Preparation method, product and application of thioureido macrocyclic peptide

Abstract

The invention relates to the technical field of organic synthesis, in particular to a preparation method, a product and application of thioureido macrocyclic peptide. The preparation method comprises the following steps: mixing polypeptide, alkali and N, N '-thiocarbonyldiimidazole with a reaction solvent, carrying out a mixing and stirring reaction at normal temperature, after the reaction is completed, diluting a diamine compound, slowly dropwise adding the diluted diamine compound into the reaction system, and after the reaction is finished, freeze-drying, separating and purifying the reaction liquid to obtain the thioureido macrocyclic peptide. According to the invention, under a mild condition, the cyclization modification of the polypeptide is realized through a thiourea-based polypeptide preparation cyclization technology, and a solid theoretical foundation is laid for the development of the polypeptide with a specific function. The polypeptide cyclization technology for preparing thioureido has an important promotion effect on research in the fields of biomedicine and biotechnology.

Description

Technical Field

[0001] The present invention relates to the technical field of organic synthesis, and in particular to a preparation method, product and application of a thiourea macrocyclic peptide. 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. Peptides, as a special biological molecule between macromolecular proteins and amino acids, have many unique characteristics and advantages. Peptide drugs have both the specific cell selectivity of macromolecular protein drugs and the low-cost characteristics of small molecule drugs. Due to their high biological activity, wide indications, small dosage, low toxicity and side effects, and significant efficacy, they have become an important direction for new drug research and development at home and abroad. At present, they have been widely used in the prevention, diagnosis and treatment of diseases such as tumors, hepatitis, diabetes, and AIDS, and have broad development prospects. However, some existing peptide drugs are easy to enzymatically hydrolyze, resulting in poor metabolic stability, short half-life, and low transmembrane efficiency and oral availability, which seriously restricts the development of peptide drugs.

[0003] Cyclic peptides are closed-loop peptide chains formed by connecting multiple amino acids through peptide bonds. Cyclic peptides have important applications in biochemistry and medicinal chemistry. Due to their unique cyclic structure, cyclic peptides can exhibit different biological activities and stability from ordinary linear peptides. This stability mainly comes from their cyclic structure, which makes cyclic peptides less susceptible to enzyme degradation in the body, thereby prolonging the duration of their biological activity. In addition, cyclic peptides can easily cross the cell membrane and enter the cell due to their small molecular weight and good cell membrane permeability, and then interact with intracellular targets to achieve their biological functions. Therefore, cyclic peptides have important potential in drug research and development and can be used to develop new drugs with characteristics such as high efficiency, low toxicity, and high bioavailability. In general, peptide chain cyclization results in cyclic peptides that are essentially different from linear peptides. Cyclicization not only improves the structural properties of the peptide chain, but also improves pharmacokinetic properties such as absorption and biomembrane permeability, which are necessary to reach protein targets. The constrained structure of the peptide gives it a lower energy barrier to adapt to the membrane environment and bind to transport proteins, thereby increasing passive diffusion and active transport. By introducing non-peptide and non-amino acid elements, cyclic peptides can also further improve their pharmacokinetic and pharmacodynamic properties.

[0004] The modification of peptide cyclization not only significantly improves its stability and activity, but also greatly enriches the diversity of peptides. This operation is undoubtedly a key strategy in peptide research. In recent years, chemists at home and abroad have made great progress in the strategy of peptide macrocyclization. The reaction using chemical cyclization has become more mature and has attracted more and more attention from chemists. For example, native cyclization does not require protective groups, pre-modification or organic solvents, has strong compatibility, and can achieve efficient cyclization of peptides from 5 to 20 amino acid residues (J.Am.Chem.Soc.,2023,145,27218–27224.); C-H bond activation strategy, selectively activates the alkyl C-H bonds on the chain peptide substrate through metal catalysis, and intramolecularly couples with the side chains of aromatic amino acids substituted with iodine to generate cyclic products (Acc.Chem. Res.2012,45,788–802.;Nat.Commun.,2023,14,39703.); click chemistry cyclization, using efficient click reactions between specific chemical groups to achieve cyclization (Nat.Commun.,2024,15,7308.;Org.Lett.,2024,26,2601–2605.); enzymatic cyclization, using specific enzymes to catalyze the intramolecular cyclization reaction of polypeptides (Organic Letters,2024,26,2601–2605.); N-terminal modification and cyclization, through chemical modification of the N-terminus (such as thiol modification) to achieve the main chain-side chain cyclization of polypeptides (Angew.Chem.Int.Ed.,2020,59,14246–14250).

[0005] However, the existing peptide chain cyclization methods often still have some limitations and shortcomings, such as harsh reaction conditions, complex operations, and high costs. These problems have greatly restricted the widespread application of peptide chain cyclization technology. The traditional amide cyclization strategy requires the formation of a specific cyclization conformation, which is an entropically unfavorable process and is difficult to form a ring (Chem. Rev., 2015, 115, 8769–8834.). Therefore, it is urgent to develop a more suitable peptide chain cyclization method to overcome the previous harsh reaction conditions and improve the efficiency of cyclization. Summary of the invention

[0006] Based on the above content, the present invention provides a preparation method, product and application of a thiourea macrocyclic peptide.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] One of the technical solutions of the present invention is a method for preparing a thiourea macrocyclic peptide, comprising the following steps:

[0009] The polypeptide, base and N,N'-thiocarbonyldiimidazole are mixed with a reaction solvent for mixed stirring reaction. After the reaction is complete, a certain amount of solvent (N,N-dimethylformamide) of the diamine compound is diluted and slowly added dropwise to the above reaction system. After the reaction is completed, the reaction solution is freeze-dried, separated and purified to obtain the thiourea macrocyclic peptide.

