Peptidoid compound, preparation method thereof, pharmaceutical composition and application thereof

Abstract

The present application provides a peptoid compound having the structure of Formula (I) or (II): X1-Q1-X2-Q2 (I); y < 1 >-Q < 3 >-Y < 2 > (II). The peptoid compound provided by the invention can inhibit the activity of integrin, the peptide targets integrin proteins such as alpha v beta 5, alpha 5 beta 1, alpha v beta 3 and the like, signal transduction is inhibited through various receptors, and angiogenesis is inhibited. Experimental results show that the IC50 of the compounds provided by the invention to Integrin alpha v beta 3 is less than 700nM, and the IC50 of the compounds provided by the invention to Integrin alpha v beta 5 is less than 700nM; the compound provided by the invention can obviously inhibit HUVEC cells from forming lumens, and part of the compound can completely inhibit the lumens from forming at the concentration of 5 mu M, which is obviously superior to AXT107; part of compounds can significantly inhibit the area and number of lumens, increase the clearance degree and reduce the length of tubules, and are obviously superior to AXT107.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical chemistry, and in particular to a peptoid compound, a preparation method thereof, a pharmaceutical composition and use thereof. Background Art

[0002] Integrin is a transmembrane glycoprotein, which is widely present in cells. It is a member of the cell adhesion molecule family and the most important molecule that mediates the interaction between cells and the extracellular matrix (ECM). It is a type of cell membrane surface receptor that can recognize and bind to the corresponding ligand in the extracellular matrix, provide attachment points for cell adhesion, regulate cell movement, proliferation and apoptosis, mediate mutual recognition and adhesion between cells and between cells and the extracellular matrix, and has the function of connecting the external action of cells with the internal structure of cells. Integrin is a heterodimer composed of two subunits, α and β, connected by non-covalent bonds in a ratio of 1:1. It consists of three parts: the extracellular region, the transmembrane region and the intracellular region. It is connected into a spherical region in the extracellular region and contains a divalent cation binding domain. According to the specificity of integrin recognition of ligands, integrins can be roughly divided into integrins that recognize arginase-glycinase-aspartase peptide (RGD tripeptide) sequence, laminin-linked integrins, leukocyte integrins and collagen-linked integrins. Integrins bind to ECM proteins such as collagen, laminin, vitronectin, fibronectin or some cell surface molecules through their extracellular regions.

[0003] Integrins are involved in many important physiological processes, including embryogenesis, coagulation, and maintaining the integrity of tissues and organs in the body. Many pathological reaction processes, such as inflammation, thrombosis, angiogenesis, and the growth, infiltration, and metastasis of malignant tumors, are all related to abnormal regulation of integrins. In the inflammatory response, integrin-mediated adhesion of leukocytes to vascular endothelial cells is a prerequisite for inflammatory cell infiltration. The main integrins expressed on leukocytes are αXβ2, αLβ2, and αMβ2. Integrin β2 mediates and enhances the adhesion of leukocytes to activated endothelial cells, further promoting the migration of leukocytes through the endothelial cell layer into the inflammatory tissue, which is conducive to the occurrence of inflammatory reactions. At the same time, integrins are also involved in regulating the degree of inflammatory response. Studies have found that integrin β2 can limit the signal transduction of Toll-like receptors by inhibiting the NF-KB pathway and enhancing the activation of p38 protein kinase, thereby regulating the degree of inflammatory response. Integrin-mediated adhesion of endothelial cells to ECM is necessary for angiogenesis. Anti-integrin antibodies and small molecule integrin inhibitors can inhibit angiogenesis and tumor growth. Various types of integrins can affect angiogenesis, and their expression is highly specific in blood vessels. Among them, αvβ3 is a receptor for fibronectin (FN), wave adhesion protein (VN), laminin (LN), collagen (COL), etc. in the extracellular matrix. It is widely distributed and found in endothelial cells, smooth muscle cells, and hematopoietic cells (such as platelets and osteoclasts). It is also expressed in large quantities in endothelial cells and tumor cells activated by stimulation of angiogenesis-promoting factors. Integrin expression is elevated in many tumors, and elevated integrin expression is correlated with the occurrence and development of tumors. The expression and / or function of integrins is involved in almost every stage of cancer development, from primary tumor formation to cancer cell extravasation and the formation of metastatic niches. In addition, integrin signaling is associated with the acquisition of drug resistance and the important role of drugs in cancer. In dry eye, integrin αLβ2 promotes the homing of dendritic cells to lymph nodes, the activation / differentiation of T cells, and the activation / differentiation of CD4 + T cells migrate from lymph nodes to the ocular surface and ocular surface antigen-presenting cells are reactivated, thereby enhancing the ocular inflammatory response.

[0004] Integrins play an important role in a variety of biological functions. In the fields of cardiovascular disease, inflammatory bowel disease / multiple sclerosis and dry eye disease, there are already several drugs targeting integrins αIIbβ3, α4β7, α4β1 and αLβ2 on the market. In addition, there are also several drug molecules for cancer, ophthalmology and osteoporosis in clinical development, especially av integrin inhibitors of the RGD binding subfamily. There are also new drug molecules targeting avβ6 and avβ1, and they are being clinically studied for the treatment of fibrotic diseases, including idiopathic pulmonary fibrosis and non-alcoholic fatty liver disease.

[0005] αv and α5β1 play an important regulatory role in the development of abnormal angiogenesis. Inhibition of αv and α5β1 can effectively inhibit the formation of new blood vessels. Molecules such as SF0166, AXT-107, and THR-687 can effectively inhibit the formation of new blood vessels, and their effectiveness and safety in macular degeneration have been evaluated in the clinical stage. Therefore, the research on integrin inhibitors is of great significance. Summary of the invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a peptidomimetic compound, a preparation method thereof, a pharmaceutical composition and use thereof. The peptidomimetic compound provided by the present invention can inhibit the activity of integrins. The peptide targets integrin proteins such as αvβ5, α5β1, αvβ3, and inhibits signal transduction through multiple receptors to inhibit angiogenesis.

