Peptidomimetics, methods for their preparation and use

Abstract

The application discloses a peptoid and a preparation method and application thereof. The peptoid preparation method is simple, low in cost and toxicity, and high in heparin binding capacity. In-vivo test results show that the peptoid can efficiently reverse the anticoagulation effect of ordinary heparin, low-molecular heparin and ultra-low-molecular-weight heparin, and can be used for preparing a medicine for treating and / or preventing adverse reactions or diseases caused by excessive anticoagulation of ordinary heparin, low-molecular heparin and ultra-low-molecular-weight heparin, and has excellent conversion prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and in particular relates to a peptide-like substance, its preparation method and application. Background Technology

[0002] Anticoagulants are a class of drugs that prevent thrombus formation by affecting the function of clotting factors. They are mainly used to prevent and treat thrombotic diseases such as cardiovascular and cerebrovascular diseases and venous thromboembolism. Their mechanisms of action are diverse, including indirect inhibition of clotting factors, antagonism of vitamin K, or direct inhibition of specific clotting factors. Heparin is a widely used non-oral anticoagulant, especially in scenarios requiring rapid onset of action or extracorporeal anticoagulation, such as cardiac surgery, extracorporeal circulation, hemodialysis, and the acute treatment of deep vein thrombosis. Heparin is a mixture of acidic mucopolysaccharides with a large number of negative charges. Through its specific pentose sequence, it binds to antithrombin III (AT III), significantly enhancing the inactivation rate of AT III on various clotting factors, thereby exerting a powerful anticoagulant effect.

[0003] However, heparin use carries a risk of bleeding, and its anticoagulant effect must be neutralized promptly after procedures such as cardiac surgery to restore normal coagulation function. Protamine sulfate is currently the only approved heparin-specific reversal agent in China. It is a strongly basic polycationic peptide extracted from salmon sperm. Its high-density positive charge can tightly bind to the strongly negatively charged heparin through ionic bonds, forming a stable neutral salt complex. This causes heparin to dissociate from antithrombin and lose its activity, and the complex is subsequently cleared by the reticuloendothelial system. Although protamine sulfate can effectively reverse unfractionated heparin, its clinical application has significant limitations. First, it may cause a series of adverse reactions, including mild allergies, severe hypotension, bradycardia, and even life-threatening anaphylactic reactions and cardiopulmonary failure. Second, its reversal effect on low molecular weight heparin is not complete; it can only partially neutralize its anti-factor Xa activity.

[0004] Therefore, developing safer and more efficient heparin reversal agents has always been an important direction. Historically, large-molecule cationic compounds, such as hexamethylenedimethylamine bromide, have been explored, but they have significant toxicity issues. In recent years, research has shifted to small-molecule drugs, such as Ciraparantag and Delparantag, but development progress has been slow. Summary of the Invention

[0005] Objectives of the invention: The first objective is to provide a peptide that can be used as a highly effective heparin reversal agent with high safety; the second objective is to provide a method for preparing the peptide; and the third objective is to provide applications of the peptide.

[0006] Technical solution: The peptide analogue of the present invention has the following characteristics: The structure is shown in equation (I): Equation (I); or the structure shown in Equation (II): Equation (II); or the structure shown in Equation (III): Formula (III); wherein R1, R2, R3, R4, R5, and R6 are the same or different from each other, and are independently selected from any one or more of optionally substituted alkyl, optionally substituted alkylamino, optionally substituted heterocyclic alkyl, optionally substituted aryl, and optionally substituted heterocyclic aryl, and all nitrogen atoms in the pseudopeptide may optionally be quaternized or oxidized.

[0007] Preferably, the peptide further includes pharmaceutically acceptable salts, esters, stereoisomers, solvates, hydrates, metabolites, metabolic precursors, or prodrugs of compounds with structures as shown in Formula (I), Formula (II), or Formula (III).

