A peptoid compound, its preparation method and application

Abstract

The application provides a kind of peptoid compound, including the sequence of formula (I) shown connected amino acid fragment: AA2-AA3-AA4 Formula (I). Compared with the prior art, the peptoid compound provided by the application can inhibit the activity of integrin, and the peptide targets integrin class protein such as αvβ5, α5β1, αvβ3, and inhibits signal transduction through various receptors, including vascular endothelial growth factor receptor (VEGFR), hepatocyte growth factor receptor (HGFR), insulin-like growth factor receptor (IGFR) and platelet-derived growth factor receptor (PDGFR), and inhibits neovascularization through the above signal pathways, so that it can be used for the pathogenesis of inflammation, wound healing, scar formation prevention, thrombosis, vitreoretinal disease.

Description

[0001] This application claims priority to Chinese Patent Application No. 202210411656.7, filed on April 19, 2022, entitled "A Peptide Compound, Its Preparation Method and Application", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of medicinal chemistry technology, and particularly relates to a peptide-like compound, its preparation method and application. Background Technology

[0003] The RGD tripeptide sequence is present in many proteins and plays a role in cell adhesion. Proteins containing the RGD tripeptide sequence include collagen, fibronectin, vitreous connexin, von Willebrand factor (VWF), certain disintegrants, and integrins. Integrins are heterodimeric cell surface receptors that mediate cell-exposed extracellular matrix (ECM) adhesion by binding to ligands with exposed RGD sequences. Normal integrin RGD binding is thought to play a role in gene expression involved in cell growth, migration, and survival. Improper regulation of these cell growth, migration, and survival can lead to various disease states, including thrombosis, inflammation, and cancer. Therefore, RGD peptides have been investigated as potential mimics of cell adhesion proteins, and their ability to bind to integrins has been studied for therapeutic purposes such as inhibiting apoptosis, angiogenesis, and tumorigenesis. Their multimeric forms are used in internal radiotherapy agents and cancer imaging agents, and they also have anticancer drug delivery capabilities.

[0004] In the eye, integrins influence numerous processes, including ocular development, cell migration, healing, and some pathological processes. Integrins can also regulate inflammation and thrombosis in ocular tissues. In animal models, injection of RGD peptides has been reported to induce posterior vitreoretinal detachment, thus potentially for the treatment of certain retinal diseases and / or to facilitate vitrectomy for vitreous removal during surgery. Therefore, research into drugs that inhibit integrins is crucial. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a peptide-like compound that can inhibit integrin activity, its preparation method and application.

[0006] This invention provides a peptide-like compound comprising amino acid fragments linked together by the sequence shown in formula (I):

[0007] AA2-AA3-AA4 (I);

[0008] Wherein, AA2 is selected from basic amino acids, basic amino acid-like substances, and N-terminal substituted derivatives of the above-mentioned amino acids or amino acid-like substances.

[0009] AA3 is selected from nonpolar amino acids, nonpolar amino acid-like substances, polar neutral amino acids, polar neutral amino acid-like substances, and N-terminal substituted derivatives of the above amino acids or amino acid-like substances.

[0010] The AA4 is selected from amino acids with hydroxyl groups in their side chains or amino acid-like substances with hydroxyl groups in their side chains.

[0011] The AA2, AA3 and AA4 are connected by condensation to form amide bonds.

[0012] Preferably, it has the structure shown in formula (II) or formula (III):

[0013] AA1-AA2-AA3-AA4-AA5-AA6 formula (II);

[0014] cycl[AA1-AA2-AA3-AA4-AA5-AA6] formula (III);

[0015] Among them, AA1, AA3 and AA6 are each independently selected from nonpolar amino acids, nonpolar amino acid-like substances, polar neutral amino acids, polar neutral amino acid-like substances, and N-terminal substituted derivatives of the above amino acids or amino acid-like substances.

[0016] The AA2 is selected from basic amino acids, basic amino acid-like substances, and N-terminal substituted derivatives of the above-mentioned amino acids or amino acid-like substances.

[0017] The AA4 is selected from amino acids with hydroxyl groups in their side chains or amino acid-like substances with hydroxyl groups in their side chains.

[0018] The AA5 is selected from polar neutral amino acids or polar neutral amino acid classes;

[0019] The AA1, AA2, AA3, AA4, AA5 and AA6 are connected by condensation into amide bonds, and AA1 is the N-terminus;

[0020] Cyc means that any one of AA1 to AA5 forms a cyclization ring with AA6 through condensation to form an amide bond.

[0021] Preferably, AA1 and AA3 are each independently selected from glycine, alanine, proline, leucine, valine, isoleucine, phenylalanine, serine, threonine, tyrosine, methyltyrosine, ethyltyrosine, methionine, amino acids or substituted amino acids or amino acids represented by any one of formulas (1) to (5):

[0022]

[0023] Wherein, -X1 is -H, substituted or unsubstituted C1-C8 alkyl, -OH, substituted or unsubstituted C2-C8 alkenyl, -NH2, -CONH2, -CHO, -COCH3, -SOCH3, -SO2CH3, -SOCH2CH3, -SO2CH2CH3, p-toluenesulfonyl, substituted or unsubstituted C3-C10 aliphatic cyclic group, substituted or unsubstituted C6-C20 aromatic cyclic group, substituted or unsubstituted C2-C20 heterocyclic group, substituted or substituted C1-C8 alkane acyl, substituted or unsubstituted C1-C8 alkane sulfonyl;

[0024] The substituents in the substituted C1-C8 alkyl, substituted C2-C8 alkenyl, substituted C3-C10 alicyclic, substituted C6-C20 aromatic, substituted C2-C20 heterocyclic, substituted C1-C8 alkane acyl, and substituted C1-C8 alkane sulfonyl groups are each independently selected from one or more of the following groups: halogen, hydroxyl, amino, carboxyl, sulfonic acid, mercapto, methyl sulfide, ethyl sulfide, phosphoric acid, nitro, -SOMe, -SO2Me, -CONH2, -COOMe, -COOEt, -NHCOH, -NHCOCH3, -NHSO2Me, p-toluenesulfonamide, phosphate ester, and sulfonate ester.

[0025] R3 to R6 are each independently selected from substituted or unsubstituted C1-C8 alkylene groups, substituted or unsubstituted C2-C8 alkenyl groups, substituted or unsubstituted C2-C8 alkyne groups, heteroatoms, substituted or unsubstituted C3-C10 alicyclic groups, substituted or unsubstituted C6-C20 aromatic cyclic groups, and substituted or unsubstituted C2-C20 heterocyclic groups; wherein the substituted C1-C8 alkylene groups, substituted C2-C8 alkenyl groups, substituted C2-C8 alkyne groups, and substituted C The substituents in the 3-C10 aliphatic ring group, the substituted C6-C20 aromatic ring group, and the substituted C2-C20 heterocyclic group are each independently selected from one or more of the following groups: halogen, hydroxyl, amino, carboxyl, sulfonic acid, mercapto, methyl sulfide, ethyl sulfide, phosphoric acid, nitro, -SOMe, -SO2Me, -CONH2, -COOMe, -COOEt, -NHCOH, -NHCOCH3, -NHSO2Me, p-toluenesulfonamide, phosphate ester, and sulfonate ester.

[0026] R7 and R8 are each independently selected from -H, C1-C8 alkyl or C1-C8 alkylamino groups;

[0027] m1 is an integer from 1 to 17; m2 is an integer from 0 to 13;

[0028] The substituents of the aforementioned substituted amino acids or amino acid-like substances are each independently selected from one or more of halogens, hydroxyl groups, and thiol groups.

[0029] Preferably, AA1 and AA3 are each independently selected from glycine, alanine, proline, leucine, valine, isoleucine, phenylalanine, serine, threonine, tyrosine, methyltyrosine, ethyltyrosine, methionine, L-configured amino acids of the above amino acids, D-configured amino acids of the above amino acids, amino acid-like substances or substituted amino acids or amino acid-like substances represented by any one of formulas (6) to (12):

[0030]

[0031] m1 is an integer from 1 to 17; m2 is an integer from 0 to 13;

[0032] The substituents of the aforementioned substituted amino acids or amino acid-like substances are each independently selected from one or more of halogens, hydroxyl groups, and thiol groups.

[0033] Preferably, the AA2 is selected from arginine, lysine, histidine, Arg(Pbf), N-terminal alkylated derivatives of the above amino acids, or D-configuration amino acids of the above amino acids.

[0034] The side chain of AA4 includes one or more of sulfonyl, boric acid, amide and carboxyl groups;

[0035] The AA5 is selected from threonine, serine, cysteine, N-terminal alkylated derivatives of the above amino acids, or D-configuration amino acids of the above amino acids.

