC tracer of the reaction solution before purification of the target product obtained in Example 4;

[0027] Figure 8 This is the LC tracer diagram of the target product obtained in Example 4 after purification;

[0028] Fig. 9 This is the LC tracer of the reaction solution before purification of the target product obtained in Example 5;

[0029] Fig.10 This is the LC tracer diagram of the target product obtained in Example 5 after purification;

[0030] Fig.11 This is the LC tracer of the reaction solution before purification of the target product obtained in Example 6;

[0031] Fig.12 This is the LC tracer diagram of the target product obtained in Example 6 after purification;

[0032] Fig.13 This is the LC tracer of the reaction solution before purification of the target product obtained in Example 7;

[0033] Fig.14 This is the LC tracer diagram of the target product obtained in Example 7 after purification;

[0034] Fig.15 This is the LC tracer of the reaction solution before purification of the target product obtained in Example 8;

[0035] Fig.16 This is the LC tracer diagram of the target product obtained in Example 8 after purification;

[0036] Fig.17 This is the LC tracer of the reaction solution before purification of the target product obtained in Example 9;

[0037] Fig.18 This is the LC tracer diagram of the target product obtained in Example 9 after purification;

[0038] Fig.19 This is the LC tracer of the reaction solution before purification of the target product obtained in Example 10;

[0039] Fig. 20 This is the LC tracer diagram of the target product obtained in Example 10 after purification;

[0040] Fig.21 This is the LC tracer of the reaction solution before purification of the target product obtained in Example 11;

[0041] Fig. 22 This is the LC tracer of the target product obtained in Example 11 after purification.

[0042] Fig.23 The cell survival rate of polypeptide A5 and its novel urea-based cyclic peptide compound B5 in Example 5 on human non-small cell lung cancer (A549-Luc);

[0043] Fig.24 The cell survival rate of polypeptide A8 and its novel urea-based cyclic peptide compound B8 in Example 8 on human non-small cell lung cancer (A549-Luc); DETAILED DESCRIPTION

[0044] 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.

[0045] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0046] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0047] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0048] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0049] The first aspect of the present invention provides a method for preparing a novel urea-based cyclic peptide compound, comprising the following steps:

[0050] The polypeptide containing an amino group or a lysine residue is dissolved in an organic solvent A to obtain a reaction solution 1, and N,N'-thiocarbonyl-diimidazole is dissolved in an organic solvent B to obtain a reaction solution 2;

[0051] The reaction solution 2 and the base are sequentially added into the reaction solution 1 to react to obtain a novel urea-based cyclic peptide compound;

[0052] The polypeptide containing an amino group or a lysine residue is a polypeptide having an N-terminal amino group and a side chain amino group or a polypeptide having a side chain amino group and a side chain amino group.

[0053] In some embodiments of the present invention, the polypeptide containing amino or lysine residues is an N-terminal amino-side chain amino polypeptide or a side chain amino-side chain amino oligopeptide, a biologically active peptide containing amino or lysine residues, or a polypeptide drug containing amino or lysine residues.

[0054] In some embodiments of the present invention, the polypeptide containing an amino group or a lysine residue includes an acetyl, tert-butyloxycarbonyl, tert-butyl, 9-fluorenylmethoxycarbonyl, benzyloxycarbonyl or methyl ester protected polypeptide.

[0055] In some embodiments of the present invention, the polypeptide having an amino group or a lysine residue includes a polypeptide having a side chain exposed guanidine group, amide group, imidazole group, hydroxyl group, phenolic hydroxyl group, indole group, sulfhydryl group or carboxyl group.

[0056] In some embodiments of the present invention, the structural formula of the polypeptide containing an amino group or a lysine residue is as shown in Formula A:

[0057]

[0058] In formula A, R 1 is hydrogen or acetyl; R 2 is hydroxy, amino, benzyl or methoxy.

[0059] In some embodiments of the present invention, the structural formula of the carbonyl diimidazole is Wherein, R is S or O.

[0060] In some embodiments of the present invention, the molar ratio of the polypeptide containing an amino group or a lysine residue to carbonyldiimidazole is 1:1-1:2.

[0061] In some embodiments of the present invention, the organic solvent A and the organic solvent B are one or a mixed solvent of two selected from acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, methanol, tetrahydrofuran, and acetone.