[0008] Preferably, the alkyl group is C1-C. 10 The terms are: straight-chain or branched alkyl; the alkylamino group represents an alkyl group containing 1 to 6 carbon atoms connected to the remainder of the molecule via an amino group, wherein the nitrogen atom of the amino group may optionally be quaternized; the heterocyclic alkyl group is a 6-membered monocyclic non-aromatic ring structure connected to the remainder of the molecule via a heteroatom, a carbon atom, or an alkyl group; the heterocyclic aryl group is a saturated 6-membered monocyclic or 6-membered heterocyclic aryl group containing one heteroatom selected from nitrogen on the ring, connected to the remainder of the molecule via a heteroatom, a carbon atom, or an alkyl group; the heteroatom may occupy an intermolecular linking position, and the heteroatom is selected from nitrogen or oxygen; the optional substitutions are selected from any one or more of halogens, C1-C5 alkyl groups, hydroxyl groups, cycloalkyl groups, heterocyclic alkyl groups, alkoxy groups, carbonyl groups, heterocyclic aryl groups, and aryl groups.

[0009] Preferably, R1 is selected from: , , , , , , , Any one of the following; the R2 is selected from: , , , , , , , Any one of the following; the R3 is selected from: , , , , , Any one of the following; the R4 is selected from: , , , Any one of the following; the R5 is selected from: , , , , , Any one of the following; the R6 is selected from: , Any one of them.

[0010] Preferably, the peptide is shown in any one of the items in Table 1, and the corresponding structure is shown in formulas (1) to (39):

[0011] Table 1. Peptide Names

[0012]

[0013] (Continued from previous table) ,

[0014] , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , .

[0015] The method for preparing the peptide described in this invention comprises the following steps: , Using methanol as the reaction solvent, the structure is as shown in formula (B) m Compounds and structures as shown in formula (C) p The compound was reacted at 40–60 °C for 0.5–1.5 h, and then the compound with the structure of formula (A) was added. n Compounds and structures as shown in formula (D) q The compound was reacted for 10-14 h to obtain a peptide with the structure shown in formula (I), wherein A n B m : C p :D q The molar ratio is (0.8~1.2):(0.8~1.2):(0.8~1.2):(0.8~1.2); or,

[0016] Using methanol as the reaction solvent, the structure is as shown in formula (E x Compounds and structures as shown in formula (C) p The compound was reacted at 40–60 °C for 0.5–1.5 h, and then the compound with the structure of formula (A) was added. n Compounds and structures as shown in formula (D) q The compound was reacted for 10-14 h to obtain a peptide with the structure shown in formula (II), wherein A n E x : C p :D q The molar ratio is (1.8~2.2):(0.8~1.2):(1.8~2.2):(1.8~2.2); or,

[0017] Using methanol as the reaction solvent, the structure is as shown in formula (F y Compounds and structures as shown in formula (C) p The compound was reacted at 40–60 °C for 0.5–1.5 h, and then the compound with the structure of formula (A) was added. n Compounds and structures as shown in formula (D) q The compound was reacted for 10-14 h to obtain a peptide with the structure shown in formula (III), wherein A n : F y : C p :D q The molar ratio is (2.8~3.2):(0.8~1.2):(2.8~3.2):(2.8~3.2); the definitions of R1, R2, R3, R4, R5, and R6 are the same as those previously defined.

[0018] The compositions of the present invention contain the aforementioned peptides or their pharmaceutically acceptable salts, esters, stereoisomers, solvates, hydrates, metabolites, metabolic precursors or prodrugs as active ingredients.

[0019] The reagent for reversing the anticoagulant activity of anticoagulants described in this invention contains the aforementioned peptides or compositions.

[0020] The application of the peptides, compositions, or reagents described in this invention in the preparation of drugs that reverse the anticoagulant activity of anticoagulants.

[0021] Preferably, the anticoagulant is unfractionated heparin (average molecular weight ~15000 Da), low molecular weight heparin (average molecular weight 3000~8000 Da), or ultra-low molecular weight heparin (average molecular weight <2000 Da).

[0022] Preferably, the drug is a drug for treating the side effects of excessive anticoagulation caused by the use of unfractionated heparin, low molecular weight heparin, and ultra-low molecular weight heparin to treat diseases, wherein the diseases treated with unfractionated heparin, low molecular weight heparin, and ultra-low molecular weight heparin are thromboembolic diseases; wherein the embolic diseases are myocardial infarction, thrombophlebitis, and pulmonary embolism.