[0007] The present application provides a peptidomimetic compound having a structure of formula (I) or (II):

[0008] X1-Q1-X2-Q2(I);

[0009] Y1-Q3-Y2(II);

[0010] Wherein, in the structure of formula (I), Q1 has the amino acid sequence shown in SEQ ID No. 1, or an amino acid sequence in which one or more amino acids are inserted, deleted, or replaced based on the amino acid sequence shown in SEQ ID NO: 1, and the function remains unchanged;

[0011] Q2 has the amino acid sequence shown in SEQ ID No. 2 or 3, or an amino acid sequence in which one or more amino acids are inserted, deleted, or substituted based on the amino acid sequence shown in SEQ ID NO: 2 or 3, and the function remains unchanged;

[0012] X1 includes 0 to 5 amino acid residues, and X2 includes 0 to 15 amino acid residues; the amino acids in X1 and X2 are independently selected from lysine, arginine, serine, tyrosine, phenylalanine, leucine, threonine, alanine, proline, glutamine or cysteine;

[0013] In the structure of formula (II), Q3 has the amino acid sequence shown in SEQ ID No. 4 or 5, or an amino acid sequence in which one or more amino acids are inserted, deleted, or replaced based on the amino acid sequence shown in SEQ ID NO: 4 or 5, and the function is maintained unchanged;

[0014] Y1 includes 2 to 8 amino acid residues, and Y2 includes 0 to 8 amino acid residues; the amino acids in Y1 and Y2 are independently selected from leucine, alanine, serine, arginine, asparagine, aspartic acid, tyrosine or tryptophan.

[0015] The present application provides a peptoid compound having a structure of formula (I):

[0016] X1-Q1-X2-Q2(I);

[0017] In the structure of formula (I), Q1 has the amino acid sequence shown in SEQ ID No.1, specifically: KRKRKKSR or -Lys-Arg-Lys-Arg-Lys-Lys-Ser-Arg-, or an amino acid sequence in which one or more amino acids are inserted, deleted, or replaced on the basis of the amino acid sequence shown in SEQ ID NO:1 and the function remains unchanged.

[0018] Specifically, the amino acid sequence in which one or more amino acids are inserted, deleted, or substituted based on the amino acid sequence shown in SEQ ID NO: 1 and the function remains unchanged can be inserted, deleted, or substituted according to the similarity of the following amino acids: polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties. For example, the 20 genetically encoded amino acids can be divided into the following six standard amino acid groups:

[0019] (1) Hydrophobicity: Met, Ala, Val, Leu, Ile;

[0020] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0021] (3) Acidic: Asp, Glu;

[0022] (4) Basicity: His>Lys>Arg;

[0023] (5) Residues that affect chain orientation: Gly, Pro; and

[0024] (6) Aromatic family: Trp, Tyr, Phe.

[0025] Insertion, deletion, substitution of one or more amino acids can be selected according to another amino acid listed in the same group of the six standard amino acid groups shown above. For example, Asp is exchanged by Glu to retain a negative charge in the polypeptide. Glycine and proline can be substituted for each other based on their ability to destroy α-helix. In some specific implementations, some preferred insertions, deletions, substitutions in the above six groups are internal exchanges in the following subgroups: (i) Ala, Val, Leu and lie; (ii) Ser and Thr; (iii) Asn and Gin; (iv) Lys and Arg; and (v) Tyr and Phe.

[0026] Q2 has the amino acid sequence shown in SEQ ID NO.2 or 3, or an amino acid sequence in which one or more amino acids are inserted, deleted or substituted based on the amino acid sequence shown in SEQ ID NO:2 or 3, and the function remains unchanged; wherein the sequence of SEQ ID No.2 is: FININNVINF or -Phe-Ile-Asn-Ile-Asn-Asn-Val-Ile-Asn-Phe-; the sequence of SEQ ID No.3 is: FIDINDVINF or -Phe-Ile-Asp-Ile-Asn-Asp-Val-Ile-Asn-Phe-. The amino acid sequence in which one or more amino acids are inserted, deleted or substituted based on the amino acid sequence shown in SEQ ID NO:2 or 3, and the function remains unchanged, can refer to the rules described above, and this application will not repeat them here.

[0027] The present application provides a peptidomimetic compound having a structure of formula (II):

[0028] Y1-Q3-Y2(II)

[0029] In the structure of formula (II), Q3 has the amino acid sequence shown in SEQ ID No.4 or 5, or an amino acid sequence in which one or more amino acids are inserted, deleted or replaced based on the amino acid sequence shown in SEQ ID No: 4 or 5, and the function remains unchanged; wherein the sequence of SEQ ID No.4 is IDINDVINFASRND or -Ile-Asp-Ile-Asn-Asp-Val-Ile-Asn-Phe-Ala-Ser-Arg-Asn-Asp; and the sequence of SEQ ID No.5 is CNINNVCNFASRND or -Cys-Asn-Ile-Asn-Asn-Val-Cys-Asn-Phe-Ala-Ser-Arg-Asn-Asp-. The amino acid sequence in which one or more amino acids are inserted, deleted or replaced based on the amino acid sequence shown in SEQ ID NO: 4 or 5, and the function remains unchanged, can refer to the rules described above, and this application will not repeat them here.

[0030] In some specific implementations, the X1 also includes pyruvate.

[0031] In some specific implementations, X1 is selected from pyruvate or glutamine;

[0032] The X2 comprises 0, 1, 2, 3, 4, 12 or 13 amino acid residues.

[0033] In some specific implementations, X2 includes more than 0 amino acid residues, wherein the amino acids include at least one or more of tyrosine, lysine and serine.

[0034] In some specific implementations, the Y1 comprises 2, 3 or 6 amino acid residues; and the Y2 comprises 0, 3 or 5 amino acid residues.