[0010] The polypeptide contains at least two -NH 2 Linear peptides.

[0011] In a preferred embodiment of the present invention, the polypeptide is a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, a hexapeptide, or the like.

[0012] In a preferred embodiment of the invention, the polypeptide contains two alkylamines.

[0013] In a preferred embodiment of the present invention, the polypeptide contains amino acid residues such as lysine, alanine, tryptophan, arginine, phenylalanine, tyrosine, valine, leucine or isoleucine.

[0014] In a preferred embodiment of the present invention, the polypeptide is an oligopeptide containing lysine, an active peptide containing lysine, or a polypeptide drug containing lysine.

[0015] In a preferred embodiment of the present invention, the polypeptide is a polypeptide having a naked amino group, sulfhydryl group, imidazole group, guanidine group, phenolic hydroxyl group or amide group on the side chain.

[0016] In a preferred embodiment of the present invention, the structural formula of the polypeptide is as shown in Formula A:

[0017]

[0018] In formula A, R 1 , R 2 Each of the following groups is independently methyl, ethyl, acetyl, tert-butyloxycarbonyl, benzyloyloxycarbonyl, 9-fluorenylmethoxycarbonyl, carboxyl or amino.

[0019] In a preferred embodiment of the present invention, the molar ratio of the polypeptide to the N,N'-thiocarbonyldiimidazole is 1:(1-5).

[0020] In a preferred embodiment of the present invention, the molar ratio of the polypeptide to the diamine compound is 1:2.

[0021] In a preferred embodiment of the present invention, the base is triethylamine, and the molar ratio of the polypeptide to the base is 1:(1-10).

[0022] In a preferred embodiment of the present invention, the reaction solvent is one or two of acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, methanol, and tetrahydrofuran. When the reaction solvent is a mixed solvent of the above two solvents, the volume ratio of the two solvents is (0.2-5):1.

[0023] The present invention does not impose any particular limitation on the amount of the reaction solvent. The amount of the reaction solvent used is sufficient to dissolve the polypeptide and N,N'-thiocarbonyldiimidazole and ensure that the reaction proceeds smoothly.

[0024] In a preferred embodiment of the present invention, the structural formula of the diamine compound is H 2 NR 3 -NH 2 ; Among them, R 3 It is trimethylene, 1,3-dimethylenebenzene, cyclohexane, benzene ring or polyethylene glycol segment.

[0025] The reaction process is shown in the following reaction formula:

[0026]

[0027] In a preferred embodiment of the present invention, the reactions are carried out at room temperature, in an air atmosphere, and under non-direct exposure conditions, and the reaction time is 12 to 24 hours.

[0028] In the present invention, the reaction liquid is continuously monitored by mass spectrometry during the entire reaction process to ensure that the raw material polypeptide is completely converted.

[0029] After the reaction is completed, the steps of freeze drying and separation and purification are also included. Preparative HPLC is used for fine separation and purification. The purpose of freeze drying is to remove the solvent.

[0030] In the reaction formula, (2) means diluting the diamine compound with an organic solvent and then slowly adding it to the reaction system. The amount of the organic solvent used for dilution in the present invention is not particularly limited, and the amount commonly used in the art can be used, such as diluting to a diamine compound concentration of 2 to 20 mmol / L.

[0031] The present invention does not impose any particular limitation on freeze-drying parameter settings such as pressure, temperature, and time, and the freeze-drying parameter settings commonly used by those skilled in the art may be adopted.

[0032] The polypeptide cyclization product (i.e., thiourea macrocyclic peptide) obtained by the above-mentioned thiourea-based polypeptide cyclization technology can be used to develop polypeptide drugs with specific functions and study the structural functions of compounds. This method can be used for the cyclization modification of various natural polypeptides, as well as for the cyclization modification of various non-protected peptides, and for the study of anti-tumor and membrane permeability.

[0033] The second technical solution of the present invention is a thiourea macrocyclic peptide prepared according to the above-mentioned preparation method.

[0034] The third technical solution of the present invention is the use of the above-mentioned thiourea macrocyclic peptide in the preparation of drugs for treating anti-tumor, wherein the anti-tumor cells are Coca2 cells and / or TE-1 cells.

[0035] The present invention discloses the following technical effects:

[0036] (1) The present invention belongs to the field of chemical synthesis and can be easily reacted at room temperature without the need for high temperature, high pressure or other harsh reaction conditions.

[0037] (2) The present invention is convenient and simple to operate, has high substrate applicability, does not require a directing group, does not involve metal, and can be exposed to the air for reaction without the need for inert gas protection.

[0038] (3) In terms of applicability, the present invention can cover the requirements for macrocyclization modification from rings with more than ten members to rings with more than thirty members, thereby effectively solving the difficult problems currently faced by macrocyclization.