[0023] Preferably, the drug is a drug used to treat surgical procedures requiring the reversal of the anticoagulant activity of unfractionated heparin, low molecular weight heparin, and ultra-low molecular weight heparin; the surgical procedures are hemodialysis, extracorporeal circulation, catheterization, and microvascular surgery.

[0024] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the peptide preparation method is simple, low in cost, low in toxicity, and has a strong binding ability with heparin. In vivo test results show that it can effectively reverse the anticoagulant effect of unfractionated heparin, low molecular weight heparin, and ultra-low molecular weight heparin. It can be used to prepare drugs for the treatment and / or prevention of adverse reactions or diseases caused by excessive anticoagulation of unfractionated heparin, low molecular weight heparin, and ultra-low molecular weight heparin, and has excellent translational prospects. Attached Figure Description

[0025] Figure 1 A statistical graph showing the results of the anti-FXa activity reversal test of the peptide; Figure 2 A statistical graph showing the results of peptide reversing unfractionated heparin in mouse tail-disconnection experiments; Figure 3 The results of the reversal of three types of heparin by peptides 2 and 16 in the mouse tail disconnection experiment are statistically shown in Figure A, which shows the blood loss (top) and bleeding time (bottom) of the unfractionated heparin group; Figure B shows the blood loss (top) and bleeding time (bottom) of the low molecular weight heparin group; and Figure C shows the blood loss (top) and bleeding time (bottom) of the ultra-low molecular weight heparin group. Figure 4 This is a statistical graph of body weight in mice during an acute toxicity test of peptide-16 in vivo. Figure 5 Organ coefficient statistics for acute toxicity experiments of peptide-16 in mice. Detailed Implementation

[0026] The technical solution of the present invention will be further described below.

[0027] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Experimental methods not specifically described in the examples are generally performed under standard conditions or as recommended by the manufacturer.

[0028] The compounds in the following examples are all based on structures as shown in formula (I), formula (II), or formula (III): Formula (I); Formula (II); Equation (III). The synthetic route is as follows: ;or ;or ; R1, R2, R3, R4, R5, and R6 may be the same as or different from each other, and are independently selected from one or more of optionally substituted alkyl, optionally substituted alkylamino, optionally substituted heterocyclic alkyl, optionally substituted aryl, and optionally substituted heterocyclic aryl groups. All nitrogen atoms in the pseudopeptide may optionally be quaternized or oxidized. The alkyl group is C1-C. 10 The terms are: straight-chain or branched alkyl groups; the alkylamino group represents an alkyl group containing 1 to 6 carbon atoms connected to the remainder of the molecule via an amino group, wherein the nitrogen atom of the amino group may optionally be quaternized; the heterocyclic alkyl group is a 6-membered monocyclic non-aromatic ring structure connected to the remainder of the molecule via a heteroatom, a carbon atom, or an alkyl group; the heterocyclic aryl group is a saturated 6-membered monocyclic or 6-membered heterocyclic aryl group containing one heteroatom selected from nitrogen on the ring, connected to the remainder of the molecule via a heteroatom, a carbon atom, or an alkyl group; the heteroatom may occupy an intermolecular linking position, and the heteroatom is selected from nitrogen or oxygen; the optional substitutions are selected from one or more of halogens, C1-C5 alkyl groups, hydroxyl groups, cycloalkyl groups, heterocyclic alkyl groups, alkoxy groups, carbonyl groups, heterocyclic aryl groups, and aryl groups.