[0062] The present invention does not impose any particular limitation on the amount of organic solvent A and organic solvent B, as long as the amount of solvents required for the reaction can be ensured and the polypeptide containing amino or lysine residues and carbonyldiimidazole can be fully dissolved.

[0063] In some embodiments of the present invention, 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 used is 1 to 5 eq.

[0064] In some embodiments of the present invention, the reaction temperature is room temperature, and the reaction time is 1-6 hours; the reaction atmosphere is air atmosphere, and it is a non-direct open reaction.

[0065] In some embodiments of the present invention, after the reaction is completed, the separation and purification step comprises: concentrating and drying the reaction solution, and then separating and purifying by preparative chromatography.

[0066] In the present invention, the separation and purification is preferably performed by preparative high performance liquid separation and purification. The present invention does not specifically limit the freeze drying parameters, and the freeze drying parameters commonly used by those skilled in the art can be used.

[0067] The reaction formula of the preparation method of the above-mentioned urea-containing macrocyclic peptide compound is as follows:

[0068]

[0069] The second aspect of the present invention provides a novel urea cyclic peptide compound prepared by the above preparation method.

[0070] The third aspect of the present invention provides a use of the novel ureido cyclic peptide compound in the preparation of a drug for treating human non-small cell lung cancer (A549-Luc).

[0071] The fourth aspect of the present invention provides a drug for treating human non-small cell lung cancer (A549-Luc), the raw materials of which include the above-mentioned novel urea cyclic peptide compound and pharmaceutically acceptable excipients.

[0072] 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.

[0073] 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.

[0074] Example 1

[0075] 0.01mmol of polypeptide A1 (snake venom tripeptide) was added to a 25mL single-necked round-bottomed spherical bottle containing a stirring magnet, and 10mL of dimethyl sulfoxide was added thereto as a reaction solvent, and the mixture was placed at room temperature. Subsequently, 0.015mmol of N,N'-thiocarbonyl-diimidazole was diluted in 10mL of dimethyl sulfoxide and transferred to a 25mL constant pressure dropping funnel. The diluted N,N'-thiocarbonyl-diimidazole and triethylamine (1.6eq.) were added dropwise to the aforementioned single-necked round-bottomed spherical bottle in a slow and uniform manner through the funnel, and the reaction was carried out for 5h at room temperature and in an air atmosphere (non-direct open reaction). After the reaction was completed, the obtained reaction solution was freeze-dried using a freeze dryer. Subsequently, the obtained residue was subjected to high-performance liquid phase analysis, preparative high-performance liquid phase separation and purification, and finally a 13-membered ring polypeptide final product B1 was obtained. The LC yield was 65.5%.

[0076] The structural characterization data of the obtained product are shown below:

[0077]

[0078] HRMS-ESI(m / z):calcd for[MH] + C 20 H 26 N 5 O 3 S 1 :416.1762; found:416.1763.

[0079] Example 2

[0080] 0.01mmol of polypeptide A2 (Ac-Lys-Gly-His-Lys-OMe) was added to a 25mL single-necked round-bottomed spherical bottle containing a stirring magnet, and 10mL of dimethyl sulfoxide was added thereto as a reaction solvent, and the mixture was placed at room temperature. Subsequently, 0.015mmol of N,N'-thiocarbonyl-diimidazole was diluted in 10mL of dimethyl sulfoxide and transferred to a 25mL constant pressure dropping funnel. The diluted N,N'-thiocarbonyl-diimidazole and triethylamine (1.6eq.) were added dropwise to the aforementioned single-necked round-bottomed spherical bottle in a slow and uniform manner through the funnel, and the reaction was carried out for 5h at room temperature and in an air atmosphere (non-direct open reaction). After the reaction was completed, the obtained reaction solution was freeze-dried using a freeze dryer. Subsequently, the obtained residue was subjected to high-performance liquid phase analysis and separation and purification by preparative high-performance liquid phase, and finally a 21-membered ring polypeptide final product B2 was obtained. The LC yield was 72%.

[0081] The structural characterization data of the obtained product are shown below:

[0082]

[0083] HRMS-ESI(m / z):calcd for[MH] + C 23 H 36 N 9 O 5 S 1 :550.2566; found:550.2567.