[0036] Preferably, the AA4 is selected from sulfonylalanine and any one of the amino acids represented by formulas (13) to (16):

[0037]

[0038] R1 and R2 are each independently selected from H or C1-C8 alkyl groups;

[0039] n1 to n7 are each independently selected from integers from 0 to 13;

[0040] L1 to L3 are each independently selected from substituted or unsubstituted C1 to C8 alkylene groups and substituted or unsubstituted C6 to C20 arylene groups;

[0041] The substituents in the substituted C1-C8 alkylene group and the substituted C6-C20 aryl group are each independently selected from one or more of the following groups: halogen, hydroxyl, amino, carboxyl, sulfonic acid, mercapto, methyl sulfide, ethyl sulfide, phosphoric acid, nitro, -SOMe, -SO2Me, -CONH2, -COOMe, -COOEt, -NHCOH, -NHCOCH3, -NHSO2Me, p-toluenesulfonamide, phosphate ester, and sulfonate ester.

[0042] Y1 to Y3 are each independently selected from C or S=O.

[0043] Preferably, the AA6 is selected from one of the structures shown in formulas (17) to (24):

[0044]

[0045] The R9 and R 10 Each is independently selected from H, halogen, C1-C8 alkyl, C1-C8 alkylamino, amino, hydroxyl, and C6-C20 aromatic cyclic groups;

[0046] The R 11 With R 13 Each is independently selected from -B(OH)2, halogens, borate ester groups, sulfonic acid or sulfonic acid ester groups, phosphoric acid or phosphate ester groups;

[0047] The R 12 Selected from H, C1-C8 alkyl, hydroxyl, C1-C8 alkane acyl, C1-C8 alkane sulfonyl, benzene sulfonyl, p-toluene sulfonyl, C6-C20 aryl sulfonyl group, C1-C8 alkane sulfinyl group, C1-C8 alkane phosphoryl group, C6-C20 aryl phosphoryl group, and C6-C20 aromatic ring group;

[0048] The R 14 Selected from H, C1-C8 alkyl, amino, and C6-C20 aryl cyclogroups;

[0049] The R 15 Selected from H, C1-C8 alkyl groups;

[0050] The R 16 It is selected from H, C1-C8 alkyl, C1-C8 alkoxy, C6-C20 aryloxy, and C6-C20 arylcyclogroup.

[0051] The present invention also provides the use of the above-mentioned peptide-like compound in the preparation of a drug that inhibits integrin.

[0052] The present invention also provides the use of the above-mentioned peptide-like compound in the preparation of a drug for inhibiting cell adhesion at the Arg-Gly-Asp binding site.

[0053] This invention provides a peptide-like compound comprising amino acid fragments linked together by the sequence shown in formula (I): AA2-AA3-AA4 (I); wherein, AA2 is selected from basic amino acids, basic amino acid-like compounds, or N-terminal substituted derivatives of the above amino acids or amino acid-like compounds; AA3 is selected from nonpolar amino acids, nonpolar amino acid-like compounds, polar neutral amino acids, polar neutral amino acid-like compounds, or N-terminal substituted derivatives of the above amino acids or amino acid-like compounds; AA4 is selected from amino acids or amino acid-like compounds with hydroxyl groups in their side chains; AA2, AA3, and AA4 are linked by an amide bond formed by condensation. Compared with existing technologies, the peptide-like compounds provided by this invention can inhibit the activity of integrins. The peptides target integrin-like proteins such as αvβ5, α5β1, and αvβ3, and inhibit signal transduction through multiple receptors, including vascular endothelial growth factor receptor (VEGFR), hepatocyte growth factor receptor (HGFR), insulin-like growth factor receptor (IGFR), and platelet-derived growth factor receptor (PDGFR). By inhibiting angiogenesis through these signaling pathways, they can be used for the prevention of inflammation, wound healing, and scar formation; thrombosis; and the pathogenesis of vitreoretinal diseases such as floaters, idiopathic macular holes, vitreous-macular traction, age-related macular degeneration, and wet macular degeneration. They are also used in choroidal neovascularization, vitreoretinal surgery, venous occlusion, corneal neovascularization, ischemic optic nerve, and prevention of iris redness and scar formation during glaucoma surgery. Attached Figure Description

[0054] Figure 1 The LCMS spectrum of compound TM obtained in Example 1 of this invention;

[0055] Figure 2 The LCMS spectrum of compound TM10 obtained in Example 2 of this invention;

[0056] Figure 3 The LCMS spectrum of compound TM19 obtained in Example 3 of this invention;

[0057] Figure 4 The image shows the LCMS spectrum of compound TM20 obtained in Example 4 of this invention. Detailed Implementation

[0058] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0059] This invention provides a peptide-like compound comprising amino acid fragments linked together by the sequence shown in formula (I):

[0060] AA2-AA3-AA4 (I);

[0061] Wherein, AA2 is a basic amino acid, a basic amino acid-like amino acid, or an N-terminal substituted derivative of the above-mentioned amino acid or amino acid-like amino acid; when the above-mentioned amino acid is a chiral amino acid, its configuration can be either R configuration or S configuration, without any special restrictions; preferably, AA2 is arginine, lysine, histidine, Arg(Pbf), an N-terminal alkylated derivative of the above-mentioned amino acid, or a D-configuration amino acid of the above-mentioned amino acid.

[0062] AA3 is a nonpolar amino acid, a nonpolar amino acid-like substance, a polar neutral amino acid, a polar neutral amino acid-like substance, or an N-terminal substituted derivative of the above-mentioned amino acid or amino acid-like substance; when the above-mentioned amino acid is a chiral amino acid, its configuration can be either R configuration or S configuration, without any special limitation; preferably, the AA3 is glycine, alanine, proline, leucine, valine, isoleucine, phenylalanine, serine, threonine, tyrosine, methyltyrosine, ethyltyrosine, methionine, the D configuration amino acid of the above-mentioned amino acid, the L configuration amino acid of the above-mentioned amino acid, an amino acid-like substance or a substituted amino acid-like substance shown in any one of formulas (1) to (5); when the above-mentioned amino acid or amino acid-like substance includes a chiral structure, the above-mentioned amino acid and the structure in the general formula have chirality, including its S and R configurations; in the present invention, it is further preferred that the AA3 is glycine, alanine, proline, leucine, valine, isoleucine, phenylalanine, serine, threonine, tyrosine, methyltyrosine, ethyltyrosine and methionine as its D configuration amino acid.

[0063]

[0064] Wherein, -X1 is -H, substituted or unsubstituted C1-C8 alkyl, -OH, substituted or unsubstituted C2-C8 alkenyl, -NH2, -CONH2, -CHO, -COCH3, -SOCH3, -SO2CH3, -SOCH2CH3, -SO2CH2CH3, p-toluenesulfonyl, substituted or unsubstituted C3-C10 aliphatic cyclic group, substituted or unsubstituted C6-C20 aromatic cyclic group, substituted or unsubstituted C2-C20 heterocyclic group, substituted or substituted C1-C8 alkane acyl, substituted or unsubstituted C1-C8 alkane. Sulfonyl group; preferably -H, substituted or unsubstituted C1-C5 alkyl, -OH, substituted or unsubstituted C2-C5 alkenyl, -NH2, -CONH2, -CHO, -COCH3, -SOCH3, -SO2CH3, -SOCH2CH3, -SO2CH2CH3, p-toluenesulfonyl, substituted or unsubstituted C4-C8 aliphatic cyclic group, substituted or unsubstituted C6-C15 aromatic cyclic group, substituted or unsubstituted C4-C15 heterocyclic group, substituted or substituted C1-C5 alkane acyl group, substituted or unsubstituted C1-C5 alkane sulfonyl group. Acyl group; more preferably -H, substituted or unsubstituted C1-C3 alkyl, -OH, substituted or unsubstituted C2-C3 alkenyl, -NH2, -CONH2, -CHO, -COCH3, -SOCH3, -SO2CH3, -SOCH2CH3, -SO2CH2CH3, p-toluenesulfonyl, substituted or unsubstituted C5-C6 aliphatic cyclic group, substituted or unsubstituted C6-C10 aromatic cyclic group, substituted or unsubstituted C5-C10 heterocyclic group, substituted or substituted C1-C3 alkane acyl, substituted or unsubstituted C1-C3 alkane sulfonyl Acyl group; preferably -H, substituted or unsubstituted C1-C2 alkyl, -OH, substituted or unsubstituted C2-C3 alkenyl, -NH2, -CONH2, -CHO, -COCH3, -SOCH3, -SO2CH3, -SOCH2CH3, -SO2CH2CH3, p-toluenesulfonyl, substituted or unsubstituted C5-C6 aliphatic cyclic group, substituted or unsubstituted C6-C9 aromatic cyclic group, substituted or unsubstituted C5-C9 heterocyclic group, substituted or substituted C1-C2 alkane acyl, substituted or unsubstituted C1-C2 alkane sulfonyl.

[0065] The substituents in the substituted C1-C8 alkyl, substituted C2-C8 alkenyl, substituted C3-C10 aliphatic cyclic, substituted C6-C20 aromatic cyclic, substituted C2-C20 heterocyclic, substituted C1-C8 alkane acyl, and substituted C1-C8 alkane sulfonyl groups are each independently one or more of the following: halogen, hydroxyl, amino, carboxyl, sulfonic acid, mercapto, methyl sulfide, ethyl sulfide, phosphate, nitro, -SOMe, -SO2Me, -CONH2, -COOMe, -COOEt, -NHCOH, -NHCOCH3, -NHSO2Me, p-toluenesulfonamide, phosphate ester, and sulfonate ester.