[0029] Example 1: Preparation of peptide 3 with the structure shown in formula (3) 2-[bis(2-aminoethyl)amino]ethyl-1-amine (41 mg, 0.28 mmol) and pyridine-3-carboxaldehyde (91 mg, 0.85 mmol) were added sequentially to a 10 mL centrifuge tube, dissolved in 1 mL of methanol, and stirred at 50 °C for 1 hour. Then, N,N-dimethylglycine (87 mg, 0.85 mmol) and 3-(isocyanomethyl)pyridine (100 mg, 0.85 mmol) were added sequentially, and the mixture was stirred at 50 °C for another 12 hours. Thin-layer chromatography (TLC) confirmed the completion of the reaction. The mixture was concentrated under reduced pressure and purified by silica gel column chromatography to give 140 mg of a yellow solid, which was identified as peptide 3. Its melting point (mp) was 80–82 °C. Characterization and purity data are as follows: 1 H NMR (400 MHz, Chloroform- d ) δ 8.69-8.34 (m, 12H), 7.96-7.58 (m, 6H), 7.37-7.18 (m, 8H), 5.98-5.36 (m, 3H), 4.63-4.23 (m, 6H), 3.68-2.82 (m, 12H), 2.53-2.14 (m, 24H). 13 C NMR (101 MHz, Deuterium Oxide) δ 168.15, 166.96, 147.86,146.40, 145.88, 142.12, 139.97, 139.71, 138.38, 133.77, 127.49, 127.21,62.91, 58.21, 51.12, 44.31, 44.18, 40.65. HRMS (ESI): m / z [M+H]+ Calcd forC 57 H 73 N 16 O6: 1077.5899, Found: 1077.5586. HPLC: 99.76%.

[0030] Example 2: Preparation of peptide 1 with the structure shown in formula (1) 3-[4-(3-aminopropyl)piperazin-1-yl]propyl-1-amine (85 mg, 0.43 mmol) and pyridine-4-carboxaldehyde (91 mg, 0.85 mmol) were added sequentially to a 10 mL centrifuge tube, dissolved in 1 mL of methanol, and stirred at 50 °C for 1 hour. Then, N,N-dimethylglycine (87 mg, 0.85 mmol) and 3-(isocyanomethyl)pyridine (100 mg, 0.85 mmol) were added sequentially, and the mixture was stirred at 50 °C for another 12 hours. Thin-layer chromatography (TLC) confirmed the completion of the reaction. The mixture was concentrated under reduced pressure and purified by silica gel column chromatography to give 178 mg of a yellow solid, which was identified as peptide 1. HRMS (ESI): m / z [M+H] + =905.515.

[0031] Example 3: Preparation of peptide 2 with the structure shown in formula (2) Referring to the method in Example 2, N,N-dimethylglycine was replaced with an equimolar amount of 1,4-oxazacyclohexyl-4-ylacetic acid, and pyridine-4-carboxaldehyde was replaced with an equimolar amount of pyridine-3-carboxaldehyde, to obtain peptide 2. Its characterization and purity data are as follows: 1 H NMR (400 MHz, Chloroform- d ) δ 8.61-8.54 (m, 6H), 8.50-8.47 (m, 2H),7.85 (d, J = 8.1 Hz, 2H), 7.65 (d, J = 7.8 Hz, 2H), 7.39-7.22 (m, 4H), 6.90 (t, J =6.1 Hz, 2H), 5.61 (s, 2H), 4.51-4.43 (m, 4H), 3.72 (t, J = 4.5 Hz, 8H), 3.48(s, 4H), 3.34-3.20 (m, 4H), 2.62-2.46 (m, 12H), 2.28-2.07 (m, 8H), 1.74-1.39(m, 4H). 13C NMR (101 MHz, Deuterium Oxide) δ 168.47, 165.83, 147.81, 145.89,142.07, 141.48, 139.97, 139.81, 138.42, 134.21, 127.64, 127.20, 63.34, 62.78,57.00, 53.33, 52.96, 48.81, 46.14, 40.62, 23.02. HRMS (ESI): m / z [M+H]+ Calcdfor C 48 H 65 N 12 O6: 905.5133, Found: 905.5150. HPLC: 98.54%.

[0032] Example 4: Preparation of peptide 4 with the structure shown in formula (4) Referring to the method of Example 2, N,N-dimethylglycine was replaced with an equimolar amount of (4-methylpiperazin-1-yl)acetic acid, pyridine-4-carboxaldehyde was replaced with an equimolar amount of benzaldehyde, and 3-(isocyanomethyl)pyridine was replaced with an equimolar amount of 4-(isocyanomethyl)-1-methoxybenzene to obtain peptide 4. HRMS (ESI): m / z [M+H] + =987.6184.

[0033] Example 5: Preparation of peptide 5 with the structure shown in formula (5) Referring to the method of Example 2, N,N-dimethylglycine was replaced with an equimolar amount of (4-methylpiperazin-1-yl)acetic acid, and pyridine-4-carboxaldehyde was replaced with an equimolar amount of naphthalene-2-carboxaldehyde, to obtain peptide 5. HRMS (ESI): m / z [M+H] + =1029.6191.