[0035] In some specific implementations, the Y1 includes at least phenylalanine;

[0036] The Y2 includes non-zero amino acid residues, wherein the amino acids include at least one or more of tyrosine and serine.

[0037] In some specific implementations, the peptoid compound has an amino acid sequence as shown in SEQ ID No. 6 to 23, or an amino acid sequence in which one or more amino acids are inserted, deleted, or replaced based on the amino acid sequence as shown in SEQ ID NO: 6 to 23 and the function is maintained unchanged.

[0038] Specifically, the amino acid sequences shown in SEQ ID No.6 to 23 are:

[0039] SEQ ID No.6:

[0040] H-Pyr-Lys-Arg-Lys-Arg-Lys-Lys-Ser-Arg-Tyr-Lys-Ser-Phe-Ile-Asn-Ile-Asn

[0041] -Asn-Val-Ile-Asn-Phe-OH

[0042] SEQ ID No.7:

[0043] H-Pyr-Lys-Arg-Lys-Arg-Lys-Lys-Ser-Arg-Tyr-Lys-Phe-Ile-Asn-Ile-Asn-Asn

[0044] -Val-Ile-Asn-Phe-OH

[0045] SEQ ID No.8:

[0046] H-Pyr-Lys-Arg-Lys-Arg-Lys-Lys-Ser-Arg-Tyr-Phe-Ile-Asn-Ile-Asn-Asn-Val

[0047] -Ile-Asn-Phe-OH

[0048] SEQ ID No.9:

[0049] H-Pyr-Lys-Arg-Lys-Arg-Lys-Lys-Ser-Arg-Phe-Ile-Asn-Ile-Asn-Asn-Val-Ile-Asn-Phe-OH

[0050] SEQ ID No.10:

[0051] H-Pyr-Lys-Arg-Lys-Arg-Lys-Lys-Ser-Arg-Tyr-Lys-Ser-Leu-Arg-Arg-Phe-Ser

[0052] -Thr-Ala-Pro-Phe-Ala-Phe-Ile-Asp-Ile-Asn-Asp-Val-Ile-Asn-Phe-OH

[0053] SEQ ID No.11:

[0054] H-Gln-Lys-Arg-Lys-Arg-Lys-Lys-Ser-Arg-Tyr-Lys-Ser-Leu-Arg-Arg-Phe-Ser

[0055] -Thr-Ala-Pro-Phe-Ala-Phe-Ile-Asp-Ile-Asn-Asp-Val-Ile-Asn-Phe-OH

[0056] SEQ ID No.12:

[0057] H-Gln-Lys-Arg-Lys-Arg-Lys-Lys-Ser-Arg-Tyr-Lys-Ser-Phe-Ala-Phe-Ile-Asn

[0058] -Ile-Asn-Val-Ile-Asn-Phe-OH

[0059] SEQ ID No.13:

[0060] H-Gln-Lys-Arg-Lys-Arg-Lys-Lys-Ser-Arg-Tyr-Lys-Phe-Ile-Asn-Ile-Asn-Asn-Val

[0061] -Asn-Phe-OH

[0062] SEQ ID No.14:

[0063] H-Gln-Lys-Arg-Lys-Arg-Lys-Lys-Ser-Arg-Tyr-Lys-Ser-Leu-Arg-Arg-Phe-Ser-Thr

[0064] -Ala-Pro-Phe-Ala-Phe-Ile-Asp-Ile-Asn-Asp-Val-Ile-Asn-Phe-OH

[0065] SEQ ID No.15:

[0066] H-Phe-Ala-Phe-Ile-Asp-Ile-Asn-Asp-Val-Ile-Asn-Phe-Ala-Ser-Arg-Asn-Asp-Tyr

[0067] -Ser-Tyr-OH

[0068] SEQ ID No.16:

[0069] H-Thr-Ala-Pro-Phe-Ala-Phe-Ile-Asp-Ile-Asn-Asp-Val-Ile-Asn-Phe-Ala-Ser-Arg

[0070] -Asn-Asp-OH

[0071] SEQ ID No.17:

[0072] H-Phe-cyc[Cys-Asn-Ile-Asn-Asn-Val-Cys]-Asn-Phe-Ala-Ser-Arg-Asn-Tyr-Ser-Tyr-Trp-Leu-OH

[0073] SEQ ID No.18:

[0074] H-Pyr-Lys-Arg-Lys-Arg-Lys-Lys-Ser-Arg-Tyr-Lys-Phe-Ile-Asn-Ile-Asn

[0075] -Val-Ile-Asn-Phe-NH2

[0076] SEQ ID No.19:

[0077] H-Pyr-Lys-Arg-Lys-Arg-Lys-Lys-Ser-Arg-Tyr-Phe-Ile-Asn-Ile-Asn-Asn-Val

[0078] -Ile-Asn-Phe-NH2

[0079] SEQ ID No.20:

[0080] H-Pyr-Lys-Arg-Lys-Arg-Lys-Lys-Ser-Arg-Phe-Ile-Asn-Ile-Asn-Asn-Val-Ile-Asn-Phe-NH2

[0081] SEQ ID No.21:

[0082] H-Pyr-Lys-Arg-Lys-Arg-Lys-Lys-Ser-Arg-Tyr-Lys-Ser-Leu-Arg-Arg-Phe-Ser

[0083] -Thr-Ala-Pro-Phe-Ala-Phe-Ile-Asp-Ile-Asn-Asp-Val-Ile-Asn-Phe-NH2

[0084] SEQ ID No.22:

[0085] H-Gln-Lys-Arg-Lys-Arg-Lys-Lys-Ser-Arg-Tyr-Lys-Ser-Phe-Ala-Phe-Ile-Asn-Ile-Asn-Asn-Val-Ile-Asn-Phe-NH2.