[0066] R3 to R6 are each independently a substituted or unsubstituted C1 to C8 alkylene group, a substituted or unsubstituted C2 to C8 alkenyl group, a substituted or unsubstituted C2 to C8 alynyl group, a heteroatom, a substituted or unsubstituted C3 to C10 alicyclic group, a substituted or unsubstituted C6 to C20 aromatic cyclic group, or a substituted or unsubstituted C2 to C20 heterocyclic group; preferably, each is independently a substituted or unsubstituted C1 to C6 alkylene group, a substituted or unsubstituted C2 to C6 alkenyl group, or a substituted or unsubstituted C2 to C6 alynyl group. The group may contain: a radical, a heteroatom, a substituted or unsubstituted C3-C9 aliphatic cyclic group, a substituted or unsubstituted C6-C15 aromatic cyclic group, or a substituted or unsubstituted C2-C15 heterocyclic group; more preferably, each of the following is independently substituted or unsubstituted: a C1-C4 alkylene group, a substituted or unsubstituted C2-C4 alkenylene group, a substituted or unsubstituted C2-C4 alkyneylene group, a heteroatom, a substituted or unsubstituted C5-C6 aliphatic cyclic group, a substituted or unsubstituted C6-C10 aromatic cyclic group, or a substituted or unsubstituted C2-C10 heterocyclic group.

[0067] The substituents in the substituted C1-C8 alkylene group, substituted C2-C8 alkenyl group, substituted C2-C8 alkyne group, substituted C3-C10 aliphatic cyclic group, substituted C6-C20 aromatic cyclic group, and substituted C2-C20 heterocyclic group are each preferably halogen, hydroxyl, amino, carboxyl, sulfonic acid group, mercapto, methyl sulfide group, ethyl sulfide group, phosphate group, nitro group, -SOMe, -SO2Me, -CONH2, -COOMe, -COOEt, -NHCOH, -NHCOCH3, -NHSO2Me, p-toluenesulfonamide group, phosphate ester and sulfonate group, or one or more of these.

[0068] R7 and R8 are each independently H, C1-C8 alkyl or C1-C8 alkylamino, preferably each independently H, C1-C6 alkyl or C1-C6 alkylamino, more preferably each independently H, C1-C4 alkyl or C1-C4 alkylamino, and even more preferably each independently H, C1-C2 alkyl or C1-C2 alkylamino.

[0069] m1 is an integer from 1 to 17, preferably an integer from 1 to 15, more preferably an integer from 1 to 12, even more preferably an integer from 1 to 10, even more preferably an integer from 1 to 8, even more preferably an integer from 1 to 5, even more preferably an integer from 1 to 4, even more preferably an integer from 1 to 3, and most preferably 1 or 2; m2 is an integer from 0 to 13, preferably an integer from 0 to 10, more preferably an integer from 0 to 8, even more preferably an integer from 0 to 5, even more preferably an integer from 0 to 4, even more preferably an integer from 0 to 3, and most preferably 0 to 2, which can be 0, 1, or 2.

[0070] The substituents of the aforementioned substituted amino acids or amino acid-like substances are each preferably one or more of halogens, hydroxyl groups, and thiol groups. In this invention, the substituted amino acids or amino acid-like substances refer to the fact that the hydrogen in the methylene group of the amino acid and amino acid-like substances of the aforementioned formula can be replaced, introducing one or more of halogens, hydroxyl groups, and thiol groups to facilitate subsequent optimization of pharmaceutical molecules. The halogens are preferably fluorine, chlorine, or bromine.

[0071] More preferably, the AA3 is glycine, alanine, proline, leucine, valine, isoleucine, phenylalanine, serine, threonine, tyrosine, methyltyrosine, ethyltyrosine, methionine, an L-configured amino acid of the above amino acids, a D-configured amino acid of the above amino acids, an amino acid-like substance or a substituted amino acid or amino acid-like substance represented by any one of formulas (6) to (12):

[0072]

[0073] m1 is an integer from 1 to 17; m2 is an integer from 0 to 13; m1 and m2 are the same as described above, and will not be repeated here.

[0074] The substituents of the aforementioned substituted amino acids or amino acid-like substances are each independently selected from one or more of halogens, hydroxyl groups, and thiol groups.

[0075] The AA4 is an amino acid or an amino acid-like substance with a hydroxyl group in its side chain; preferably, the side chain of AA4 includes one or more of sulfonyl, borate, amide, and carboxyl groups; more preferably, the AA4 is selected from sulfonyl alanine and any one of the amino acids shown in formulas (13) to (16):

[0076]

[0077] R1 and R2 are each independently H or C1 to C8 alkyl groups, preferably each independently H or C1 to C6 alkyl groups, more preferably each independently H or C1 to C4 alkyl groups, and even more preferably each independently H or C1 to C2 alkyl groups.

[0078] n1 to n7 are each independently selected from integers from 0 to 13, preferably each independently selected from integers from 0 to 10, more preferably each independently selected from integers from 0 to 8, even more preferably each independently selected from integers from 0 to 6, even more preferably each independently selected from integers from 0 to 4, and most preferably each independently selected from 0, 1, 2 or 3.

[0079] L1 to L3 are each independently a substituted or unsubstituted C1 to C8 alkylene group or a substituted or unsubstituted C6 to C20 arylene group; preferably a substituted or unsubstituted C1 to C5 alkylene group or a substituted or unsubstituted C6 to C15 arylene group; more preferably a substituted or unsubstituted C1 to C3 alkylene group or a substituted or unsubstituted C6 to C10 arylene group; even more preferably a substituted or unsubstituted C1 to C2 alkylene group or a substituted or unsubstituted C6 to C8 arylene group.

[0080] The substituents in the substituted C1-C8 alkylene groups and the substituted C6-C20 aryl groups are each preferably one or more of the following: halogen, hydroxyl, amino, carboxyl, sulfonic acid, mercapto, methyl sulfide, ethyl sulfide, phosphoric acid, nitro, -SOMe, -SO2Me, -CONH2, -COOMe, -COOEt, -NHCOH, -NHCOCH3, -NHSO2Me, p-toluenesulfonamide, phosphate ester, and sulfonate ester.

[0081] Y1 to Y3 are each independently C or S = O.

[0082] In this invention, AA4 can be a chiral amino acid or an amino acid-like substance, or a chiral amino acid or an amino acid-like substance, without any particular limitation. Specifically, in this invention, AA4 has the following fragment, where the left nitrogen terminus of the fragment is connected to the carboxylic acid of the AA3 amino acid, and the right carbonyl terminus is connected to the nitrogen terminus of the AA5 amino acid. The specific fragment is as follows:

[0083]

[0084] In addition to the amino acid fragments linked by the above-mentioned sequence, the peptide-like compounds provided by the present invention may also include other amino acids linked by amide bonds at both ends, and the number of amino acids is not limited, and may be one or more.

[0085] According to the present invention, preferably, the peptide compound has the structure shown in formula (II) or formula (III):

[0086] AA1-AA2-AA3-AA4-AA5-AA6 formula (II);

[0087] cycl[AA1-AA2-AA3-AA4-AA5-AA6] formula (III);

[0088] Among them, AA1, AA3 and AA6 are each independently nonpolar amino acids, nonpolar amino acid-like amino acids, polar neutral amino acids, polar neutral amino acid-like amino acids, or N-terminal substituted derivatives of the above amino acids or amino acid-like amino acids; when the above amino acids are chiral amino acids, their configuration can be either R configuration or S configuration, without any special restrictions.

[0089] Preferably, AA1 and AA3 are each independently glycine, alanine, proline, leucine, valine, isoleucine, phenylalanine, serine, threonine, tyrosine, methyltyrosine, ethyltyrosine, methionine, D-configured amino acids of the above amino acids, L-configured amino acids of the above amino acids, amino acid-like substances or substituted amino acids or amino acid-like substances represented by any one of formulas (1) to (5); when the above amino acids or amino acid-like substances include chiral structures, the above amino acids and the structures in the general formulas include their S and R configurations when they are chiral.