[0034] Example 6: Preparation of peptide 6 with the structure shown in formula (6) Following the method of Example 2, N,N-dimethylglycine was replaced with an equimolar amount of (4-methylpiperazin-1-yl)acetic acid to obtain peptide 6. Its characterization and purity data are as follows: 1 H NMR (400 MHz, Chloroform- d) δ 8.74-8.42 (m, 8H), 7.77-7.62 (m, 2H), 7.41-7.06 (m, 8H), 5.79-5.40 (m, 2H), 4.89-4.26 (m, 4H), 3.73-3.24 (m, 8H), 2.76-2.12 (m, 34H). 13 C NMR (101 MHz, Deuterium Oxide) δ 167.80, 165.70, 153.82, 145.92, 141.45, 139.99, 139.81,138.33, 127.53, 127.23, 64.24, 64.18, 56.60, 53.30, 49.72, 48.78, 46.28,42.85, 40.63, 23.09. HRMS (ESI): m / z [M+H]+ Calcd for C 50 H 71 N 14 O4: 931.5760, Found: 931.5783. HPLC: 99.46%.

[0035] Example 7: Preparation of peptide 7 with the structure shown in formula (7) Following the method of Example 1, N,N-dimethylglycine was replaced with an equimolar amount of 1,4-oxazacyclohexyl-4-ylacetic acid to obtain peptide 7. HRMS (ESI): m / z [M+H] + =1203.6216.

[0036] Example 8: Preparation of peptide 8 with the structure shown in formula (8) Following the method of Example 1, N,N-dimethylglycine was replaced with an equimolar amount of (4-methylpiperazin-1-yl)acetic acid to obtain peptide 8. HRMS (ESI): m / z [M+H] + =1242.7165.

[0037] Example 9: Preparation of peptide 9 with the structure shown in formula (9) Following the method of Example 1, N,N-dimethylglycine was replaced with an equimolar amount of 1,4-oxazacyclohexyl-4-ylacetic acid, and pyridine-3-carboxaldehyde was replaced with an equimolar amount of pyridine-4-carboxaldehyde, yielding peptide 9. Its characterization and purity data are as follows: 1 H NMR (400 MHz, Chloroform- d) δ 8.69-8.35 (m, 12H), 8.28-7.96 (m, 3H),7.64 (d, J = 21.7 Hz, 3H), 7.37-6.99 (m, 11H), 5.94-5.34 (m, 3H), 4.73-4.30 (m,6H), 3.82-2.81 (m, 24H), 2.56-2.32 (m, 18H). 13 C NMR (101 MHz, DeuteriumOxide) δ 167.48, 166.30, 153.27, 146.42, 145.92, 141.42, 139.98, 127.59,127.19, 63.23, 57.06, 52.98, 51.58, 51.15, 40.62. HRMS (ESI): m / z [M+H]+Calcd for C 63 H 79 N 16 O9: 1203.6216, Found: 1203.6195. HPLC: 98.91%.

[0038] Example 10: Preparation of peptide 10 with the structure shown in formula (10) Referring to the method of Example 1, N,N-dimethylglycine was replaced with an equimolar amount of 1,4-oxazacyclohexyl-4-ylacetic acid, and 3-(isocyanomethyl)pyridine was replaced with an equimolar amount of 4-(isocyanomethyl)-1-methoxybenzene to obtain peptide 10. HRMS (ESI): m / z [M+H] + =1290.6676.

[0039] Example 11: Preparation of peptide 11 with the structure shown in formula (11) Referring to the method of Example 2, N,N-dimethylglycine was replaced with an equimolar amount of (4-methylpiperazin-1-yl)acetic acid, and 3-(isocyanomethyl)pyridine was replaced with an equimolar amount of 4-(isocyanomethyl)-1-methoxybenzene to obtain peptide 11. HRMS (ESI): m / z [M+H] + =989.6089.