[0084] Example 3

[0085] 0.01mmol of polypeptide A3 (Ac-Lys-Trp-Lys-OMe) was added to a 25mL single-necked round-bottomed spherical bottle containing a stirring magnet, and 10mL of dimethyl sulfoxide was added thereto as a reaction solvent, and the mixture was placed at room temperature. Subsequently, 0.015mmol of N,N'-thiocarbonyl-diimidazole was diluted in 10mL of dimethyl sulfoxide and transferred to a 25mL constant pressure dropping funnel. The diluted N,N'-thiocarbonyl-diimidazole and triethylamine (1.6eq.) were added dropwise to the aforementioned single-necked round-bottomed spherical bottle in a slow and uniform manner through the funnel, and the reaction was carried out for 5h at room temperature and in an air atmosphere (non-direct open reaction). After the reaction was completed, the obtained reaction solution was freeze-dried using a freeze dryer. Subsequently, the obtained residue was subjected to high-performance liquid phase analysis and separation and purification by preparative high-performance liquid phase, and finally the 18-membered ring polypeptide final product B3 was obtained. The LC yield was 16%.

[0086] The structural characterization data of the obtained product are shown below:

[0087]

[0088] HRMS-ESI(m / z):calcd for[MH] + C 27 H 39 N 6 O 5 S 1 :559.2697found:559.2685

[0089] Example 4

[0090] 0.01mmol of polypeptide A4 (Ac-Ala-Lys-Lys-OMe) was added to a 25mL single-necked round-bottomed spherical bottle containing a stirring magnet, and 10mL of dimethyl sulfoxide was added thereto as a reaction solvent, and the mixture was placed at room temperature. Subsequently, 0.015mmol of N,N'-thiocarbonyl-diimidazole was diluted in 10mL of dimethyl sulfoxide and transferred to a 25mL constant pressure dropping funnel. The diluted N,N'-thiocarbonyl-diimidazole and triethylamine (1.6eq.) were added dropwise to the aforementioned single-necked round-bottomed spherical bottle in a slow and uniform manner through the funnel, and the reaction was carried out for 5h at room temperature and in an air atmosphere (non-direct open reaction). After the reaction was completed, the reaction solution was freeze-dried using a freeze dryer. Subsequently, the obtained residue was subjected to high-performance liquid phase analysis and separation and purification by preparative high-performance liquid phase, and finally a 15-membered ring polypeptide final product B4 was obtained. The LC yield was 44%.

[0091] The structural characterization data of the obtained product are shown below:

[0092]

[0093] HRMS-ESI(m / z):calcd for[MH] + C 19 H 34 N 5 O 5 S 1 :444.2275found:444.2271

[0094] Example 5

[0095] 0.01mmol of polypeptide A5 (Ac-Lys-Ala-Ala-Trp-Ile--Lys-OMe) was added to a 25mL single-necked round-bottomed spherical bottle containing a stirring magnet, and 10mL of dimethyl sulfoxide was added thereto as a reaction solvent, and the mixture was placed at room temperature. Subsequently, 0.015mmol of N,N'-thiocarbonyl-diimidazole was diluted in 10mL of dimethyl sulfoxide and transferred to a 25mL constant pressure dropping funnel. The diluted N,N'-thiocarbonyl-diimidazole and triethylamine (1.6eq.) were added dropwise to the aforementioned single-necked round-bottomed spherical bottle in a slow and uniform manner through the funnel, and the reaction was carried out for 5h at room temperature and in an air atmosphere (non-direct open reaction). After the reaction was completed, the obtained reaction solution was freeze-dried using a freeze dryer. Subsequently, the obtained residue was subjected to high-performance liquid phase analysis and separation and purification by preparative high-performance liquid phase, and finally a 27-membered ring polypeptide final product B5 was obtained. LC yield 77%.