[0090]

[0091] Wherein, -X1 is -H, substituted or unsubstituted C1-C8 alkyl, -OH, substituted or unsubstituted C2-C8 alkenyl, -NH2, -CONH2, -CHO, -COCH3, -SOCH3, -SO2CH3, -SOCH2CH3, -SO2CH2CH3, p-toluenesulfonyl, substituted or unsubstituted C3-C10 aliphatic cyclic group, substituted or unsubstituted C6-C20 aromatic cyclic group, substituted or unsubstituted C2-C20 heterocyclic group, substituted or substituted C1-C8 alkane acyl, substituted or unsubstituted C1-C8 alkane. Sulfonyl group; preferably -H, substituted or unsubstituted C1-C5 alkyl, -OH, substituted or unsubstituted C2-C5 alkenyl, -NH2, -CONH2, -CHO, -COCH3, -SOCH3, -SO2CH3, -SOCH2CH3, -SO2CH2CH3, p-toluenesulfonyl, substituted or unsubstituted C4-C8 aliphatic cyclic group, substituted or unsubstituted C6-C15 aromatic cyclic group, substituted or unsubstituted C4-C15 heterocyclic group, substituted or substituted C1-C5 alkane acyl group, substituted or unsubstituted C1-C5 alkane sulfonyl group. Acyl group; more preferably -H, substituted or unsubstituted C1-C3 alkyl, -OH, substituted or unsubstituted C2-C3 alkenyl, -NH2, -CONH2, -CHO, -COCH3, -SOCH3, -SO2CH3, -SOCH2CH3, -SO2CH2CH3, p-toluenesulfonyl, substituted or unsubstituted C5-C6 aliphatic cyclic group, substituted or unsubstituted C6-C10 aromatic cyclic group, substituted or unsubstituted C5-C10 heterocyclic group, substituted or substituted C1-C3 alkane acyl, substituted or unsubstituted C1-C3 alkane sulfonyl Acyl group; preferably -H, substituted or unsubstituted C1-C2 alkyl, -OH, substituted or unsubstituted C2-C3 alkenyl, -NH2, -CONH2, -CHO, -COCH3, -SOCH3, -SO2CH3, -SOCH2CH3, -SO2CH2CH3, p-toluenesulfonyl, substituted or unsubstituted C5-C6 aliphatic cyclic group, substituted or unsubstituted C6-C9 aromatic cyclic group, substituted or unsubstituted C5-C9 heterocyclic group, substituted or substituted C1-C2 alkane acyl, substituted or unsubstituted C1-C2 alkane sulfonyl.

[0092] The substituents in the substituted C1-C8 alkyl, substituted C2-C8 alkenyl, substituted C3-C10 alicyclic, substituted C6-C20 aromatic, substituted C2-C20 heterocyclic, substituted C1-C8 alkane acyl, and substituted C1-C8 alkane sulfonyl groups are each independently one or more of the following: halogen, hydroxyl, amino, carboxyl, sulfonic acid, mercapto, methyl sulfide, ethyl sulfide, phosphoric acid, nitro, -SOMe, -SO2Me, -CONH2, -COOMe, -COOEt, -NHCOH, -NHCOCH3, -NHSO2Me, p-toluenesulfonamide, phosphate ester, and sulfonate ester.

[0093] R3 to R6 are each independently a substituted or unsubstituted C1 to C8 alkylene group, a substituted or unsubstituted C2 to C8 alkenyl group, a substituted or unsubstituted C2 to C8 alynyl group, a heteroatom, a substituted or unsubstituted C3 to C10 alicyclic group, a substituted or unsubstituted C6 to C20 aromatic cyclic group, or a substituted or unsubstituted C2 to C20 heterocyclic group; preferably, each is independently a substituted or unsubstituted C1 to C6 alkylene group, a substituted or unsubstituted C2 to C6 alkenyl group, or a substituted or unsubstituted C2 to C6 alynyl group. The group may contain: a radical, a heteroatom, a substituted or unsubstituted C3-C9 aliphatic cyclic group, a substituted or unsubstituted C6-C15 aromatic cyclic group, or a substituted or unsubstituted C2-C15 heterocyclic group; more preferably, each of the following is independently substituted or unsubstituted: a C1-C4 alkylene group, a substituted or unsubstituted C2-C4 alkenylene group, a substituted or unsubstituted C2-C4 alkyneylene group, a heteroatom, a substituted or unsubstituted C5-C6 aliphatic cyclic group, a substituted or unsubstituted C6-C10 aromatic cyclic group, or a substituted or unsubstituted C2-C10 heterocyclic group.

[0094] The substituents in the substituted C1-C8 alkylene group, substituted C2-C8 alkenyl group, substituted C2-C8 alkyne group, substituted C3-C10 aliphatic cyclic group, substituted C6-C20 aromatic cyclic group, and substituted C2-C20 heterocyclic group are each preferably halogen, hydroxyl, amino, carboxyl, sulfonic acid group, mercapto, methyl sulfide group, ethyl sulfide group, phosphate group, nitro group, -SOMe, -SO2Me, -CONH2, -COOMe, -COOEt, -NHCOH, -NHCOCH3, -NHSO2Me, p-toluenesulfonamide group, phosphate ester and sulfonate group, or one or more of these.

[0095] R7 and R8 are each independently H, C1-C8 alkyl or C1-C8 alkylamino, preferably each independently H, C1-C6 alkyl or C1-C6 alkylamino, more preferably each independently H, C1-C4 alkyl or C1-C4 alkylamino, and even more preferably each independently H, C1-C2 alkyl or C1-C2 alkylamino.

[0096] m1 is an integer from 1 to 17, preferably an integer from 1 to 15, more preferably an integer from 1 to 12, even more preferably an integer from 1 to 10, even more preferably an integer from 1 to 8, even more preferably an integer from 1 to 5, even more preferably an integer from 1 to 4, even more preferably an integer from 1 to 3, and most preferably 1 or 2; m2 is an integer from 0 to 13, preferably an integer from 0 to 10, more preferably an integer from 0 to 8, even more preferably an integer from 0 to 5, even more preferably an integer from 0 to 4, even more preferably an integer from 0 to 3, and most preferably 0 to 2, which can be 0, 1, or 2.

[0097] The substituents of the aforementioned substituted amino acids or amino acid-like substances are each preferably one or more of halogens, hydroxyl groups, and thiol groups. In this invention, the substituted amino acids or amino acid-like substances refer to the fact that the hydrogen in the methylene group of the amino acid and amino acid-like substances of the aforementioned formula can be replaced, introducing one or more of halogens, hydroxyl groups, and thiol groups to facilitate subsequent optimization of pharmaceutical molecules. The halogens are preferably fluorine, chlorine, or bromine.

[0098] More preferably, AA1 and AA3 are each independently glycine, alanine, proline, leucine, valine, isoleucine, phenylalanine, serine, threonine, tyrosine, methyltyrosine, ethyltyrosine, methionine, an L-configured amino acid of the above amino acids, a D-configured amino acid of the above amino acids, an amino acid-like substance or a substituted amino acid or amino acid-like substance represented by any one of formulas (6) to (12):

[0099]

[0100] m1 is an integer from 1 to 17; m2 is an integer from 0 to 13; m1 and m2 are the same as described above, and will not be repeated here.

[0101] The substituents of the aforementioned substituted amino acids or amino acid-like substances are each independently selected from one or more of halogens, hydroxyl groups, and thiol groups.

[0102] The AA2 is a basic amino acid, a basic amino acid-like substance, or an N-terminal substituted derivative of the above-mentioned amino acid or amino acid-like substance; the AA2 is the same as described above and will not be repeated here.

[0103] The AA4 is an amino acid or an amino acid-like substance with a hydroxyl side chain; the AA4 is the same as described above and will not be repeated here.

[0104] The AA5 is a polar neutral amino acid or a polar neutral amino acid-like amino acid, preferably threonine, serine, cysteine, N-terminal alkylated derivatives of the above amino acids, or D-configuration amino acids of the above amino acids.

[0105] Preferably, the AA6 is one of the structures shown in formulas (17) to (24):

[0106]

[0107] The R9 and R 10 Each of the following components is independently H, a halogen, a C1-C8 alkyl group, a C1-C8 alkylamino group, an amino group, a hydroxyl group, or a C6-C20 aromatic ring group, preferably H, a halogen, a C1-C6 alkyl group, a C1-C6 alkylamino group, an amino group, a hydroxyl group, or a C6-C15 aromatic ring group, more preferably H, a halogen, a C1-C4 alkyl group, a C1-C4 alkylamino group, an amino group, a hydroxyl group, or a C6-C10 aromatic ring group, and even more preferably H, a halogen, a C1-C2 alkyl group, a C1-C2 alkylamino group, an amino group, a hydroxyl group, or a C6-C9 aromatic ring group; the halogen is preferably F, Cl, or Br.

[0108] The R 11 With R 13 Preferably, it is -B(OH)2, halogen, borate ester group, sulfonic acid or sulfonic acid ester group, phosphoric acid or phosphate ester group.

[0109] The R 12 It can be H, C1-C8 alkyl, hydroxyl, C1-C8 alkane acyl, C1-C8 alkane sulfonyl, benzene sulfonyl, p-toluene sulfonyl, C6-C20 aryl sulfonyl group, C1-C8 alkane sulfinyl, C1-C8 alkane phosphoryl group, C6-C20 aryl phosphoryl group or C6-C20 aromatic cyclic group; preferably H, C1-C6 alkyl, hydroxyl, C1-C6 alkane acyl, C1-C6 alkane sulfonyl, benzene sulfonyl, p-toluene sulfonyl, C6-C15 aryl sulfonyl group, C1-C6 alkane sulfinyl, C1-C6 alkane phosphoryl group, C6-C15 aryl phosphoryl group or C6-C15 aromatic cyclic group; More preferably, it is H, C1-C4 alkyl, hydroxyl, C1-C4 alkane acyl, C1-C4 alkane sulfonyl, benzene sulfonyl, p-toluene sulfonyl, C6-C10 aryl sulfonyl group, C1-C4 alkane sulfinyl, C1-C4 alkane phosphoryl group, C6-C10 aryl phosphoryl group or C6-C10 aromatic ring group; even more preferably, it is H, C1-C2 alkyl, hydroxyl, C1-C2 alkane acyl, C1-C2 alkane sulfonyl, benzene sulfonyl, p-toluene sulfonyl, C6-C8 aryl sulfonyl group, C1-C2 alkane sulfinyl, C1-C2 alkane phosphoryl group, C6-C8 aryl phosphoryl group or C6-C8 aromatic ring group.