[0040] Example 12: Preparation of peptide 12 with the structure shown in formula (12) Following the method of Example 1, N,N-dimethylglycine was replaced with an equimolar amount of (4-methylpiperazin-1-yl)acetic acid, and pyridine-3-carboxaldehyde was replaced with an equimolar amount of pyridine-4-carboxaldehyde, yielding peptide 12. Its characterization and purity data are as follows:1 H NMR (400 MHz, Chloroform- d ) δ 8.66-8.36 (m, 12H), 7.82-6.99 (m, 15H), 5.85-5.40 (m, 3H), 4.59-4.12 (m, 6H), 3.50-2.86 (m, 12H), 2.62-2.15 (m, 40H). 13 C NMR (101 MHz, Deuterium Oxide) δ 167.43, 166.20, 153.25, 146.51, 145.94,141.47, 139.99, 138.23, 127.60, 127.22, 64.55, 56.70, 56.43, 49.72, 49.47,43.81, 42.85, 40.65. HRMS (ESI): m / z [M+H]+ Calcd for C 66 H 88 N 19 O6: 1242.7165, Found: 1242.7128. HPLC: 99.50%.

[0041] Example 13: Preparation of peptide 13 with the structure shown in formula (13) Following the method of Example 2, N,N-dimethylglycine was replaced with an equimolar amount of (4-methylpiperazin-1-yl)acetic acid to obtain peptide 13. Its characterization and purity data are as follows: 1 H NMR (400 MHz, Chloroform- d ) δ 8.67-8.39 (m, 8H), 7.91-7.56 (m, 4H), 7.42-7.03 (m, 6H), 6.05-5.57 (m, 2H), 4.61-4.34 (m, 4H), 3.56-3.02 (m, 8H), 2.71-2.04 (m, 29H), 1.48-1.09 (m, 4H). 13CNMR (101 MHz, Deuterium Oxide) δ 168.07, 165.22, 147.71, 145.71, 141.80,141.07, 139.69, 139.47, 138.19, 133.94, 127.37, 127.01, 62.49, 56.39, 52.76,49.64, 49.34, 46.29, 42.72, 40.43, 39.92, 23.11. HRMS (ESI): m / z [M+H]+ Calcdfor C 47 H 66 N 13 O4: 876.5361, Found: 876.5331. HPLC: 98.53%.

[0042] Example 14: Preparation of peptide 14 with the structure shown in formula (14) Following the method of Example 2, N,N-dimethylglycine was replaced with an equimolar amount of (4-methylpiperazin-1-yl)acetic acid, and pyridine-4-carboxaldehyde was replaced with an equimolar amount of pyridine-3-carboxaldehyde, yielding peptide 14. Its characterization and purity data are as follows: 1 H NMR (400 MHz, Chloroform- d ) δ 8.71-8.37 (m, 8H), 7.77-7.57 (m, 2H), 7.42-7.10 (m, 6H), 5.90-5.36 (m, 2H), 4.90-4.25 (m, 4H), 3.73-3.08 (m, 8H), 2.66-1.87 (m, 29H), 1.72-1.22 (m, 4H). 13 C NMR (101 MHz, Deuterium Oxide) δ 167.76,165.53, 153.83, 145.90, 141.36, 139.95, 139.77, 138.32, 127.49, 127.20,64.26, 56.60, 52.99, 49.82, 49.64, 46.49, 42.84, 40.61, 39.79, 23.35. HRMS(ESI): m / z [M+H]+ Calcd for C 47 H 66 N 13 O4: 876.5361, Found: 876.5345. HPLC: 98.66%.

[0043] Example 15: Preparation of peptide 15 with the structure shown in formula (15) Referring to the method of Example 1, N,N-dimethylglycine was replaced with an equimolar amount of 1,4-oxazacyclohexyl-4-ylacetic acid, pyridine-3-carboxaldehyde was replaced with an equimolar amount of pyridine-4-carboxaldehyde, and 3-(isocyanomethyl)pyridine was replaced with an equimolar amount of 4-(isocyanomethyl)-1-methoxybenzene to obtain peptide 15. HRMS (ESI): m / z [M+H] + =1290.6676.