[0096] The structural characterization data of the obtained product are shown below:

[0097]

[0098] HRMS-ESI(m / z):calcd for[MH] - C 39 H 58 N 9 O 8 S 1 :812.4135found:812.4138

[0099] Example 6

[0100] 0.01mmol of polypeptide A6 (Ipamorelin) was added to a 25mL single-necked round-bottomed spherical bottle containing a stirring magnet, and 10mL of dimethyl sulfoxide was added thereto as a reaction solvent, and the mixture was placed at room temperature. Subsequently, 0.015mmol of N,N'-thiocarbonyl-diimidazole was diluted in 10mL of dimethyl sulfoxide and transferred to a 25mL constant pressure dropping funnel. The diluted N,N'-thiocarbonyl-diimidazole and triethylamine (1.6eq.) were added dropwise to the aforementioned single-necked round-bottomed spherical bottle in a slow and uniform manner through the funnel, and the reaction was carried out for 5h at room temperature and in an air atmosphere (non-direct open reaction). After the reaction was completed, the obtained reaction solution was freeze-dried using a freeze dryer. Subsequently, the obtained residue was subjected to high-performance liquid phase analysis and separation and purification by preparative high-performance liquid phase, and finally a 20-membered ring polypeptide final product B6 was obtained. The LC yield was 62.6%.

[0101] The structural characterization data of the obtained product are shown below:

[0102]

[0103] HRMS-ESI(m / z):calcd for[MH] + C 39 H 46 N 9 O 5 S 1 :752.3348found:752.3349

[0104] Example 7

[0105] Peptide A7 (H 2 N-Lys-Lys-Ala-Leu-Arg-Arg-Gln-Glu-Thr-Val-Asp-Ala-Leu-NH 2 )0.01mmol was added to a 25mL single-necked round-bottomed spherical bottle containing a stirring magnet, 10mL of dimethyl sulfoxide was added thereto as a reaction solvent, and placed at room temperature. Subsequently, 0.015mmol N,N'-thiocarbonyl-diimidazole and triethylamine (1.6eq.) were sequentially added dropwise to the aforementioned single-necked round-bottomed spherical bottle, and reacted for 5h at room temperature and air atmosphere (non-direct open reaction). After the reaction was completed, the obtained reaction solution was freeze-dried using a freeze dryer. Subsequently, the obtained residue was subjected to high-performance liquid chromatography analysis and separation and purification by preparative high-performance liquid chromatography, and finally a 15-membered ring polypeptide final product B7 was obtained. The LC yield was 63.2%.

[0106] The structural characterization data of the obtained product are shown below:

[0107]

[0108] HRMS-ESI(m / z):calcd for[MH] + / 2C 66 H 119 N 23 O 19 S 1 :784.9381found:784.9384

[0109] Example 8

[0110] Peptide A8(H 2 0.01mmol of N-Lys-Arg-Trp-Trp-Lys-Trp-Trp-Arg-Arg-OH) was added to a 25mL single-necked round-bottomed spherical bottle containing a stirring magnet, and 10mL of dimethyl sulfoxide was added thereto as a reaction solvent, and the mixture was placed at room temperature. Subsequently, 0.015mmol of N,N'-thiocarbonyl-diimidazole was diluted in 10mL of dimethyl sulfoxide and transferred to a 25mL constant pressure dropping funnel. The diluted N,N'-thiocarbonyl-diimidazole and triethylamine (1.6eq.) were added dropwise to the aforementioned single-necked round-bottomed spherical bottle in a slow and uniform manner through the funnel, and the reaction was carried out for 5h at room temperature and air atmosphere (non-direct open reaction). After the reaction was completed, the obtained reaction solution was freeze-dried using a freeze dryer. Subsequently, the obtained residue was subjected to high-performance liquid phase analysis and separation and purification by preparative high-performance liquid phase, and finally a 24-membered ring polypeptide final product B8 was obtained. LC yield 60%.

[0111] The structural characterization data of the obtained product are shown below:

[0112]

[0113] HRMS-ESI(m / z):calcd for[MH] + / 2C 75 H 102 N 24 O 10 S 1 :765.3960found:765.3961