[0110] The R 14It is H, C1-C8 alkyl, amino or C6-C20 aryl cyclogroup; preferably H, C1-C6 alkyl, amino or C6-C15 aryl cyclogroup; more preferably H, C1-C4 alkyl, amino or C6-C10 aryl cyclogroup; even more preferably H, C1-C2 alkyl, amino or C6-C8 aryl cyclogroup.

[0111] The R 15 It is an alkyl group of H or C1 to C8, preferably an alkyl group of H or C1 to C6, more preferably an alkyl group of H or C1 to C4, and even more preferably an alkyl group of H or C1 to C2.

[0112] The R 16 It is H, C1-C8 alkyl, C1-C8 alkoxy, C6-C20 aryloxy, or C6-C20 arylcycloyl; preferably H, C1-C6 alkyl, C1-C6 alkoxy, C6-C15 aryloxy, or C6-C15 arylcycloyl; more preferably H, C1-C4 alkyl, C1-C4 alkoxy, C6-C10 aryloxy, or C6-C10 arylcycloyl; even more preferably H, C1-C2 alkyl, C1-C2 alkoxy, C6-C8 aryloxy, or C6-C8 arylcycloyl.

[0113] More preferably, the AA6 is one of the following structures:

[0114]

[0115] The AA1, AA2, AA3, AA4, AA5 and AA6 are connected by condensation into amide bonds, and AA1 is the N-terminus;

[0116] Cyc means that any one of AA1 to AA5 forms a cyclization ring with AA6 through condensation to form an amide bond.

[0117] In this invention, the most preferred peptide compound is one of those shown as TM1 to TM72:

[0118]

[0119]

[0120]

[0121]

[0122]

[0123] This invention also provides a method for preparing the above-mentioned peptide-like compound. The preparation can be carried out according to polypeptide synthesis methods well-known to those skilled in the art, without any special limitations. In this invention, a solid-phase synthesis method is preferred. The C-terminus of the first amino acid at the C-terminus of the peptide-like compound is covalently linked to a solid-phase support. Then, using the N-terminus of this amino acid as the starting point for synthesis, a condensation reaction is performed by removing the amino protecting group and adding an excess of the activated second amino acid to lengthen the peptide chain. This process is repeated until the desired peptide chain length is achieved. Finally, the peptide is cleaved from the resin, separated, and purified to obtain the peptide-like compound. In the embodiments provided by this invention, the Fmoc polypeptide synthesis method is specifically used as an example. The resin used is well-known to those skilled in the art and is not particularly limited. In the embodiments provided by this invention, CTC resin is specifically used as an example. In this invention, an organic base and a condensing agent are preferably used to activate the amino acid. The organic base is preferably one or more of DIEA, TEA, and NMM. The condensing agent is selected from one or more of DCC, DIC, EDC, BOP, pyBOP, AOP, TBTU, HBTU, and HATU.

[0124] The present invention also provides the use of the above-mentioned peptide-like compound in the preparation of a drug that inhibits integrin.

[0125] The present invention also provides the use of the above-mentioned peptide compound in the preparation of a drug for inhibiting cell adhesion at the Arg-Gly-Asp binding site.

[0126] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, describes a peptide-like compound, its preparation method, and its applications.

[0127] All reagents used in the following examples are commercially available.

[0128] The English names, Chinese names, and letter representations of the 20 amino acids are shown in the table below.

[0129]

[0130]

[0131] Compound synthesis process

[0132] The synthesis of all compounds was carried out in parallel according to Examples 1, 2, and 3. Detailed synthetic strategies were selected as follows:

[0133] The linear peptide type was selected and the synthesis method was carried out according to Example 1 or Example 2;

[0134] The cyclic peptide was synthesized according to Example 3 or Example 4.

[0135] The detailed synthesis method is as follows:

[0136] Example 1: Synthesis of Positive-1 (TM)

[0137] Synthetic route

[0138]

[0139] Step 1: Synthesize M1

[0140] Weigh 0.503 g of CTC resin (sub=1.0 mmol / g) and add it to a peptide synthesis tube. Add DCM (10 ml) and allow it to swell for 0.5 h. Weigh 0.202 g of Fmoc-Pro-OH (0.6 mmol) and dissolve it in DCM (10 ml) solution. Add DIEA (0.2 ml, 1.2 mmol) and react for 1 h. Wash the resin three times with DCM (10 ml). Add 10 ml of blocking buffer (V / V ratio DCM:MeOH:DIEA = 70:25:5) and block for 15 min. Wash the resin six times with DMF to obtain intermediate M1, which can be used directly in the next reaction.

[0141] Step 2: Synthesize M2

[0142] Deprotection with Fmoc: Add 10 ml of piperidine / DMF (1 / 4 v / v) solution, deprotect for 5 min, dry under vacuum and add 10 ml of piperidine / DMF (1 / 4 v / v) solution again, deprotect for 10 min, dry under vacuum and wash the resin 6 times with DMF, take a small amount of resin and test with 5% ninhydrin ethanol solution (take a small amount of resin and add 1 ml of 5% ninhydrin solution and heat at 100℃ for 5 min). The resin turns deep blue and can be used directly for the next reaction.

[0143] Coupling: Fmoc-Thr(tBu)-OH (0.598 g, 1.5 mmol) was dissolved in 5 ml of DMF. TBTU (0.492 g, 1.5 mmol) and DIEA (0.391 g, 3 mmol) were added to the reaction solution under ice bath conditions and stirred for 10 min. After the reaction, the activating solution was added to the Fmoc-free resin, and the reaction was allowed to proceed for 1 h. A small amount of resin was tested with a 5% ninhydrin ethanol solution (a small amount of resin was added to 1 ml of 5% ninhydrin solution and heated at 100 °C for 5 min). The resin showed a colorless reaction. The reaction solution was dried under vacuum and washed 6 times with 10 ml of DMF. The resulting resin was used directly in the next reaction step.

[0144] The coupling cycle is repeated sequentially with Fmoc-Cysteic-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH and Fmoc-Gly-OH, finally yielding intermediate M2, which is directly used in the next reaction.

[0145] Step 3: Synthesize M3

[0146] Deprotection with Fmoc: Add 10 ml of piperidine / DMF (1 / 4 v / v) solution, deprotect for 5 min, dry under vacuum, and add 10 ml of piperidine / DMF (1 / 4 v / v) solution again, deprotect for 10 min, dry under vacuum, and wash the resin 6 times with DMF. Take a small amount of resin and test it with 5% ninhydrin ethanol solution (take a small amount of resin, add 1 ml of 5% ninhydrin solution and heat at 100℃ for 5 min). The resin turns dark blue. Wash with 10 ml each of DCM and MeOH, alternately 3 times, dry under vacuum, transfer to a vacuum oven, and dry at 30℃ for 2 h to obtain 0.983 g of resin, which can be directly used for the next reaction.

[0147] Step 4: Synthesize TM (Positive Result 1)

[0148] Add 10 ml of lysis buffer (TFA:H2O:Tis = 90:5:5) to 0.983 g of resin, react for 2 h, filter, and wash the resin with 5 ml of TFA. Combine the filtrates, add dropwise 100 ml of methyl tert-butyl ether at approximately -5 °C, allow to settle for 1 h, centrifuge, and remove the supernatant. Add 100 ml of MTBE, wash, centrifuge, and remove the supernatant. Repeat this operation 3 times to obtain a white solid. Transfer the solid to a vacuum drying oven and dry at 30 °C for 2 h to obtain 0.254 g of crude peptide. Purify by high pressure to obtain a purified solution. Concentrate the purified solution to remove most of the organic solvent, and freeze-dry to obtain 56.21 mg of TM with a purity of 97.4%, yield 17.6%.

[0149] The obtained TM was analyzed by mass spectrometry, and the MS(ESI) value was 638.4 [M+H]. + Its spectrum is as follows Figure 1 As shown.

[0150] The TM obtained in Example 1 was analyzed using nuclear magnetic resonance, and the results were obtained. 1 H NMR(600MHz,D2O)δ4.58–4.52(m,1H),4.36–4.27(m,2H),4.07(p,J=6.4Hz,1H),3.97–3.87(m,2H),3.87–3.73(m,3H),3.67(d,J=8.5Hz, 1H),3.34–3.24(m,2H),3.15(t,J=6.9Hz,2H),2.25(s,1H),2.03–1.88(m,3H),1.87–1.69(m,3H),1.66–1.54(m,2H),1.23–1.16(m,3H).