[0039] (4) The present invention is used for polypeptide diversity modification, which can provide a theoretical basis for the development of specific functional polypeptide drugs and the study of compound structure and function.

[0040] In summary, the present invention realizes the cyclization modification of polypeptides under mild conditions through a thiourea-based polypeptide cyclization technology, laying a solid theoretical foundation for the development of specific functional polypeptides. The present invention has an important role in promoting research in the fields of biomedicine and biotechnology. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is the LC tracer of the reaction solution before purification in Example 1;

[0042] Figure 2 This is the LC tracer of the target product obtained after purification in Example 1;

[0043] Figure 3 This is the LC tracer of the reaction solution before purification in Example 2;

[0044] Figure 4 This is the LC tracer of the target product obtained after purification in Example 2;

[0045] Figure 5 This is the LC tracer of the reaction solution before purification in Example 3;

[0046] Figure 6 This is the LC tracer of the target product obtained after purification in Example 3;

[0047] Figure 7This is the LC tracer of the reaction solution before purification in Example 4;

[0048] Figure 8 This is the LC tracer of the target product obtained after purification in Example 4;

[0049] Fig. 9 This is the LC tracer of the reaction solution before purification in Example 5;

[0050] Fig.10 This is the LC tracer of the target product obtained after purification in Example 5;

[0051] Fig.11 This is the LC tracer of the reaction solution before purification in Example 6;

[0052] Fig.12 This is the LC tracer of the target product obtained after purification in Example 6;

[0053] Fig.13 This is the LC tracer of the reaction solution before purification in Example 7;

[0054] Fig.14 This is the LC tracer of the target product obtained after purification in Example 7;

[0055] Fig.15 This is the LC tracer of the reaction solution before purification in Example 8;

[0056] Fig.16 This is the LC tracer of the target product obtained after purification in Example 8;

[0057] Fig.17 This is the LC tracer of the reaction solution before purification in Example 9;

[0058] Fig.18 This is the LC tracer of the target product obtained after purification in Example 9;

[0059] Fig.19 This is the LC tracer of the reaction solution before purification in Example 10;

[0060] Fig. 20 This is the LC tracer of the target product obtained after purification in Example 10;

[0061] Fig.21 This is the LC tracer of the reaction solution before purification in Example 11;

[0062] Fig. 22 This is the LC tracer of the target product obtained after purification in Example 11;

[0063] Fig.23 This is the LC tracer of the reaction solution before purification in Example 12;

[0064] Fig.24This is the LC tracer of the target product obtained after purification in Example 12;

[0065] Fig.25 This is the LC tracer of the reaction solution before purification in Example 13;

[0066] Fig.26 This is the LC tracer of the target product obtained after purification in Example 13;

[0067] Fig. 27 This is the LC tracer of the reaction solution before purification in Example 14;

[0068] Fig.28 This is the LC tracer of the target product obtained after purification in Example 14;

[0069] Fig.29 This is the LC tracer of the reaction solution before purification in Example 15;

[0070] Fig.30 This is the LC tracer of the target product obtained after purification in Example 15.

[0071] Fig.31 This is the anticancer cell activity test diagram of Effect Example 1.

[0072] Fig.32 Example 1: Anticancer Activity IC 50 Result graph.

[0073] Fig.33 This is the anticancer cell activity test diagram of Effect Example 2.

[0074] Fig.34 Example 2: Anticancer Activity IC 50 Result graph. DETAILED DESCRIPTION

[0075] 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.

[0076] In view of the shortcomings and deficiencies of the prior art methods for preparing cyclic peptides, the present invention provides a method for preparing a thiourea-based macrocyclic peptide. The method is divided into two steps: first, a thiourea-based macrocyclic peptide containing at least two -NH 2The peptide is used as a raw material, and reacts with N,N'-thiocarbonyldiimidazole under the action of common bases and solvents to generate isothiocyanate-modified peptides; then, a simple diamine (including but not limited to 1,3-propylenediamine, m-cyclohexyldiamine and o-cyclohexyldiamine) diluted with an appropriate solvent is introduced into the established reaction system and slowly added dropwise into the reaction system to generate the target cyclization product. This reaction exhibits the advantages of mild reaction conditions, simple operation, and high reaction efficiency, and has good application prospects.

[0077] 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.

[0078] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0079] Example 1

[0080] 0.02mmol of polypeptide A1 (Ac-Lys-Trp-Lys-OMe), 0.02mmol of triethylamine and 0.08mmol of N,N'-thiocarbonyldiimidazole were added to a 25mL glass test tube containing a stirring magnet. Subsequently, 5mL of N,N-dimethylformamide was added as a reaction solvent, and stirring was continued for 12 hours at room temperature until the reaction was complete. After the reaction was completed, 0.04mmol of 1,3-propylenediamine was diluted in 20mL of N,N-dimethylformamide and transferred to a 25mL dropping funnel. Subsequently, the diluted 1,3-propylenediamine was slowly and evenly added to the aforementioned reaction tube through the funnel. After the reaction was completed, the reaction solution was freeze-dried using a freeze dryer. Subsequently, the obtained residue was separated and purified by preparative HPLC to finally obtain a 24-membered ring polypeptide final product B1, and the purified product was dried using a freeze dryer. A small amount of the residue before purification and the pure sample after purification were taken and analyzed by LC using acetonitrile and water to make it 10 mM. The LC yield was 37%.