[0086] SEQ ID No.23:

[0087] H-Thr-Ala-Pro-Phe-Ala-Phe-Ile-Asp-Ile-Asn-Asp-Val-Ile-Asn-Phe-Ala-Ser-Arg

[0088] -Asn-Asp-NH2

[0089] The amino acid sequences in which one or more amino acids are inserted, deleted or replaced based on the amino acid sequences shown in SEQ ID NOs: 6 to 23 and the functions are maintained unchanged can refer to the rules described above, and the present application will not elaborate on them here.

[0090] The present application also provides the use of the above-mentioned peptoid compound in the preparation of integrin inhibitors.

[0091] In some specific implementations, the integrin inhibitor includes an integrin αvβ5 inhibitor, an integrin α5β1 inhibitor, or an integrin αvβ3 inhibitor.

[0092] The present application also provides the use of the above-mentioned peptoid compound in the preparation of angiogenesis inhibitors.

[0093] The present application provides a peptoid compound having a structure of formula (I) or (II): X1-Q1-X2-Q2 (I); Y1-Q3-Y2 (II). The peptoid compound provided in the present application can inhibit the activity of integrins, and the peptide targets integrin proteins such as αvβ5, α5β1, and αvβ3, and inhibits signal transduction through multiple receptors to inhibit angiogenesis. The experimental results show that the IC50 of the compounds provided in the present application for Integrinαvβ3 is less than 700nM, and the IC50 of Integrinαvβ5 is less than 700nM; the compounds provided in the present application can significantly inhibit the formation of lumen by HUVEC cells, and some compounds completely inhibit lumen formation at a concentration of 5uM, which is significantly better than AXT107; some compounds can significantly inhibit the formation of lumen area, lumen number, increase lumen gap and reduce lumen length, which is significantly better than AXT107. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Figure 1 HPLC spectrum of compound 1 prepared in Example 1;

[0095] Figure 2 This is the LCMS spectrum of compound 1 prepared in Example 1.

[0096] Figure 3 HPLC spectrum of compound 12 prepared in Example 12 of the present application;

[0097] Figure 4 LCMS spectrum of compound 12 prepared in Example 12;

[0098] Figure 5 This is the lumen image obtained in Example 15 of the present application;

[0099] Figure 6 It is a statistical graph of the effect of each compound on the lumen area;

[0100] Figure 7 It is a statistical graph of the effect of each compound on the number of lumens;

[0101] Figure 8 It is a statistical graph of the effect of each compound on the lumen gap;

[0102] Fig. 9 This is a statistical graph of the effect of each compound on the lumen length. DETAILED DESCRIPTION

[0103] The present invention provides a peptoid compound, a preparation method thereof, a pharmaceutical composition and its use. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0104] The peptoid compound, preparation method, pharmaceutical composition and use thereof provided by the present invention are described in detail below with reference to the examples.

[0105] Embodiment 1:

[0106] synthesis:

[0107] H-Pyr-Lys-Arg-Lys-Arg-Lys-Lys-Ser-Arg-Tyr-Lys-Ser-Phe-Ile-Asn-Ile-Asn-Asn-Val-Ile-Asn-Phe-OH (Compound 1)

[0108] Its structural formula is as follows:

[0109]

[0110] Step 1: Synthesis of Fmoc-Phe-resin

[0111] 2-CTC Resin (1.0 g, 1.0 mmol) was swollen with DCM (10 ml) in a peptide synthesis tube at room temperature for 15 minutes, the solvent was drained, and a mixed solution of Fmoc-Phe-OH (193 mg, 0.5 mmol) and DIEA (191 mg, 1.5 mmol) in DCM (10 mL) was added, and the mixture was reacted with nitrogen gas at room temperature for 2 hours; after the reaction was completed, 0.5 ml of methanol and 0.3 ml of DIEA were added to continue the reaction for 15 minutes, the reaction solution was drained, and the mixture was washed three times with DCM (10 mL), and finally washed three times with DMF (10 mL), and the solvent was drained to obtain Fmoc-Phe-resin, which was directly used in the next reaction.

[0112] Step 2: Synthesis

[0113] H-Pyr-Lys(Boc)-Arg(Pbf)-Lys(Boc)-Arg(Pbf)-Lys(Boc)-Lys(Boc)-Ser(tBu)

[0114] -Arg(Pbf)-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Phe-Ile-Asn(Trt)-Ile-Asn(Trt)-Asn(Trt)-Val-Ile-Asn(Trt)-Phe-Resin

[0115] Removal of Fmoc protecting group: piperidine / DMF (volume ratio 1 / 4, 10 mL) was added to the Fmoc-Phe-resin obtained above, reacted at room temperature for 5 minutes, drained, and piperidine / DMF (volume ratio 1 / 4, 10 mL) was added again and reacted at room temperature for 5 minutes. After the reaction was completed, the reaction solution was drained and washed with DMF (10 mL) 6 times, drained and waited for the next reaction.

[0116] Coupling of amino acids: HOBT (138 mg, 1 mmol), HBTU (385 mg, 1 mmol) and DIEA (193 mg, 1.5 mmol) were added to a DMF (10 ml) solution of Fmoc-Asn(Trt)-OH (610 mg, 1 mmol) respectively, and the reaction was activated in an ice bath for 15 minutes; after the activation was completed, the activation solution was added to the resin prepared above, and the reaction was carried out at room temperature for 1 hour; the reaction solution was drained, and the resin was washed 6 times with DMF (10 mL) to obtain Fmoc-Asn(Trt)-Phe-resin.

[0117] Repeat the above steps to couple amino acids Fmoc-Ile-OH, Fmoc-Val-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ile-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Ser(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc- oc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, H-Pyr-OH, get H-Pyr-Lys(Boc)-Arg(Pbf)-Lys(Boc)-Arg(Pbf)-Lys (Boc)-Lys(Boc)-Ser(tBu)-Arg(Pbf)-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Phe-Ile-Asn(Trt)-Ile-Asn(Trt)-Asn(Trt)-Val-Ile-Asn(Trt)-Phe-resin.