[0044] Example 16: Preparation of peptide 16 with the structure shown in formula (16) Following the method of Example 1, N,N-dimethylglycine was replaced with an equimolar amount of 1,4-oxazacyclohexyl-4-ylacetic acid, and pyridine-3-carboxaldehyde was replaced with an equimolar amount of naphthalene-2-carboxaldehyde, yielding peptide 16. Its characterization and purity data are as follows: 1 H NMR (400 MHz, Chloroform- d ) δ 8.57-8.31 (m, 6H), 7.96-7.06 (m, 27H), 6.75-6.40 (m, 3H), 6.14-5.41 (m, 3H), 4.67-4.18 (m, 6H), 3.77-2.85 (m, 24H),2.58-2.19 (m, 18H). 13 C NMR (101 MHz, Deuterium Oxide) δ 169.57, 165.47,145.98, 145.47, 140.27, 139.95, 139.41, 138.47, 138.14, 132.92, 132.50,129.74, 129.00, 127.84, 127.37, 127.20, 63.39, 62.83, 56.94, 52.92, 40.19,39.96, 39.52. HRMS (ESI): m / z [M+2H] / 2+ Calcd for C 78 H 89 N 13 O9 / 2: 675.8453, Found: 675.8449. HPLC: 99.33%.

[0045] Example 17: Preparation of peptide 17 with the structure shown in formula (17) Following the method of Example 1, pyridine-3-carboxaldehyde was replaced with an equimolar amount of pyridine-4-carboxaldehyde to obtain peptide 17. Its characterization and purity data are as follows: 1 H NMR (400 MHz, Chloroform- d ) δ 8.73-8.32 (m, 12H),7.82-7.59 (m, 6H), 7.37-7.15 (m, 8H), 5.83-5.14 (m, 3H), 4.93-4.14 (m, 6H), 3.85-2.88 (m, 12H), 2.48-1.97 (m, 24H). 13 C NMR (101 MHz, Deuterium Oxide) δ168.15, 166.96, 147.86, 146.40, 145.88, 142.12, 139.97, 139.71, 138.38,133.77, 127.49, 127.21, 62.91, 58.21, 51.12, 44.31, 44.18, 40.65. HRMS (ESI):m / z [M+H]+ Calcd for C 57 H 73 N 16 O6: 1077.5899, Found: 1077.5571. HPLC: 98.62%.

[0046] Example 18: Preparation of peptide 18 with the structure shown in formula (18) Referring to the method of Example 2, N,N-dimethylglycine was replaced with an equimolar amount of 1,4-oxazacyclohexyl-4-ylacetic acid, and 3-(isocyanomethyl)pyridine was replaced with an equimolar amount of 4-(isocyanomethyl)-1-methoxybenzene to obtain peptide 18. HRMS (ESI): m / z [M+H] + =963.5456.

[0047] Example 19: Preparation of peptide 19 with the structure shown in formula (19) Referring to the method of Example 2, N,N-dimethylglycine was replaced with an equimolar amount of (4-methylpiperazin-1-yl)acetic acid, pyridine-4-carboxaldehyde was replaced with an equimolar amount of pyridine-3-carboxaldehyde, and 3-(isocyanomethyl)pyridine was replaced with an equimolar amount of 4-(isocyanomethyl)-1-methoxybenzene, yielding peptide 19. HRMS (ESI): m / z [M+H] + =989.6089.

[0048] Example 20: Preparation of peptide 20 with the structure shown in formula (20) Referring to the method of Example 2, N,N-dimethylglycine was replaced with an equimolar amount of 1,4-oxazacyclohexyl-4-ylacetic acid, pyridine-4-carboxaldehyde was replaced with an equimolar amount of pyridine-3-carboxaldehyde, and 3-(isocyanomethyl)pyridine was replaced with an equimolar amount of 4-(isocyanomethyl)-1-methoxybenzene to obtain peptide 20. HRMS (ESI): m / z [M+H] + =963.5456.

[0049] Example 21: Preparation of peptide 21 with the structure shown in formula (21) Referring to the method of Example 2, N,N-dimethylglycine was replaced with 1,4-oxazacyclohexyl-4-ylacetic acid, pyridine-4-carboxaldehyde was replaced with an equimolar amount of naphthalene-2-carboxaldehyde, and 3-(isocyanomethyl)pyridine was replaced with an equimolar amount of 4-(isocyanomethyl)-1-methoxybenzene to obtain peptide 21. HRMS (ESI): m / z [M+H] + =1061.5864.