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

[0082]

[0083] HRMS-ESI(m / z):calcd for[MH] - C 31 H 45 N 8 O 5 S 2 -:673.2960; found:673.2964.

[0084] The LC tracer of the reaction solution before purification in this example is as follows Figure 1 As shown, the LC tracer of the target product obtained after purification is as follows Figure 2 shown.

[0085] Example 2

[0086] 0.02mmol of peptide A1 (Ac-Lys-Trp-Lys-OMe), 0.02mmol of triethylamine and 0.08mmol of N,N'-thiocarbonyldiimidazole were added to a 25mL glass test tube containing a stirring magnet. Subsequently, 5mL of N,N-dimethylformamide was added as the reaction solvent, and stirring was continued at room temperature until the reaction was complete. After the reaction was completed, 0.04mmol of 1,3-phenylenediamine was diluted in 20mL of N,N-dimethylformamide and transferred to a 25mL dropping funnel. Subsequently, the diluted 1,3-phenylenediamine was slowly and evenly added to the aforementioned reaction tube through the funnel. After the reaction was completed, the reaction solution was freeze-dried using a freeze dryer. Subsequently, the obtained residue was separated and purified by preparative HPLC, and the purified product was dried using a freeze dryer. Finally, a 26-membered ring polypeptide final product B2 was obtained. LC yield 70%.

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

[0088]

[0089] HRMS-ESI(m / z):calcd for[M+H] + C 36 H 49 N 8 O 5 S 2 + :737.3262; found:737.3253.

[0090] The LC tracer of the reaction solution before purification in this example is as follows Figure 3 As shown, the LC tracer of the target product obtained after purification is as follows Figure 4 shown.

[0091] Example 3

[0092] 0.02mmol of polypeptide A2 (Ac-Lys-Leu-Ala-Phe-Lys-OMe), 0.02mmol of triethylamine and 0.08mmol of N,N'-thiocarbonyldiimidazole were sequentially added to a 25mL glass test tube containing a stirring magnet. Subsequently, 5mL of N,N-dimethylformamide was added as a reaction solvent, and stirring was continued at room temperature until the reaction was complete. After the reaction was completed, 0.04mmol of 1,3-propylenediamine was diluted in 20mL of N,N-dimethylformamide and transferred to a 25mL dropping funnel. Subsequently, the diluted 1,3-propylenediamine was slowly and evenly added to the aforementioned reaction tube through the funnel. After the reaction was completed, the reaction solution was freeze-dried using a freeze dryer. Subsequently, the obtained residue was subjected to a separation and purification step of preparative HPLC, and the purified product was dried using a freeze dryer. Finally, a 30-membered ring polypeptide final product B3 was obtained. LC yield 37%.

[0093] The reaction formula and the structural characterization data of the obtained product are shown below:

[0094]

[0095] HRMS-ESI(m / z):calcd for[MH] - C 38 H 60 N 9 O 7 S 2 - :818.4063; found:818.4064.

[0096] The LC tracer of the reaction solution before purification in this example is as follows Figure 5 As shown, the LC tracer of the target product obtained after purification is as follows Figure 6 shown.

[0097] Example 4

[0098] 0.02mmol of peptide A2 (Ac-Lys-Leu-Ala-Phe-Lys-OMe), 0.02mmol of triethylamine and 0.08mmol of N,N'-thiocarbonyldiimidazole were sequentially added to a 25mL glass test tube containing a stirring magnet. Subsequently, 5mL of N,N-dimethylformamide was added as a reaction solvent, and stirring was continued at room temperature until the reaction was complete. After the reaction was completed, 0.04mmol of 1,3-phenylenediamine was diluted in 20mL of N,N-dimethylformamide and transferred to a 25mL dropping funnel. Subsequently, the diluted 1,3-phenylenediamine was slowly and evenly added to the aforementioned reaction tube through the funnel. After the reaction was completed, the reaction solution was freeze-dried using a freeze dryer. Subsequently, the obtained residue was subjected to a separation and purification step of preparative HPLC, and the purified product was dried using a freeze dryer. Finally, a 33-membered ring polypeptide final product B4 was obtained. LC yield 67%.

[0099] The reaction formula and the structural characterization data of the obtained product are shown below:

[0100]

[0101] HRMS-ESI(m / z):calcd for[MH] - C 43 H 62 N 9 O 7 S 2 - :880.4219; found:880.4218.

[0102] The LC tracer of the reaction solution before purification in this example is as follows Figure 7 As shown, the LC tracer of the target product obtained after purification is as follows Figure 8 shown.