[0118] Resin shrinkage: wash twice with DCM (5 mL) and MeOH (5 mL) alternately, and finally wash twice with MeOH (5 mL); dry the obtained resin in a vacuum oven at 30°C for 3 h to obtain 3.231 g of H-Pyr-Lys(Boc)-Arg(Pbf)-Lys(Boc)-Arg(Pbf)-Lys(Boc)-Lys(Boc)-Ser(tBu)-Arg(Pbf)-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Phe-Ile-Asn(Trt)-Ile-Asn(Trt)-Asn(Trt)-Val-Ile-Asn(Trt)-Phe-resin.

[0119] Step 3: Synthesis

[0120] H-Pyr-Lys-Arg-Lys-Arg-Lys-Lys-Ser-Arg-Tyr-Lys-Ser-Phe-Ile-Asn-Ile-Asn-Asn-Val-Ile-Asn-Phe-OH (Compound 1)

[0121] The above prepared

[0122] H-Pyr-Lys(Boc)-Arg(Pbf)-Lys(Boc)-Arg(Pbf)-Lys(Boc)-Lys(Boc)-Ser(tBu)

[0123] -Arg(Pbf)-Tyr(tBu)-Lys(Boc)-Ser(tBu)-Phe-Ile-Asn(Trt)-Ile-Asn(Trt)-Asn(Trt)-Val-Ile-Asn(Trt)-Phe-resin (1.513 g) and a mixed solution of TFA:TIS:H2O (volume ratio 95:2.5:2.5, 10 ml) were added to a 50 ml reaction bottle respectively and reacted for 2 h at room temperature; after the reaction, the mixture was filtered and washed three times with 5 ml of TFA, the solution was combined and concentrated to about 2 ml; the concentrated solution was added dropwise to 40 ml of methyl tert-butyl ether, settled in an ice bath for 30 minutes, centrifuged, and washed three times with methyl tert-butyl ether (10 ml); the solid was placed in a vacuum drying oven and dried overnight to obtain 612 mg of a white solid, which was directly purified by Pre-HPLC to obtain a purified solution, which was concentrated and lyophilized to obtain 74 mg of compound 1 as a white solid.

[0124] The obtained compound 1 was analyzed, and the results are shown in Figure 1 and Figure 2 , Figure 1The HPLC spectrum of compound 1 prepared in Example 1 is Figure 1 It can be seen that the purity of compound 1 is 98.43%. Figure 2 This is the LCMS spectrum of compound 1 prepared in Example 1. MS (ESI): m / z 1383.4 [M / 2+H] + .

[0125] Embodiments 2 to 11

[0126] According to the same method as Example 1, different amino acids or polypeptides were used to prepare compounds 2 to 11 in Table 1.

[0127] Example 12

[0128] synthesis

[0129] H-Phe-cyc[Cys-Asn-Ile-Asn-Asn-Val-Cys]-Asn-Phe-Ala-Ser-Arg-Asn-Asp-Tyr-Ser-Tyr-Trp-Leu-OH (Compound 12)

[0130] Straight chain peptide

[0131] H-Phe-Cys-Asn-Ile-Asn-Asn-Val-Cys-Asn-Phe-Ala-Ser-Arg-Asn-Asp-Tyr-Ser

[0132] The synthesis method of crude -Tyr-Trp-Leu-OH (M1) is the same as that in Example 1.

[0133] The crude product of M1 (235 mg) was dissolved in 40 ml of 5% DMSO aqueous solution, and air was blown into the solution for 24 h. The reaction solution was directly purified by Pre-HPLC to obtain a purified solution, which was concentrated and freeze-dried to obtain 3.55 mg of a white solid. The sample was dissolved in DMSO and then the spectrum was analyzed. The results are shown in Figure 3 , Figure 3 The HPLC spectrum of compound 12 prepared in Example 12 of the present application is Figure 3 It can be seen that the purity of compound 12 is 95.35%.

[0134] The obtained compound 12 was subjected to mass spectrometry analysis, and the results are shown in Figure 4 , Figure 4 This is the LCMS spectrum of compound 12 prepared in Example 12. MS (ESI): m / z (M+2H + ) / 2=1221.5, (M+NH4 + ) / 2=1229.6,(M+3H +) / 3=814.6.

[0135] Preparation method of compound 13 to compound 18: Compound 13 to compound 18 were prepared by using Rink Amide MBHA Resin as raw material and carrying out the synthesis method of step 2 and step 3 of compound 1.

[0136] Table 1 Synthesis information of compounds 1 to 18

[0137]

[0138]

[0139] Example 13 Integrin αvβ3 ELISA assay

[0140] 1. Coating solution preparation (50 ml): Take 5 ml of Solebow coating buffer (10x) + 50 μl (1M CaCl2) + 25 μl (1M MgCl2) + 44.8 ml of pure water, and use a pH meter to adjust the pH value to about 8 (original pH = 9.6), so that the final concentration of the liquid contains 1 mM Ca 2 + / 0.5mM Mg 2+ ion;

[0141] 2. Coating: dilute αvβ3 from 600ug / ml to 1ug / ml with Coating Buffer (i.e., pH of coating solution = 8.0) to a final concentration of 1ug / ml. Prepare X ml volume of coating solution. Add (1*X) / 600 volume of protein stock solution to X-(1*X) / 600 volume of coating solution. Mix by blowing with a 1ml gun to evenly distribute the protein in the solution. Pipette 100ul into a 96-well plate. Put the 96-well plate into a plastic bag and seal it to prevent evaporation. Place it at 4°C overnight.

[0142] 3. Prepare Washing Buffer (PBSTC): Take 500ml volume as an example. PBSTC includes: Tween 250ul + 500μl (1M CaCl2) + 250μl (1M MgCl2). Use PBS to make the volume to 500ml, so that the final concentration of the liquid contains 1mM Ca 2+ / 0.5mm 2+ ion.