[0151] Example 2: HTPM7002-041(TM10)

[0152] Synthetic route

[0153]

[0154] Step 1: Synthesize M1

[0155] Weigh 0.503 g of CTC resin (sub=1.0 mmol / g) and add it to a peptide synthesis tube. Add DCM (10 ml) and allow it to swell for 0.5 h. Weigh 0.202 g of Fmoc-Pro-OH (0.6 mmol) and dissolve it in DCM (10 ml) solution. Add DIEA (0.2 ml, 1.2 mmol) and react for 1 h. Wash the resin three times with DCM (10 ml). Add 10 ml of blocking buffer (V / V ratio DCM:MeOH:DIEA = 70:25:5) and block for 15 min. Wash the resin six times with DMF to obtain intermediate M1, which can be used directly in the next reaction.

[0156] Step 2: Synthesize M2

[0157] Deprotection with Fmoc: Add 10 ml of piperidine / DMF (1 / 4 v / v) solution, deprotect for 5 min, dry under vacuum and add 10 ml of piperidine / DMF (1 / 4 v / v) solution again, deprotect for 10 min, dry under vacuum and wash the resin 6 times with DMF, take a small amount of resin and test with 5% ninhydrin ethanol solution (take a small amount of resin and add 1 ml of 5% ninhydrin solution and heat at 100℃ for 5 min). The resin turns deep blue and can be used directly for the next reaction.

[0158] Coupling: Fmoc-Thr(tBu)-OH (0.598 g, 1.5 mmol) was dissolved in 5 ml of DMF. TBTU (0.492 g, 1.5 mmol) and DIEA (0.391 g, 3 mmol) were added to the reaction solution under ice bath conditions and stirred for 10 min. After the reaction, the activating solution was added to the Fmoc-free resin, and the reaction was allowed to proceed for 1 h. A small amount of resin was tested with a 5% ninhydrin ethanol solution (a small amount of resin was added to 1 ml of 5% ninhydrin solution and heated at 100 °C for 5 min). The resin showed a colorless reaction. The reaction solution was dried under vacuum and washed 6 times with 10 ml of DMF. The resulting resin was used directly in the next reaction step.

[0159] The coupling cycle is repeated sequentially with Fmoc-Asp(OAll)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH and Fmoc-Gly-OH, finally yielding intermediate M2, which is directly used in the next reaction.

[0160] Step 3: Synthesize M3

[0161] All removal: Add phenylsilane (0.738 ml, 6.0 mmol), add DCM (5 ml), react for 3 min, then add 5 ml of DCM solution containing tetrakis(triphenylphosphine)palladium (57 mg, 0.05 mmol), react for 2 h, wash the resin 8 times with DCM (10 ml); use directly for the next reaction.

[0162] Step 4: Synthesize M4

[0163] Coupling: Weigh 66 mg (0.6 mmol) of methanesulfonic acid and 192 mg (0.6 mmol) of TBTU, add 10 ml of DMF and 0.2 ml (1.2 mmol) of DIEA, and react for 2 h; dry the reaction solution under vacuum and wash 6 times with DMF (10 ml) to obtain the resin for direct use in the next step of the reaction.

[0164] Step 5: Synthesize M5

[0165] Deprotection with Fmoc: Add 10 ml of piperidine / DMF (1 / 4 v / v) solution, deprotect for 5 min, dry under vacuum and add 10 ml of piperidine / DMF (1 / 4 v / v) solution again, deprotect for 10 min, dry under vacuum and wash the resin 6 times with DMF. Take a small amount of resin and test it with 5% ninhydrin ethanol solution (take a small amount of resin and add 1 ml of 5% ninhydrin solution and heat at 100℃ for 5 min). The resin turns dark blue. Wash with 10 ml each of DCM and MeOH, alternately 3 times, dry under vacuum, transfer to a vacuum oven and dry at 30℃ for 2 h to obtain resin M5 (0.972 g), which can be directly used for the next reaction.

[0166] Step Six: Synthesize TM10

[0167] 10 ml of lysis buffer (TFA:H2O:Tis volume ratio 90:5:5) was added to resin M5 (0.983 g), and the reaction was carried out for 2 h. The mixture was then filtered, and the resin was washed with 5 ml of TFA. The filtrates were combined, and 100 ml of methyl tert-butyl ether (MTBE) at approximately -5 °C was added dropwise. The mixture was allowed to settle for 1 h, centrifuged, and the supernatant was removed. 100 ml of MTBE was added, and the mixture was washed, centrifuged, and the supernatant was removed. This operation was repeated 3 times to obtain a white solid. The solid was transferred to a vacuum drying oven and dried at 30 °C for 2 h to obtain 0.254 g of crude peptide. The peptide was purified by high pressure, and the purified solution was concentrated to remove most of the organic solvent. The purified solution was then freeze-dried to obtain 23.06 mg of TM10 with a purity of 96.94%, with a yield of 6.64%.

[0168] Mass spectrometry analysis yielded the following MS (ESI) value: m / z 695.4 [M+H] + Its spectrum is as follows Figure 2 As shown.

[0169] The TM10 obtained in Example 2 was analyzed using nuclear magnetic resonance (NMR) to obtain... 1 H NMR(600MHz,D2O)δ4.92(s,2H),4.57–4.51(m,1H),4.36–4.27(m,2H),4.07(p,J=6.4Hz,1H),3.88–3.74(m,3H),3.66(d,J=8.5Hz,1H),3.34–3 .25(m,2H),3.16(t,J=6.9Hz,2H),2.58–2.48(m,2H),2.26(s,1H),2.03 –1.88(m,3H),1.87–1.69(m,3H),1.66–1.54(m,2H),1.23–1.16(m,3H).

[0170] Example 3: Synthesis of compound TM19

[0171]

[0172] Step 1: Synthesize M1

[0173] Weigh 0.503 g of CTC resin (sub=1.0 mmol / g) and add it to a peptide synthesis tube. Add DCM (10 ml) and allow it to swell for 0.5 h. Weigh 0.202 g of Fmoc-Pro-OH (0.6 mmol) and dissolve it in DCM (10 ml) solution. Add DIEA (0.2 ml, 1.2 mmol) and react for 1 h. Wash the resin three times with DCM (10 ml). Add 10 ml of blocking buffer (V / V ratio DCM:MeOH:DIEA = 70:25:5) and block for 15 min. Wash the resin six times with DMF to obtain intermediate M1, which can be used directly in the next reaction.

[0174] Step 2: Synthesize M2

[0175] Deprotection with Fmoc: Add 10 ml of piperidine / DMF (1 / 4 v / v) solution, deprotect for 5 min, dry under vacuum and add 10 ml of piperidine / DMF (1 / 4 v / v) solution again, deprotect for 10 min, dry under vacuum and wash the resin 6 times with DMF, take a small amount of resin and test with 5% ninhydrin ethanol solution (take a small amount of resin and add 1 ml of 5% ninhydrin solution and heat at 100℃ for 5 min). The resin turns deep blue and can be used directly for the next reaction.

[0176] Coupling: Fmoc-Thr(tBu)-OH (0.601 g, 1.5 mmol) was dissolved in 5 ml of DMF. TBTU (0.496 g, 1.5 mmol) and DIEA (0.393 g, 3 mmol) were added to the reaction solution under ice bath conditions and stirred for 10 min. After the reaction, the activating solution was added to the Fmoc-free resin, and the reaction was allowed to proceed for 1 h. A small amount of resin was tested with a 5% ninhydrin ethanol solution (a small amount of resin was added to 1 ml of 5% ninhydrin solution and heated at 100 °C for 5 min). The resin showed a colorless reaction. The reaction solution was dried under vacuum and washed 6 times with 10 ml of DMF. The resulting resin was used directly in the next reaction step.

[0177] The coupling cycle is repeated sequentially with Fmoc-Cysteic-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH and Fmoc-Gly-OH, finally yielding intermediate M2, which is directly used in the next reaction.

[0178] Step 3: Synthesize M3

[0179] Deprotection with Fmoc: Add 10 ml of piperidine / DMF (1 / 4 v / v) solution, deprotect for 5 min, dry under vacuum, and add another 10 ml of piperidine / DMF (1 / 4 v / v) solution, deprotect for 10 min, dry under vacuum, and wash the resin 6 times with DMF. Take a small amount of resin and test it with 5% ninhydrin ethanol solution (take a small amount of resin, add 1 ml of 5% ninhydrin solution and heat at 100℃ for 5 min). The resin turns dark blue. Wash 3 times alternately with 10 ml each of DCM and MeOH, dry under vacuum, transfer to a vacuum oven, and dry at 30℃ for 2 h to obtain 0.944 g of resin, which can be directly used for the next reaction.