[0103] Example 5

[0104] 0.02mmol of polypeptide A3 (acetyl tetrapeptide-3), 0.02mmol of triethylamine and 0.08mmol of N,N'-thiocarbonyldiimidazole were added to a 25mL glass test tube containing a stirring magnet. Subsequently, 5mL of N,N-dimethylformamide was added as a reaction solvent, and stirring was continued at room temperature until the reaction was complete. After the reaction was completed, 0.04mmol of 1,3-phenylenediamine was diluted in 20mL of N,N-dimethylformamide and transferred to a 25mL dropping funnel. Subsequently, the diluted 1,3-phenylenediamine was slowly and evenly added to the aforementioned reaction tube through the funnel. After the reaction was completed, the reaction solution was freeze-dried using a freeze dryer. Subsequently, the obtained residue was separated and purified by preparative HPLC, and the purified product was dried using a freeze dryer. Finally, a 29-membered ring polypeptide final product B5 was obtained. LC yield 63%.

[0105] The reaction formula and the structural characterization data of the obtained product are shown below:

[0106]

[0107] HRMS-ESI(m / z):calcd for[M+H] + C 32 H 48 N 11 O 5 S 2 + :730.3276; found:730.3277.

[0108] The LC tracer of the reaction solution before purification in this example is as follows Fig. 9 As shown, the LC tracer of the target product obtained after purification is as follows Fig.10 shown.

[0109] Example 6

[0110] 0.02mmol of polypeptide A3 (acetyl tetrapeptide-3), 0.02mmol of triethylamine and 0.08mmol of N,N'-thiocarbonyl diimidazole were added to a 25mL glass test tube containing a stirring magnet. Subsequently, 5mL of N,N-dimethylformamide was added as a reaction solvent, and stirring was continued at room temperature until the reaction was complete. After the reaction was completed, 0.04mmol of 1,8-amino-3,6-dioxooctane was diluted in 20mL of N,N-dimethylformamide and transferred to a 25mL dropping funnel. Subsequently, the diluted 1,8-amino-3,6-dioxooctane was slowly and evenly added to the aforementioned reaction tube through the funnel. After the reaction was completed, the reaction solution was freeze-dried using a freeze dryer. Subsequently, the obtained residue was subjected to a separation and purification step of preparative HPLC, and the purified product was dried using a freeze dryer. Finally, a 32-membered peptide final product B6 was obtained with an LC yield of 73%.

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

[0112]

[0113] HRMS-ESI(m / z):calcd for[M+H] + C 30 H 52 N 11 O 7 S 2 + :742.3487; found:742.3491.

[0114] The LC tracer of the reaction solution before purification in this example is as follows Fig.11 As shown, the LC tracer of the target product obtained after purification is as follows Fig.12 shown.

[0115] Example 7

[0116] 0.02mmol of polypeptide A3 (acetyl tetrapeptide-3), 0.02mmol of triethylamine and 0.08mmol of N,N'-thiocarbonyl diimidazole were added to a 25mL glass test tube containing a stirring magnet. Subsequently, 5mL of N,N-dimethylformamide was added as a reaction solvent, and stirring was continued at room temperature until the reaction was complete. After the reaction was completed, 0.04mmol of 4,7,10-trioxy-1,13-tridecanediamine was diluted in 20mL of N,N-dimethylformamide and transferred to a 25mL dropping funnel. Subsequently, the diluted 4,7,10-trioxy-1,13-tridecanediamine was slowly and evenly added to the aforementioned reaction tube through the funnel. After the reaction was completed, the reaction solution was freeze-dried using a freeze dryer. Subsequently, the obtained residue was subjected to a separation and purification step of preparative HPLC, and the purified product was dried using a freeze dryer. Finally, a 37-membered peptide final product B7 was obtained with an LC yield of 65%.

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

[0118]

[0119] HRMS-ESI(m / z):calcd for[M+H] + C 34 H 60 N 11 O 8 S 2 + :814.4062; found:814.4068.

[0120] The LC tracer of the reaction solution before purification in this example is as follows Fig.13 As shown, the LC tracer of the target product obtained after purification is as follows Fig.14 shown.

[0121] Example 8

[0122] 0.02mmol of polypeptide A3 (acetyl tetrapeptide-3), 0.02mmol of triethylamine and 0.08mmol of N,N'-thiocarbonyldiimidazole were sequentially added to a 25mL glass test tube containing a stirring magnet. Subsequently, 5mL of N,N-dimethylformamide was added as a reaction solvent, and stirring was continued at room temperature until the reaction was complete. After the reaction was completed, 0.04mmol of 1,3-propylenediamine was diluted in 20mL of N,N-dimethylformamide and transferred to a 25mL dropping funnel. Subsequently, the diluted 1,3-propylenediamine was slowly and evenly added to the aforementioned reaction tube through the funnel. After the reaction was completed, the reaction solution was freeze-dried using a freeze dryer. Subsequently, the obtained residue was subjected to a separation and purification step of preparative HPLC, and the purified product was dried using a freeze dryer. Finally, a 27-membered ring polypeptide final product B8 was obtained with an LC yield of 67%.

[0123] The reaction formula and the structural characterization data of the obtained product are shown below:

[0124]

[0125] HRMS-ESI(m / z):calcd for[M+H] + C 27 H 46 N 11 O 5 S 2 + :668.3119; found:668.3110.

[0126] The LC tracer of the reaction solution before purification in this example is as follows Fig.15 As shown, the LC tracer of the target product obtained after purification is as follows Fig.16 shown.