[0143] 4. Washing: Take out the 96-well plate that has been coated overnight, pour out the remaining liquid in the plate, invert it on absorbent paper, and pat dry. Add 200ul Washing Buffer (PBSTC) to each well, let it stand for 3 minutes, pour out the remaining liquid in the plate, invert it on absorbent paper, and pat dry. Repeat this operation 3 times.

[0144] 5. Prepare blocking buffer: dilute 10% BSA with PBSTC in a 1:1 ratio.

[0145] 6. Blocking: After washing, pat dry, add 150ul Blocking Buffer to each well, and block at room temperature for 60 minutes.

[0146] 7. Prepare Dilution Buffer (PBSC): Taking 50 ml volume as an example, PBSC includes 50 μl (1M CaCl2) + 25 μl (1M MgCl2) and dilute to 50 ml with PBS.

[0147] 8. Compound preparation: Take out the compound and thaw it in advance. After the compound is completely melted, prepare a 200ul volume and dilute the compound to 2x test concentration, that is, compound = 20μM, 2μM, 1μM, 0.2μM, 0.1μM, 0.02μM, 0.002μM; positive RGDFK = 200nM.

[0148] 9. Preparation of ligand protein solution: dilute Biotinylated-Vitronectin (100ug / ml) to 1ug / ml (2x) with DilutionBuffer (PBSC), calculate the required volume, knowing that each well has 50ul, volume = 50*(total number of wells + 4 wells (to balance loss)). Take protein mother solution (1*volume) / 100 and add it to the total volume - (1*volume) / 100 in a centrifuge tube and mix well. Prepare 15min before the pre-incubation is completed.

[0149] 10. Mix the compound and ligand protein: Mix the compound and ligand protein in a 1:1 ratio for later use.

[0150] 11. Wash the plate: Repeat step 4.

[0151] 12. Add the mixed solution prepared in step 10: Pipette 100ul of the mixed solution into the 96-well plate, put the 96-well plate into a plastic bag, and let it stand at room temperature for 1.5h.

[0152] 13. Add HRP-SA: dilute HRP-SA with Dilution Buffer (PBSC) at a ratio of 1:5000, mix well in a centrifuge tube, add 100ul HRP solution to each well, put into a plastic bag, and let stand at room temperature for 60 minutes.

[0153] 14. Wash the plate: Repeat step 4.

[0154] 15. Add color developing solution: Add 50ul Substrate Solution (TMB color developing solution) to each well under light-proof conditions for 3 minutes, and read the results in an ELISA reader (pay attention to the color change. When the color is darker, read the OD650. When the OD650 reading reaches between 0.85-1.0, add the stop solution);

[0155] 16. Add stop solution: Add 25ul Stop Solution to each well, and then place it in the microplate reader for reading.

[0156] The results are shown in Table 2, which shows the IC50 values ​​(nM) of the compounds prepared in the present application against αvβ3.

[0157] Table 2 IC50 values ​​(nM) of the compounds prepared in this application for αvβ3

[0158] Serial number Integrinαvβ3 IC50(nM) Compound 2 323.9 Compound 3 267.2 Compound 4 657.8 Compound 5 687.4

[0159] As shown in Table 2, the IC50 values ​​of compounds 2 to 5 against Integrin αvβ3 are all less than 700 nM.

[0160] Example 14 Integrin αvβ5 ELISA detection

[0161] 1. Coating solution preparation (50 ml): Take 5 ml of Solebow coating buffer (10x) + 50 μl (1M CaCl2) + 25 μl (1M MgCl2) + 44.8 ml of pure water, and use a pH meter to adjust the pH value to about 8 (original pH = 9.6), so that the final concentration of the liquid contains 1 mM Ca 2 + / 0.5mM Mg 2+ ion.

[0162] 2. Coating: dilute αvβ5 from 500ug / ml to 1ug / ml with Coating Buffer (i.e., pH of coating solution = 8.0), with a final concentration of 1ug / ml. Prepare X ml volume of coating solution. Add (1*X) / 500 volume of protein stock solution to X-(1*X) / 500 volume of coating solution. Mix by blowing with a 1ml gun to evenly distribute the protein in the solution. Pipette 100ul into a 96-well plate. Put the 96-well plate into a plastic bag and seal it to prevent evaporation. Place it at 4°C overnight.

[0163] 3. Prepare Washing Buffer (PBSTC): Take 500ml volume as an example. PBSTC includes: Tween 250ul + 500μl (1M CaCl2) + 250μl (1M MgCl2). Use PBS to make the volume to 500ml, so that the final concentration of the liquid contains 1mM Ca 2+ / 0.5mm 2+ ion

[0164] 4. Washing: Take out the 96-well plate that has been coated overnight, pour out the remaining liquid in the plate, invert it on absorbent paper, and pat dry. Add 200ul Washing Buffer (PBSTC) to each well, let it stand for 3 minutes, pour out the remaining liquid in the plate, invert it on absorbent paper, and pat dry. Repeat this operation 3 times.

[0165] 5. Prepare blocking buffer: dilute 10% BSA with PBSTC in a 1:1 ratio.

[0166] 6. Blocking: After washing, pat dry, add 150ul Blocking Buffer (5% BSA (diluted with PBSTC)) to each well, and incubate at room temperature for 60 minutes for blocking.

[0167] 7. Prepare Dilution Buffer (PBSC): Taking 50 ml volume as an example, PBSC includes 50 μl (1M CaCl2) + 25 μl (1M MgCl2) and dilute to 50 ml with PBS.

[0168] 8. Compound preparation: Take out the compound and thaw it in advance. After the compound is completely melted, prepare a 200ul volume and dilute the compound to 2x test concentration, that is, compound = 20μM, 2μM, 1μM, 0.2μM, 0.1μM, 0.02μM, 0.002μM; positive RGDFK = 400nM.

[0169] 9. Preparation of ligand protein solution: dilute Biotinylated-Vitronectin (100ug / ml) to 2ug / ml (2x) with DilutionBuffer (PBSC), calculate the required volume, knowing that each well has 50ul, volume = 50*(total number of wells + 4 wells (to balance loss)). Take (1*volume) / 100 of the protein stock solution and add it to the total volume - (1*volume) / 100 in a centrifuge tube and mix well. Prepare 15min before the pre-incubation is completed.