[0180] Step 4: Synthesize M4

[0181] Add 10 ml of lysis buffer (TFA:TIS:H2O volume ratio 95:2.5:2.5) to 0.944 g of resin and react for 1 h. After the reaction is complete, filter and wash the resin with 5 ml of TFA. Combine the filtrates and repeat the above operation once. After removing most of the solvent by rotary evaporation, add 100 ml of methyl tert-butyl ether at approximately -5 °C dropwise, allow to settle for 1 h, centrifuge, and remove the supernatant. Add 100 ml of MTBE, wash, centrifuge, and remove the supernatant. Repeat this operation 3 times to obtain a white solid. Transfer the solid to a vacuum drying oven and dry at 30 °C for 2 h to obtain 0.230 g of crude peptide.

[0182] Step 5: Synthesize TM19

[0183] Add DCM (10 ml) to dissolve TBTU (194 mg, 0.6 mmol), HOBT (83 mg, 0.6 mmol), and DIEA (0.2 ml, 1.2 mmol). After reacting for 10 h, remove DCM by rotary evaporation, and then purify the solution by high pressure. Concentrate the purified solution to remove most of the organic solvent, and freeze-dry it to obtain 10.03 mg of TM19 with a purity of 98.36%, yield 3.02%.

[0184] Mass spectrometry analysis yielded the following MS (ESI) value: m / z 664.6 [M+H]. + Its spectrum is as follows Figure 3 As shown.

[0185] The obtained TM19 was analyzed using nuclear magnetic resonance, and the results were obtained. 1 H NMR(600MHz,D2O)δ4.67–4.63(m,1H),4.41(dd,J=8.6,5.4Hz,1H),4.30(t,J=7.4Hz,1H),4.20( t,J=5.8Hz,1H),4.04(d,J=15.1Hz,1H),3.95–3.85(m,2H),3.82-3.76(m,2H),3.63-3.59(m,2H) ,3.52(t,J=8.8Hz,1H),3.47–3.37(m,3H),3.32–3.23(m,1H),3.12(s,2H),2.22-2.18(m,1H),1. 98~1.97(m,1H),1.92-1.83(m,2H),1.71-1.59(m,3H),1.44-1.41(m,2H),1.12(d,J=6.4Hz,3H).

[0186] Example 4: Synthesis of compound TM20

[0187]

[0188] Step 1: Synthesize M1

[0189] Weigh 0.503 g of CTC resin (sub=1.0 mmol / g) and add it to a peptide synthesis tube. Add DCM (10 ml) and allow it to swell for 0.5 h. Weigh 0.202 g of Fmoc-Pro-OH (0.6 mmol) and dissolve it in DCM (10 ml) solution. Add DIEA (0.2 ml, 1.2 mmol) and react for 1 h. Wash the resin three times with DCM (10 ml). Add 10 ml of blocking buffer (V / V ratio DCM:MeOH:DIEA = 70:25:5) and block for 15 min. Wash the resin six times with DMF to obtain intermediate M1, which can be used directly in the next reaction.

[0190] Step 2: Synthesize M2

[0191] Deprotection with Fmoc: Add 10 ml of piperidine / DMF (1 / 4 v / v) solution, deprotect for 5 min, dry under vacuum and add 10 ml of piperidine / DMF (1 / 4 v / v) solution again, deprotect for 10 min, dry under vacuum and wash the resin 6 times with DMF, take a small amount of resin and test with 5% ninhydrin ethanol solution (take a small amount of resin and add 1 ml of 5% ninhydrin solution and heat at 100℃ for 5 min). The resin turns deep blue and can be used directly for the next reaction.

[0192] Coupling: Fmoc-Thr(tBu)-OH (0.598 g, 1.5 mmol) was dissolved in 5 ml of DMF. TBTU (0.492 g, 1.5 mmol) and DIEA (0.391 g, 3 mmol) were added to the reaction solution under ice bath conditions and stirred for 10 min. After the reaction, the activating solution was added to the Fmoc-free resin, and the reaction was allowed to proceed for 1 h. A small amount of resin was tested with a 5% ninhydrin ethanol solution (a small amount of resin was added to 1 ml of 5% ninhydrin solution and heated at 100 °C for 5 min). The resin showed a colorless reaction. The reaction solution was dried under vacuum and washed 6 times with 10 ml of DMF. The resulting resin was used directly in the next reaction step.

[0193] The coupling cycle is repeated sequentially with Fmoc-Cysteic-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH and Fmoc-Gly-OH, finally yielding intermediate M2, which is directly used in the next reaction.

[0194] Step 3: Synthesize M3

[0195] Deprotection with Fmoc: Add 10 ml of piperidine / DMF (1 / 4 v / v) solution, deprotect for 5 min, dry under vacuum, and add 10 ml of piperidine / DMF (1 / 4 v / v) solution again, deprotect for 10 min, dry under vacuum, and wash the resin 6 times with DMF. Take a small amount of resin and test it with 5% ninhydrin ethanol solution (take a small amount of resin, add 1 ml of 5% ninhydrin solution and heat at 100℃ for 5 min). The resin turns dark blue. Wash with 10 ml each of DCM and MeOH, alternately 3 times, dry under vacuum, transfer to a vacuum oven, and dry at 30℃ for 2 h to obtain 0.983 g of resin, which can be directly used for the next reaction.

[0196] Step 4: Synthesize M4

[0197] Add 10 ml of lysis buffer (TFE:DCM volume ratio 1:1) to 0.941 g of resin, react for 1 h, filter, and wash the resin with 5 ml of DCM. Combine the filtrates and repeat the above operation once. After removing most of the solvent by rotary evaporation, add 100 ml of methyl tert-butyl ether at approximately -5 °C dropwise, allow to settle for 1 h, centrifuge, and remove the supernatant. Add 100 ml of MTBE, wash, centrifuge, and remove the supernatant. Repeat this operation 3 times to obtain a white solid. Transfer the solid to a vacuum drying oven and dry at 30 °C for 2 h to obtain 0.230 g of crude peptide.

[0198] Step 5: Synthesize TM20

[0199] Add DCM (10 ml) to dissolve TBTU (192 mg, 0.6 mmol), HOBT (81 mg, 0.6 mmol), and DIEA (0.2 ml, 1.2 mmol). After reacting for 10 h, remove DCM by rotary evaporation, and then purify the solution by high pressure. Concentrate the purified solution to remove most of the organic solvent, and freeze-dry it to obtain 18.37 mg of TM20 with a purity of 96.51%, yield 4.00%.

[0200] Mass spectrometry analysis was performed, yielding MS(ESI): m / z 916.6 [M+H]. + Its spectrum is as follows Figure 4 As shown.

[0201] The obtained TM20 was analyzed using nuclear magnetic resonance, and the results were obtained. 1 H NMR(600MHz,D2O)δ4.66–4.62(m,1H),4.40(dd,J=8.6,5.4Hz,1H),4.30(t,J=7.4Hz,1H),4.20(t,J=5.8Hz,1H), 4.03(d,J=15.1Hz,1H),3.95–3.85(m,2H),3.82-3.76(m,2H),3.62-3.58(m,2H),3.52(t,J=8.8Hz,1H),3.47–3.3 7(m,3H),3.32–3.23(m,1H),3.12(s,2H),3.00(s,2H),2.46(s,3H),2.40(s,3H),2.22-2.18(m,1H),2.02(s,3H) ,1.98~1.97(m,1H),1.92-1.83(m,2H),1.71-1.59(m,3H),1.44-1.41(m,2H),1.41(s,6H),1.11(d,J=6.4Hz,3H).

[0202] Bioactivity test

[0203] The inhibitory effects of the compounds from the examples on integrin αvβ3, αvβ5, α5β1 and their ligands vitronectin and fibronectin were tested using ELISA. 96-well plates were coated with vitronectin or fibronectin. 50 μl of each of the different example compounds was serially diluted and added to each well, followed by 50 μl of biotin-labeled integrin αvβ3, αvβ5, and α5β1. After incubation at room temperature for 1 hour, the plates were eluted three times with TBS buffer. 50 μl of Streptavidin-HRP was added, and the plates were incubated at room temperature for 30 minutes. After elution three times with TBS buffer, 100 μl of HRP substrate TMB was added, and the reaction was incubated at room temperature for 30 minutes. The reaction was terminated by adding 50 μl of 1M sulfuric acid solution. The absorbance of each well was read at 450 nm using a microplate reader. The absorbance was calculated using the formula (OD). top -OD detect ) / (OD top -OD bottom The inhibition rates of each test concentration of the compound in the examples against the binding of integrin αvβ3, αvβ5, α5β1 to their ligands vitronectin and fibronectin were calculated, and the IC50 of the inhibition rate at each concentration against the binding of integrin αvβ3, αvβ5, α5β1 to their ligands vitronectin and fibronectin was calculated using a four-parameter formula fitted to the inhibition rates at each concentration. 50 .

[0204] The inhibition rates of the compounds of the embodiments involved in this invention against the binding of integrin αvβ3, αvβ5, α5β1 and their ligands vitronectin and fibronectin at concentrations of 5 mM and 2 mM are shown in Table 1, as determined by ELISA. The IC50 values ​​for the inhibition of the binding of the compounds of the embodiments involved in this invention against the binding of integrin αvβ3, αvβ5, α5β1 and their ligands vitronectin and fibronectin are also shown in Table 1. 50 See Table 2.