[0127] Example 9

[0128] 0.02mmol of peptide A4 (Val-Lys) and 0.08mmol of N,N'-thiocarbonyldiimidazole were sequentially added to a 25mL glass test tube containing a stirring magnet. Subsequently, 5mL of N,N-dimethylformamide was added as a reaction solvent, and stirring was continued at room temperature until the reaction was complete. After the reaction was completed, 0.04mmol of 1,3-propylenediamine was diluted in 20mL of N,N-dimethylformamide and transferred to a 25mL dropping funnel. Subsequently, the diluted 1,3-propylenediamine was slowly and evenly added to the aforementioned reaction tube through the funnel. After the reaction was completed, the reaction solution was freeze-dried using a freeze dryer. Subsequently, the obtained residue was subjected to a separation and purification step of preparative HPLC, and the purified product was dried using a freeze dryer. Finally, a 17-membered ring polypeptide final product B9 was obtained with an LC yield of 28%.

[0129] The reaction formula and the structural characterization data of the obtained product are shown below:

[0130]

[0131] HRMS-ESI(m / z):calcd for[M+H] + C 16 H 31 N 6 O 2 S 2 + :403.1944; found:403.1942.

[0132] The LC tracer of the reaction solution before purification in this example is as follows Fig.17 As shown, the LC tracer of the target product obtained after purification is as follows Fig.18 shown.

[0133] Example 10

[0134] 0.02mmol of polypeptide A5 (snake venom tripeptide), 0.02mmol of triethylamine and 0.08mmol of N,N'-thiocarbonyldiimidazole were added to a 25mL glass test tube containing a stirring magnet. Subsequently, 5mL of N,N-dimethylformamide was added as a reaction solvent, and stirring was continued at room temperature until the reaction was complete. After the reaction was completed, 0.04mmol of 1,3-propylenediamine was diluted in 20mL of N,N-dimethylformamide and transferred to a 25mL dropping funnel. Subsequently, the diluted 1,3-propylenediamine was slowly and evenly added to the aforementioned reaction tube through the funnel. After the reaction was completed, the reaction solution was freeze-dried using a freeze dryer. Subsequently, the obtained residue was subjected to a separation and purification step of preparative HPLC, and the purified product was dried using a freeze dryer. Finally, a 19-membered ring polypeptide final product B10 was obtained with an LC yield of 75%.

[0135] The reaction formula and the structural characterization data of the obtained product are shown below:

[0136]

[0137] HRMS-ESI(m / z):calcd for[M+H] + C 24 H 36 N 7 O 3 S 2 + :534.2316; found:534.2318.

[0138] The LC tracer of the reaction solution before purification in this example is as follows Fig.19 As shown, the LC tracer of the target product obtained after purification is as follows Fig. 20 shown.

[0139] Embodiment 11

[0140] 0.02mmol of polypeptide A6 (Ac-Lys-Ala-Ala-Trp-Ile-Lys-OMe), 0.02mmol of triethylamine and 0.08mmol of N,N'-thiocarbonyldiimidazole were sequentially added to a 25mL glass test tube containing a stirring magnet. Subsequently, 5mL of N,N-dimethylformamide was added as a reaction solvent, and stirring was continued at room temperature until the reaction was complete. After the reaction was completed, 0.04mmol of 1,3-propylenediamine was diluted in 20mL of N,N-dimethylformamide and transferred to a 25mL dropping funnel. Subsequently, the diluted 1,3-propylenediamine was slowly and evenly added to the aforementioned reaction tube through the funnel. After the reaction was completed, the reaction solution was freeze-dried using a freeze dryer. Subsequently, the obtained residue was separated and purified by preparative HPLC, and the purified product was dried using a freeze dryer. Finally, a 33-membered peptide final product B11 was obtained with an LC yield of 20%.

[0141] The reaction formula and the structural characterization data of the obtained product are shown below:

[0142]

[0143] HRMS-ESI(m / z):calcd for[MH] - C 43 H 66 N 11 O 8 S 2 - :928.4543; found:928.4536.

[0144] The LC tracer of the reaction solution before purification in this example is as follows Fig.21 As shown, the LC tracer of the target product obtained after purification is as follows Fig. 22 shown.

[0145] Example 12

[0146] 0.02mmol of polypeptide A7 (Ac-Lys-Leu-Val-Lys-OMe), 0.02mmol of triethylamine and 0.08mmol of N,N'-thiocarbonyldiimidazole were added to a 25mL glass test tube containing a stirring magnet. Subsequently, 5mL of N,N-dimethylformamide was added thereto as a reaction solvent, and stirring was continued at room temperature until the reaction was complete. After the reaction was completed, 0.04mmol of 1,3-propylenediamine was diluted in 20mL of N,N-dimethylformamide and transferred to a 25mL dropping funnel. Subsequently, the diluted 1,2-diaminocyclohexane was slowly and evenly added to the aforementioned reaction tube through the funnel. After the reaction was completed, the reaction solution was freeze-dried using a freeze dryer. Subsequently, the obtained residue was separated and purified by preparative HPLC, and the purified product was dried using a freeze dryer. Finally, a 27-membered peptide final product B12 was obtained with an LC yield of 55%.

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

[0148]

[0149] HRMS-ESI(m / z):calcd for[MH] - C 31 H 55 N 8 O 6 S 2 - :699.3691; found:699.3693.