[0170] 10. Mix the compound and ligand protein: Mix the compound and ligand protein in a 1:1 ratio for later use.

[0171] 11. Wash the plate: Repeat step 4.

[0172] 12. Add the mixed solution prepared in step 10: Pipette 100ul of the mixed solution into the 96-well plate, put the 96-well plate into a plastic bag, and let it stand at room temperature for 1.5h.

[0173] 13. Add HRP-SA: dilute HRP-SA with Dilution Buffer (PBSC) at a ratio of 1:5000, mix well in a centrifuge tube, add 100ul HRP solution to each well, put into a plastic bag, and let stand at room temperature for 60 minutes.

[0174] 14. Wash the plate: Repeat step 4.

[0175] 15. Add color developing solution: In a dark environment, add 50ul Substrate Solution (TMB color developing solution) to each well for 3 minutes, and then place in a microplate reader to read the results (pay attention to the color change, when the color is darker, read the OD650, and when the OD650 reading reaches between 0.85-1.0, add the stop solution).

[0176] 16. Add stop solution: Add 25ul Stop Solution to each well, and then place it in the microplate reader for reading.

[0177] The results are shown in Table 3, which shows the IC50 values ​​(nM) of the compounds prepared in the present application against αvβ5.

[0178] Table 3 IC50 values ​​(nM) of the compounds prepared in this application for αvβ5

[0179] Serial number Integrin αvβ5 IC50(nM) Compound 2 338.7 Compound 3 301.9 Compound 4 518.1 Compound 5 629.4 Compound 7 66.85

[0180] As shown in Table 3, the IC50 values ​​of compounds 2 to 5 and compound 7 for Integrin αvβ5 are all less than 700 nM.

[0181] Example 15: Inhibition of neovascularization

[0182] 1. Preparation before the experiment

[0183] (1) Place the ice in a foam box after freezing with an ice maker, immerse Matrigel in ice and place in a refrigerator at 4 degrees overnight before use;

[0184] (2) Pre-cool the 96-well culture plate at 4 degrees in advance. Open the package of the 96-well plate in a clean bench, seal the gaps around the plate with sealing film, and place it in a 4-degree refrigerator.

[0185] (3) Fill a 50 ml centrifuge tube with 100 ul pipette tips, pre-cool it to -20 degrees, and place it on ice to maintain low temperature before use.

[0186] (4) Cells (in use: covering the entire bottom of the container, with cells in contact but not crowded) are replaced with basal culture medium 12 h in advance.

[0187] 2. Matrigel loading

[0188] (1) Replace the 96-well ELISA plate rotor in the centrifuge in advance and set it to precool at 4 degrees;

[0189] (2) Before use, place the 96-well plate flat on ice in a foam box. On ice, use tweezers to grab a cold gun tip and pipette 50 μL of matrix gel into the 96-well culture plate to avoid bubbles.

[0190] (3) Centrifuge the 96-well plate with gel at 1200 g for 10 min at 4°C;

[0191] (4) Place the 96-well plate in a 37°C incubator for 30 min to allow the gel to solidify;

[0192] 3. Cell plating

[0193] (1) Cell resuspension

[0194] ①Discard the culture medium.

[0195] ② Add 2 ml PBS buffer to wash the cells and then discard the PBS buffer.

[0196] ③After adding 2 ml of digestion solution, observe under a microscope until the cells appear round and fall off by tapping lightly.

[0197] ④ Immediately add 4 ml of complete culture medium to terminate digestion, blow the cells to suspend them individually, transfer the cell solution to a 15 ml centrifuge tube, and centrifuge at 3000 rpm for 2 min.

[0198] ⑤Discard the supernatant, add 1ml PBS, resuspend the cells, take 10ul of cell solution and add 10ul of dye to mix, then add 10ul volume to the counting plate for counting, and dilute the cells to 5*10 5 / ml.

[0199] (2) Dilution compound

[0200] Compounds were diluted to the desired concentration in basal medium.

[0201] (3) Incubation

[0202] First, add 50ul (3×) compound, 50μl (3x) VEGF165 protein (i.e., 80ng / ml) to each well, and then add 50ul cell solution (the cells need to be mixed with a pipette from time to time during the process, and then the liquid is pipetted and added to the plate), put it in the incubator, and incubate it at 37°C and 5% CO2.

[0203] 4. Take photos

[0204] After 10 h of incubation, photos were taken and recorded using a microscope (objective lens magnification was 4).

[0205] 5. Results

[0206] Results of intuitive image analysis: Lumen images such as Figure 5 As shown, Figure 5 The lumen image obtained in Example 15 of the present application, wherein 001 is the positive reference AXT107 compound, Figure 5 It can be seen that compounds 2, 3, 4, 5, 7 and 11 (referred to as 014, 015, 016, 024, 027 and 033, respectively) significantly inhibited the formation of tubes in HUVEC cells. Through intuitive analysis of imaging pictures, 014, 015 and 027 completely inhibited tube formation at a concentration of 5uM, which was significantly better than 001 (AXT107).

[0207] The lumen area was analyzed and the results are shown in Figure 6 , Figure 6 is a statistical graph of the effect of each compound on the lumen area, wherein 1 is the positive reference AXT107 compound, Figure 6 It can be seen that compounds 2, 3, 4, 5, 7 and 11 (referred to as 14, 15, 16, 24, 27 and 33, respectively) significantly inhibited the formation of tubes in HUVEC cells. According to data analysis, the inhibitory effects of 15, 16, 24, 27 and 33 at 5uM were significantly better than 1 (AXT107).