[0205] Table 1. Inhibition rate of the compounds in the examples on the binding of integrin to its ligands

[0206]

[0207]

[0208]

[0209]

[0210] As shown in Table 1, the compounds TM21, TM22, TM11, TM13, and TM14 in the examples showed higher inhibition rates of binding of integrin αvβ3 to its ligand Vitronectin at concentrations of 5 mM and 2 mM than the positive compound.

[0211] In the examples, TM22 and TM30 showed higher inhibition rates of binding of integrin αvβ5 to its ligand Vitronectin at concentrations of 5 mM and 2 mM, respectively, than the positive compound.

[0212] In the examples, compounds TM1, TM10, TM21, TM11, TM30, TM66, and TM69 showed higher inhibition rates of binding to integrin α5β1 and its ligand Fibronectin at concentrations of 5 mM and 2 mM than the positive compounds.

[0213] Table 2. IC50 values ​​of the compounds in the examples for inhibiting the binding of integrin and its ligands. 50

[0214]

[0215] As shown in Table 2, the IC50 values ​​of TM22 (HTPM7002-043), TM13 (HTPM7002-057), TM14 (HTPM7002-058), and TM34 (HTPM7002-092) in the examples inhibited the binding of integrin αvβ3 to its ligand Vitronectin. 50 All were lower than those of positive compounds.

[0216] The IC50 of TM34 (HTPM7002-092) in the example compound inhibited the binding of integrin αvβ5 to its ligand Vitronectin. 50 Lower than positive compounds.

[0217] The IC50 of the compound TM10 (HTPM7002-041) in the examples inhibited the binding of integrin α5β1 to its ligand Fibronectin. 50 Lower than positive compounds.

[0218] Cell adhesion experiment

[0219] Dilute the protein to 2.5 μg / mL Fibronectin or 10 μg / mL Vitronectin using 1× coating buffer (Solarbio / C1055), add 100 μL / well to a 96-well plate, and incubate overnight at 4°C. Discard the coating protein, wash twice with 200 μL PBS each time, and aspirate dry. Digest logarithmic-phase HUVEC cells with 1 ml trypsin-EDTA (Solarbio / T1300). Observe the cells under a microscope to ensure they are round. Immediately dilute the cells to 6.66 × 10⁶ using complete culture medium (45 ml DMEM basal medium (gibco / C11965500BT + 4.5 ml FBS (Sigma-F8318) + 0.5 ml penicillin-streptomycin solution (Biosharp-BL505A))). 5 / ml, 75μL per well (containing 5×10⁻⁶ ml) 4 Cells were seeded in 96-well plates, and 25 μL of the test compound was added to bring the final concentrations to 100, 25, 6.25, 1.56, 0.69, 0.098, 0.024, and 0 μM. After incubation for 1 h, the 96-well plates were removed, the culture medium was aspirated, and each well was washed twice with 200 μL of PBS. The PBS was discarded, and the plates were aspirated. 100 μL of CellTiter-Glo reagent (Beyotime / C0069XL) was added to each well, shaken for 2 min, and incubated at room temperature for 10 min. The chemiluminescence value was measured using a microplate reader, and the cell adhesion rate at different time points was calculated. The results are shown in Table 3.

[0220] Table 3 Results of cell adhesion assay

[0221]

[0222] Eye Comparison

[0223] Five μL of blank rat vitreous homogenate was taken and mixed with 45 μL of physiological saline, 50 μL of HTPM7002-041(TM10) standard series solutions (10 ng / mL, 100 ng / mL, 4000 ng / mL), 25 μL of internal standard, and 150 μL of 10% TCA. The mixture was vortexed for 2 min, centrifuged at 3800 rpm for 20 min at 4℃, and the supernatant was used for LC-MS / MS analysis to prepare a standard curve. Subsequently, five μL of the rat vitreous homogenate was taken and mixed with 45 μL of physiological saline. The mixture was sonicated for 2 min, vortexed for 1 min, and then 50 μL of methanol, 25 μL of internal standard, and 150 μL of 10% TCA were added. The mixture was vortexed for 2 min, centrifuged at 3800 rpm for 20 min at 4℃, and the supernatant was used for LC-MS / MS analysis. The results are shown in Table 4.

[0224] Chromatographic conditions

[0225] Instrumentation: SHIMADZU LC-20AD High Performance Liquid Chromatography System

[0226] Column: Hypersil GOLD Aq, 3 μm, 3 x 100 mm

[0227] Mobile phase A: 0.2% formic acid solution

[0228] Mobile phase B: Acetonitrile

[0229] Gradient elution procedure:

[0230]

[0231]

[0232] Flow rate: 0.5 mL / min

[0233] Column temperature: 40℃

[0234] Injection volume: 10 μL

[0235] Internal standard: Gliclazide (100 ng / mL methanol solution)

[0236] Retention time: HTPM7002-041: 2.80 min; Griezmann: 3.28 min.

[0237] Mass spectrometry conditions

[0238] Instrumentation: API4000 triple quadrupole mass spectrometer

[0239]

[0240] Data Acquisition: Analyst 1.6

[0241] Table 4 LC-MS / MS Analysis Results of Samples

[0242] HTPM7002-041 120.62968 0.42182034 41.449169

[0243] Liver microsomal stability

[0244] Information on liver microsomes is shown in Table 5.

[0245] Table 5 Liver microsome information

[0246]

[0247] Microsome working solution: Prepare an appropriate concentration of microsome working solution in 100 mM potassium phosphate buffer. Stop solution preparation: At 4°C, use methanol (MeOH) containing 200 ng / mL tolbutamide and 200 ng / mL labetalol as internal standards (IS) as the stop solution. Using the Apricot automated workstation, add 2 μL / well of the composite working solution to all 96-well reaction plates (T0, T5, T15, T30, T40, T45, T60, NCF60), except for the blank plate. Then add 100 μL / well of microsome solution to all reaction plates (Blank, T0, T5, T15, T30, T45, T60, NCF60). All reactions containing the compound and microsome mixture are pre-incubated at 37°C for 10 minutes. Add 98 μL / well of 100 mM potassium phosphate buffer to the NCF60 reaction plate, incubate the NCF60 reaction plate at 37°C, and start the timer.

[0248] NCF60 culture

[0249]

[0250] After pre-incubation, except for NCF60, 98 μL of NADPH regeneration system was added to each reaction plate (Blank, T0, T5, T15, T30, T45, T60) to start the reaction.

[0251] Final concentrations of each component in the culture medium

[0252]

[0253] The reaction plate was incubated at 37°C, and a timer was started.

[0254] Reaction plate culture

[0255]

[0256] At the appropriate endpoint time point for each reaction plate, 200 μL / well of 4% phosphoric acid and 800 μL / well of stop solution were added to terminate the reaction. Each plate was sealed and shaken for 10 min, then centrifuged at 4000 rpm for 20 min at 4 degrees Celsius. Finally, 300 μL of each plate was transferred to a new 96-well plate for LC-MS / MS analysis. The results are shown in Table 6.

[0257] Table 6. Liver microsomal stability T1 / 2 (min)

[0258] TM10 HTPM7002-041 143.2 101.9 101.4 35.4 35.5

[0259] plasma stability

[0260] Before the experiment, frozen plasma was thawed in a 37°C water bath. The plasma was centrifuged at 4000 rpm for 5 min. Using an Apricot automated workstation, 98 μL / well of blank plsma was added to all 96-well reaction plates (blank control, T0, T10, T30, T60, and T120); 2 μL / well of working solution (100 μM) was added to all wells except the blank control (T0, T10, T30, T60, and T120). The plates were incubated in a 37°C water bath, and the timer was started. At the end of the incubation, 500 μL of stop solution (dissolving HTPM7002-041 compound in CAN solution containing 200 ng / mL tolbutamide and 200 ng / mL labetalol 0.1% FA) was added to precipitate the protein. The plates were sealed and shaken for 20 min, then centrifuged at 4°C at 4000 rpm for 20 min. The supernatant in the reaction plate was transferred to a new plate, sealed and shaken for 10 min, and analyzed by LC-MS / MS. The results are shown in Table 7.

[0261] Table 7 Results of plasma stability test

[0262]

Claims

1. A peptide-like compound, characterized in that, The structure shown in equation (II): AA1-AA2-AA3-AA4-AA5-AA6 formula (II); The AA1, AA2, AA3, AA4, AA5 and AA6 are connected by condensation into amide bonds, and AA1 is the N-terminus; The AA1 is as shown in formula (10); AA3 is selected from glycine; Equation (10); m2 is an integer from 0 to 5; The AA2 is selected from arginine; The AA5 is selected from threonine; The AA4 is as shown in equation (13): Equation (13); R1 and R2 are each independently selected from H; n1 is 0, n2 is 1, and n3 is 1; The AA6 is selected from the structure shown in equation (17): Equation (17); R9 is selected from H; R 14 Selected from H.

2. The peptide-like compound according to claim 1, characterized in that, The peptide-like compounds are as shown in TM10 or TM28: 。

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