[0150] The LC tracer of the reaction solution before purification in this example is as follows Fig.23 As shown, the LC tracer of the target product obtained after purification is as follows Fig.24 shown.

[0151] Embodiment 13

[0152] 0.02mmol of polypeptide A8 (Ac-Lys-Ala-Ala-Ile-Phe-Lys-Ala-OMe), 0.02mmol of triethylamine and 0.08mmol of N,N'-thiocarbonyldiimidazole were sequentially added to a 25mL glass test tube containing a stirring magnet. Subsequently, 5mL of N,N-dimethylformamide was added thereto as a reaction solvent, and stirring was continued at room temperature until the reaction was complete. After the reaction was completed, 0.04mmol of 1,3-propylenediamine was diluted in 20mL of N,N-dimethylformamide and transferred to a 25mL dropping funnel. Subsequently, the diluted 1,3-propylenediamine was slowly and evenly added to the aforementioned reaction tube through the funnel. After the reaction was completed, the reaction solution was freeze-dried using a freeze dryer. Subsequently, the obtained residue was separated and purified by preparative HPLC, and the purified product was dried using a freeze dryer. Finally, a 33-membered peptide final product B13 was obtained with an LC yield of 70%.

[0153] The reaction formula and the structural characterization data of the obtained product are shown below:

[0154]

[0155] HRMS-ESI(m / z):calcd for[M+H] + C 44 H 72 N 11 O 9 S 2 + :962.4950; found:962.4949.

[0156] The LC tracer of the reaction solution before purification in this example is as follows Fig.25 As shown, the LC tracer of the target product obtained after purification is as follows Fig.26 shown.

[0157] Embodiment 14

[0158] 0.02mmol of polypeptide A9 (Anoplin), 0.02mmol of triethylamine and 0.08mmol of N,N'-thiocarbonyldiimidazole were sequentially added to a 25mL glass test tube containing a stirring magnet. Subsequently, 5mL of N,N-dimethylformamide was added as a reaction solvent, and stirring was continued at room temperature until the reaction was complete. After the reaction was completed, 0.04mmol of 1,3-phenylenediamine was diluted in 20mL of N,N-dimethylformamide and transferred to a 25mL dropping funnel. Subsequently, the diluted 1,3-propylenediamine was slowly and evenly added to the aforementioned reaction tube through the funnel. After the reaction was completed, the reaction solution was freeze-dried using a freeze dryer. Subsequently, the obtained residue was separated and purified by preparative HPLC, and the purified product was dried using a freeze dryer. Finally, a 27-membered ring polypeptide final product B14 was obtained with an LC yield of 33%.

[0159] The reaction formula and the structural characterization data of the obtained product are shown below:

[0160]

[0161] HRMS-ESI(m / z):calcd for[(M+2H) 2+ / 2]C 65 H 116 N 18 O 10 S 2 2+ / 2:668.4276; found:668.4283.

[0162] The LC tracer of the reaction solution before purification in this example is as follows Fig. 27 As shown, the LC tracer of the target product obtained after purification is as follows Fig.28 shown.

[0163] Embodiment 15

[0164] 0.02mmol of polypeptide A9 (Anoplin), 0.02mmol of triethylamine and 0.08mmol of N,N'-thiocarbonyldiimidazole were added in sequence to a 25mL glass test tube containing a stirring magnet. Subsequently, 5mL of N,N-dimethylformamide was added as a reaction solvent, and stirring was continued at room temperature until the reaction was complete. After the reaction was completed, 0.04mmol of 1,3-propylenediamine was diluted in 20mL of N,N-dimethylformamide and transferred to a 25mL dropping funnel. Subsequently, the diluted 1,3-propylenediamine was slowly and evenly added to the aforementioned reaction tube through the funnel. After the reaction was completed, the reaction solution was freeze-dried using a freeze dryer. Subsequently, the obtained residue was separated and purified by preparative HPLC, and the purified product was dried using a freeze dryer. Finally, a 27-membered ring polypeptide final product B15 was obtained with an LC yield of 30%.

[0165] The reaction formula and the structural characterization data of the obtained product are shown below:

[0166]

[0167] HRMS-ESI(m / z):calcd for[(M+2H) 2+ / 2]C 60 H 114 N 18 O 10 S 2 2+ / 2:655.4198; found:655.4195.

[0168] The LC tracer of the reaction solution before purification in this example is as follows Fig.29 As shown, the LC tracer of the target product obtained after purification is as follows Fig.30 shown.

[0169] Effect Example 1

[0170] In vitro antitumor experiment of a thiourea macrocyclic peptide compound:

[0171] 1 Experimental methods:

[0172] 1.1 Cell culture

[0173] Human colorectal adenocarcinoma (Caco-2) cells were cultured in a medium containing 10% FBS (Fetalbovine serum) and 1% Penicillin-Streptomycin and 89% RPMI Medium 1640basic (Roswell Park Memorial Institute) (containing (L)-glutamine) at 37°C, 5% CO2, and saturated humidity. Cells in the logarithmic growth phase were used for the experiment.

[0174] 1.2 CCK8 colorimetric assay to detect the effect of drugs on cell viability

[0175] (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.

[0176] (2) The cell concentration was adjusted to 5 x 104 / mL and inoculated into a 96-well plate, with 100 μl per well, i.e. 5,000 cells per well.