[0208] The number of lumens was analyzed and the results are shown in Figure 7 , Figure 7 is a statistical graph of the effect of each compound on the number of lumens, wherein 1 is the positive reference AXT107 compound, Figure 7 It can be seen that compounds 2, 3, 4, 5, 7 and 11 (referred to as 14, 15, 16, 24, 27 and 33, respectively) significantly inhibited the formation of tubes in HUVEC cells. At 5uM, compounds 2, 3, 4, 5, 7 and 11 were better than AXT107.

[0209] The lumen clearance was analyzed and the results are shown in Figure 8 , Figure 8 is a statistical graph of the effect of each compound on the lumen clearance, wherein 1 is the positive reference AXT107 compound, Figure 8 It can be seen that compounds 2, 3, 4, 5, 7 and 11 (referred to as 14, 15, 16, 24, 27 and 33, respectively) significantly inhibited the formation of tubes in HUVEC cells. At 5uM, compounds 2, 3, 4, 5, 7 and 11 were better than AXT107.

[0210] The lumen length was analyzed and the results are shown in Fig. 9 , Fig. 9 is a statistical graph of the effect of each compound on the lumen length, wherein 1 is the positive reference AXT107 compound, Fig. 9It can be seen that compounds 2, 3, 4, 5, 7 and 11 (referred to as 14, 15, 16, 24, 27 and 33, respectively) significantly inhibited the formation of tubes in HUVEC cells. At 5uM, compounds 2, 3, 4, 5, 7 and 11 were better than AXT107.

[0211] Comprehensive evaluation: The comprehensive data of compounds 2, 3, 4, 5, 7 and 11 in inhibiting neovascularization were better than AXT107.

[0212] Example 16 Cytotoxicity

[0213] HUVEC cells in the logarithmic growth phase were inoculated into black-edged transparent-bottom well plates to a volume of 2 × 10 cells per well. 4 cells were cultured in a carbon dioxide incubator at 37°C and 5% CO2 concentration for 24 hours in a complete culture medium (4.5 ml FBS + 45 ml ECM basal culture medium (ScienCell) + 0.5 ml penicillin-streptomycin solution (Biosharp-BL505A)). Before the experiment, the required compounds were prepared with blank basal culture medium at concentrations of 200, 100, 50, 25, 12.5, 6.25, 3.125, and 1.5625 μM, and the culture medium was replaced with a drug-containing culture medium. After incubation in the incubator for 24 hours, the 96-well plate to be tested was taken out and equilibrated at room temperature for 10 minutes. 100 μL of celltiter solution was added to each well, and the plate was oscillated on an oscillator for 2 minutes. After the oscillation was completed, it was allowed to stand for 10 minutes, and then transferred to a full black plate for detection using an ELISA reader. The results are shown in Table 4, which shows the cytotoxicity of the compounds provided in the examples of the present application.

[0214] Table 4 Cytotoxicity of compounds provided in the examples of this application

[0215] Serial number EC50(uM) Compound 2 4.8 Compound 3 6.9 Compound 4 8.1 Compound 5 13 Compound 7 7.56 Compound 11 327.6

[0216] The above are only preferred embodiments 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 principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A peptoid compound having a structure of formula (I) or (II): X1-Q1-X2-Q2(I); Y1-Q3-Y2(II); in, In the structure of formula (I), Q1 has the amino acid sequence shown in SEQ ID No. 1, or an amino acid sequence in which one or more amino acids are inserted, deleted, or replaced based on the amino acid sequence shown in SEQ ID NO: 1, and the function remains unchanged; Q2 has the amino acid sequence shown in SEQ ID No. 2 or 3, or an amino acid sequence in which one or more amino acids are inserted, deleted, or substituted based on the amino acid sequence shown in SEQ ID NO: 2 or 3, and the function remains unchanged; X1 includes 0 to 5 amino acid residues, and X2 includes 0 to 15 amino acid residues; the amino acids in X1 and X2 are independently selected from lysine, arginine, serine, tyrosine, phenylalanine, leucine, threonine, alanine, proline, glutamine or cysteine; In the structure of formula (II), Q3 has the amino acid sequence shown in SEQ ID No. 4 or 5, or an amino acid sequence in which one or more amino acids are inserted, deleted, or replaced based on the amino acid sequence shown in SEQ ID NO: 4 or 5, and the function is maintained unchanged; Y1 includes 2 to 8 amino acid residues, and Y2 includes 0 to 8 amino acid residues; the amino acids in Y1 and Y2 are independently selected from leucine, alanine, serine, arginine, asparagine, aspartic acid, tyrosine or tryptophan.

2. The peptoid compound according to claim 1, characterized in that The X1 also includes pyruvic acid.

3. The peptoid compound according to claim 2, characterized in that The X1 is selected from pyruvate or glutamine; The X2 comprises 0, 1, 2, 3, 4, 12 or 13 amino acid residues.

4. The peptoid compound according to claim 3, characterized in that The X2 includes non-zero amino acid residues, wherein the amino acids include at least one or more of tyrosine, lysine and serine.

5. The peptoid compound according to claim 1, characterized in that The Y1 comprises 2, 3 or 6 amino acid residues; the Y2 comprises 0, 3 or 5 amino acid residues.

6. The peptoid compound according to claim 5, characterized in that The Y1 at least includes phenylalanine; The Y2 includes non-zero amino acid residues, wherein the amino acids include at least one or more of tyrosine and serine.

7. The peptoid compound according to any one of claims 1 to 6, characterized in that It has the amino acid sequence shown in SEQ ID NOs. 6 to 23, or an amino acid sequence in which one or more amino acids are inserted, deleted or replaced based on the amino acid sequence shown in SEQ ID NOs: 6 to 23, and the function is maintained unchanged.

8. Use of the peptoid compound according to any one of claims 1 to 7 in the preparation of integrin inhibitors.

9. The use according to claim 8, wherein the integrin inhibitor comprises an integrin αvβ5 inhibitor, an integrin α5β1 inhibitor or an integrin αvβ3 inhibitor.

10. Use of the peptoid compound according to any one of claims 1 to 7 in the preparation of angiogenesis inhibitors.

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