[0114] Example 9

[0115] Peptide A9 (fmoc-HN-Leu-Ile-Lys-Arg-Ile-Lys-Val-Ile-Leu-CONH 2)0.01mmol was added to a 25mL single-necked round-bottomed spherical bottle containing a stirring magnet, 10mL of dimethyl sulfoxide was added thereto as a reaction solvent, and it was kept at room temperature. Subsequently, 0.015mmol N,N'-thiocarbonyl-diimidazole was diluted in 10mL of dimethyl sulfoxide and transferred to a 25mL constant pressure dropping funnel. Through the funnel, the diluted N,N'-thiocarbonyl-diimidazole and triethylamine (1.6eq.) were added dropwise to the aforementioned single-necked round-bottomed spherical bottle in a slow and uniform manner, and reacted for 5h at room temperature and air atmosphere (non-direct open reaction). After the reaction was completed, the obtained reaction solution was freeze-dried using a freeze dryer. Subsequently, the obtained residue was subjected to high-performance liquid phase analysis and separation and purification by preparative high-performance liquid phase, and finally a 24-membered ring polypeptide final product B9 was obtained. The LC yield was 78.7%.

[0116] The structural characterization data of the obtained product are shown below:

[0117]

[0118] HRMS-ESI(m / z):calcd for[MH] + / 2C 56 H104N16O 10 S 1 :597.3994found:597.4003

[0119] Example 10

[0120] 0.01mmol of polypeptide A10 (Ac-Lys-Leu-Val-Lys-OMe) was added to a 25mL single-necked round-bottomed spherical bottle containing a stirring magnet, and 10mL of dimethyl sulfoxide was added thereto as a reaction solvent, and the mixture was placed at room temperature. Subsequently, 0.015mmol of N,N'-carbonyl-diimidazole was diluted in 10mL of dimethyl sulfoxide and transferred to a 25mL constant pressure dropping funnel. The diluted N,N'-carbonyl-diimidazole and triethylamine (1.6eq.) were added to the single-necked round-bottomed spherical bottle in a slow and uniform manner through the funnel, and the reaction was carried out for 5h at room temperature and in an air atmosphere (non-direct open reaction). After the reaction was completed, the reaction solution was freeze-dried using a freeze dryer. Subsequently, the residue was subjected to high-performance liquid phase analysis and separation and purification by preparative high-performance liquid phase, and finally a 21-membered ring polypeptide final product B10 was obtained. The LC yield was 64%.

[0121] The structural characterization data of the obtained product are shown below:

[0122]

[0123] HRMS-ESI(m / z):calcd for[MH] + C 27 H 48 N 6 O 7 :569.3657found:569.3651

[0124] Embodiment 11

[0125] 0.01mmol of polypeptide A11 (Ac-Lys-Trp-Lys-OMe) was added to a 25mL single-necked round-bottomed spherical bottle containing a stirring magnet, and 10mL of dimethyl sulfoxide was added thereto as a reaction solvent, and the mixture was placed at room temperature. Subsequently, 0.015mmol of N,N'-carbonyl-diimidazole was diluted in 10mL of dimethyl sulfoxide and transferred to a 25mL constant pressure dropping funnel. The diluted N,N'-carbonyl-diimidazole and triethylamine (1.6eq.) were added dropwise to the aforementioned single-necked round-bottomed spherical bottle in a slow and uniform manner through the funnel, and the reaction was carried out for 5h at room temperature and in an air atmosphere (non-direct open reaction). After the reaction was completed, the reaction solution was freeze-dried using a freeze dryer. Subsequently, the residue was subjected to high-performance liquid phase analysis and separation and purification by preparative high-performance liquid phase, and finally the 18-membered ring polypeptide final product B11 was obtained. The LC yield was 45.3%.

[0126] The structural characterization data of the obtained product are shown below:

[0127]

[0128] HRMS-ESI(m / z):calcd for[MH] + C 27 H 39 N 6 O 6 :543.2926found:543.2916

[0129] Example 12

[0130] In vitro antitumor test of the novel urea cyclic peptide compound of the present invention

[0131] 1 Experimental methods:

[0132] 1.1 Cell culture

[0133] A549-Luc cells were cultured in RPMI Medium 1640 basic (Roswell Park Memorial Institute) culture medium (containing (L)-glutamine) containing 10% FBS (Fetal bovine serum) and 1% Penicillin-Streptomcin, at 37°C, 5% CO2, and saturated humidity, and cells in the logarithmic growth phase were used for the experiment.

[0134] 1.2 CCK8 colorimetric assay to detect the effect of drugs on cell viability

[0135] (1) Collect cells in the logarithmic growth phase, centrifuge at 1000 rpm for 3 minutes, discard the old culture medium, and add an appropriate amount of fresh culture medium for cell counting.