[0177] (3) The 96-well plate was incubated at 37°C, 5% CO2, and saturated humidity for 24 h.

[0178] (4) Prepare the drug at four concentrations of 256 μM, 64 μM, 50 μM, 32 μM, and 16 μM, and add 100 μl of each drug to the experimental wells to make the concentrations in the wells 128 μM, 32 μM, 25 μM, 16 μM, and 8 μM; add 100 μl of culture medium to the control wells and blank wells.

[0179] (5) The cells were cultured at 37°C, 5% CO2, and saturated humidity for 24 h; after the culture was completed, 10 μl was added to each well.

[0180] CCK-8 (Cell Counting Kit-8), after culturing for 2h-4h, the light absorption value at 450nm was detected using a microplate reader.

[0181] The inhibition rate of the drug was calculated according to the following formula: inhibition rate = (OD experimental - OD blank) / OD control * 100%.

[0182] 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).

[0183] 2 The results of in vitro antitumor experiments of urea-containing macrocyclic peptide compound B15 are shown in Fig.31 , 32As shown in FIG. 1 , it can be seen that the compound B15 containing the urea macrocyclic peptide of the present invention greatly improves its inhibitory activity against Caco-2 cells (IC 50 =19.17 μM).

[0184] Effect Example 2

[0185] In vitro antitumor experiment of a thiourea macrocyclic peptide compound:

[0186] 1 Experimental methods:

[0187] 1.1 Cell culture

[0188] Human esophageal cancer cells (TE-1) were cultured in a medium containing 10% FBS (Fetalbovine serum) and 1% Penicillin-Streptomycin and 89% RPMI Medium 1640 basic (Roswell Park Memorial Institute) (containing (L)-glutamine) at 37°C, 5% CO2, and saturated humidity. Cells in the logarithmic growth phase were used for the experiment.

[0189] 1.2 CCK8 colorimetric assay to detect the effect of drugs on cell viability

[0190] (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.

[0191] (2) The cell concentration was adjusted to 5 x 104 / mL and inoculated into a 96-well plate, with 100 μl per well, i.e. 5,000 cells per well.

[0192] (3) The 96-well plate was incubated at 37°C, 5% CO2, and saturated humidity for 24 h.

[0193] (4) Prepare the drug into four concentrations of 400 μM, 200 μM, 100 μM, 50 μM, and 13 μM, and add 100 μl of each to the experimental well.

[0194] The concentrations in the wells were adjusted to 200 μM, 100 μM, 50 μM, 25 μM, and 6.25 μM; 100 μl of culture medium was added to the control wells and blank wells.

[0195] (5) The cells were cultured at 37°C, 5% CO2, and saturated humidity for 24 h. After the culture, 10 μl of

[0196] CCK-8 (Cell Counting Kit-8), after culturing for 2h-4h, the light absorption value at 450nm was detected using a microplate reader.

[0197] The inhibition rate of the drug was calculated according to the following formula: inhibition rate = (OD experimental - OD blank) / OD control * 100%.

[0198] 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).

[0199] 2 The results of in vitro antitumor experiments of urea-containing macrocyclic peptide compound B15 are shown in Fig.33 , 34 As shown, the IC of TE-1 cells was measured. 50 =34.33μM.

[0200] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a thiourea macrocyclic peptide, characterized in that: The following steps are involved: The polypeptide, the base and the N,N'-thiocarbonyldiimidazole are mixed with a reaction solvent for mixed stirring reaction. After the reaction is complete, the diamine compound is diluted with a solvent and slowly added dropwise to the above reaction system. After the reaction is completed, the reaction solution is freeze-dried, separated and purified to obtain the thiourea macrocyclic peptide; The polypeptide is a linear polypeptide containing at least two -NH2.

2. The method for preparing the thiourea macrocyclic peptide according to claim 1, characterized in that: The molar ratio of the polypeptide to the N,N'-thiocarbonyldiimidazole is 1:(1-5).

3. The method for preparing the thiourea macrocyclic peptide according to claim 1, characterized in that: The molar ratio of the polypeptide to the diamine compound is 1:

2.

4. The method for preparing the thiourea macrocyclic peptide according to claim 1, characterized in that: The base is triethylamine.

5. The method for preparing the thiourea macrocyclic peptide according to claim 1, characterized in that: The reaction solvent is one or two of acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, N,N-dimethylacetamide, methanol and tetrahydrofuran.

6. The method for preparing the thiourea macrocyclic peptide according to claim 1, characterized in that: The structural formula of the diamine compound is H2N-R3-NH2, wherein R3 is trimethylene, 1,3-dimethylenebenzene, cyclohexane, benzene ring or polyethylene glycol segment.

7. The method for preparing the thiourea macrocyclic peptide according to claim 1, characterized in that: The reactions are all carried out at room temperature, in an air atmosphere, and without direct exposure, and the reaction time is 12 to 24 hours.

8. The thiourea macrocyclic peptide prepared according to the preparation method according to any one of claims 1 to 7.

9. Use of the thiourea macrocyclic peptide according to claim 8 in the preparation of a drug for treating anti-tumor, characterized in that: The anti-tumor cells are Coca2 cells and / or TE-1 cells.

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