[0136] (2) Adjust the cell concentration to 5x10 4 / mL inoculated in a 96-well plate, 100 μl per well, then 5000 cells

[0137] (3) The 96-well plate was incubated at 37°C, 5% CO2, and saturated humidity for 24 h.

[0138] (4) The drug was prepared into nine concentrations of 400 μM, 256 μM, 200 μM, 128 μM, 100 μM, 64 μM, 50 μM, 32 μM, and 16 μM, and 100 μl of each was added to the experimental wells to make the concentrations in the wells 200 μM, 128 μM, 100 μM, 64 μM, 50 μM, 32 μM, 25 μM, 16 μM, and 8 μM; 100 μl of culture medium was added to the control wells and blank wells.

[0139] (5) The cells were cultured at 37° C., 5% CO 2 , and saturated humidity for 24 h. After the culture was completed, 10 μl of CCK-8 (Cell Counting Kit-8) was added to each well. After culturing for 1.5-2 hours, the light absorption value at 450 nm was detected using an ELISA reader.

[0140] (6) Calculate the inhibition rate of the drug according to the following formula: Inhibition rate = (OD 实验 -OD 空白 ) / (OD control-OD 空白 )*100%. As:OD 实验 : Experimental group (culture medium containing cells, CCK-8, test substance), OD 空白 :Blank wells (medium without cells and test substances, CCK-8), OD 对照 : Control wells (containing cell culture medium, CCK-8, and no test substance).

[0141] The results of the in vitro anti-A549-Luc experiment of the new ureido cyclic peptide compound are shown in Fig.23 , 24 As shown, it can be seen that the urea skeleton macrocyclic peptide compounds (B5, B8) synthesized by the present invention greatly improve their inhibitory activity against A549-Luc cells.

[0142] The present invention also verified the in vitro anti-tumor experiments of the novel urea cyclic peptide compounds (i.e., target products) in Examples 1-4, 6, 7, and 9-11. The results showed that the novel urea cyclic peptide compounds of the present invention greatly improved their inhibitory activity against A549-Luc cells.

[0143] 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 novel urea-based cyclic peptide compound, characterized in that: The following steps are involved: The polypeptide containing an amino group or a lysine residue is dissolved in an organic solvent A to obtain a reaction solution 1, and carbonyl diimidazole is dissolved in an organic solvent B to obtain a reaction solution 2; The reaction solution 2 and the base are sequentially added into the reaction solution 1 to react to obtain a novel urea-based cyclic peptide compound; The polypeptide containing an amino group or a lysine residue is a polypeptide having an N-terminal amino group and a side chain amino group or a polypeptide having a side chain amino group and a side chain amino group.

2. The method for preparing the novel ureido cyclic peptide compound according to claim 1, characterized in that: The molar ratio of the polypeptide containing amino or lysine residues to carbonyldiimidazole is 1:1-1:

2.

3. The method for preparing the novel urea cyclic peptide compound according to claim 1, characterized in that: The structural formula of the carbonyl diimidazole is: Wherein, R is S or O.

4. The method for preparing the novel urea cyclic peptide compound according to claim 1, characterized in that: The organic solvent A and the organic solvent B are one or a mixed solvent of two selected from acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, methanol, tetrahydrofuran and acetone.

5. The method for preparing the novel ureido cyclic 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 to 5 eq.

6. The method for preparing the novel ureido cyclic peptide compound according to claim 1, characterized in that: The reaction temperature is room temperature, and the reaction time is 1-6 hours; the reaction atmosphere is air atmosphere, and it is not a direct open reaction.

7. The method for preparing the novel ureido cyclic peptide compound according to claim 1, characterized in that: After the reaction is completed, the separation and purification steps are: concentrating and drying the reaction solution, and then separating and purifying by preparative chromatography.

8. A novel ureido cyclic peptide compound prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the novel ureido cyclic peptide compound according to claim 8 in the preparation of a drug for treating human non-small cell lung cancer A549-Luc.

10. A drug for treating human non-small cell lung cancer A549-Luc, characterized in that: The raw materials include the novel urea cyclic peptide compound described in claim 8 and pharmaceutically acceptable excipients.

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