[0050] Example 22: Preparation of peptide 22 with the structure shown in formula (22) Referring to the method in Example 2, N,N-dimethylglycine was replaced with an equimolar amount of 1,4-oxazacyclohexyl-4-ylacetic acid, and pyridine-4-carboxaldehyde was replaced with an equimolar amount of pyridine-3-carboxaldehyde, to obtain peptide 22. HRMS (ESI): m / z [M+H] + =850.4728.

[0051] Example 23: Preparation of peptide 23 with the structure shown in formula (23) Referring to the method of Example 2, N,N-dimethylglycine was replaced with an equimolar amount of (4-methylpiperazin-1-yl)acetic acid, and pyridine-4-carboxaldehyde was replaced with an equimolar amount of pyridine-3-carboxaldehyde, to obtain peptide 23. HRMS (ESI): m / z [M+H] + =931.5783.

[0052] Example 24: Preparation of peptide 24 with the structure shown in formula (24) Following the method of Example 1, N,N-dimethylglycine was replaced with an equimolar amount of (4-methylpiperazin-1-yl)acetic acid, pyridine-3-carboxaldehyde was replaced with an equimolar amount of pyridine-4-carboxaldehyde, and 3-(isocyanomethyl)pyridine was replaced with an equimolar amount of 4-(isocyanomethyl)-1-methoxybenzene to obtain peptide 24. HRMS (ESI): m / z [M+H] + =1329.7625.

[0053] Example 25: Preparation of peptide 25 with the structure shown in formula (25) Following the method of Example 2, 3-(isocyanomethyl)pyridine was replaced with an equimolar amount of 4-(isocyanomethyl)-1-methoxybenzene to obtain peptide 25. HRMS (ESI): m / z [M+H] + =879.524.

[0054] Example 26: Preparation of peptide 26 with the structure shown in formula (26) Following the method of Example 1, N,N-dimethylglycine was replaced with an equimolar amount of (4-methylpiperazin-1-yl)acetic acid, and pyridine-3-carboxaldehyde was replaced with an equimolar amount of naphthalene-2-carboxaldehyde, yielding peptide 26. HRMS (ESI): m / z [M+H] + =1389.7777.

[0055] Example 27: Preparation of peptide 27 with the structure shown in formula (27) Following the method of Example 2, N,N-dimethylglycine was replaced with an equimolar amount of isonicotinic acid to obtain peptide 27. HRMS (ESI): m / z [M+H] + =861.4313.

[0056] Example 28: Preparation of peptide 28 with the structure shown in formula (28) Referring to the method of Example 2, N,N-dimethylglycine was replaced with an equimolar amount of 1,4-oxazacyclohexyl-4-ylacetic acid, and pyridine-4-carboxaldehyde was replaced with an equimolar amount of naphthalene-2-carboxaldehyde, yielding peptide 28. HRMS (ESI): m / z [M+H] + =1003.5558.

[0057] Example 29: Preparation of peptide 29 with the structure shown in formula (29) Following the method of Example 2, 3-(isocyanomethyl)pyridine was replaced with an equimolar amount of tert-butylisocyanate to obtain peptide 29. HRMS (ESI): m / z [M+H] + =751.5341.

[0058] Example 30: Preparation of peptide 30 with the structure shown in formula (30) Referring to the method of Example 2, pyridine-4-carboxaldehyde was replaced with an equimolar amount of naphthalene-2-carboxaldehyde, and 3-(isocyanomethyl)pyridine was replaced with an equimolar amount of 4-(isocyanomethyl)-1-methoxybenzene to obtain peptide 30. HRMS (ESI): m / z[M+H] + =977.5653.

[0059] Example 31: Preparation of peptide 31 with the structure shown in formula (31) Referring to the method of Example 2, N,N-dimethylglycine was replaced with an equimolar amount of (4-methylpiperazin-1-yl)acetic acid, and pyridine-4-carboxaldehyde was replaced with an equimolar amount of pyridine-2-carboxaldehyde, to obtain peptide 31. HRMS (ESI): m / z [M+H] + =931.5783.

[0060] Example 32: Preparation of peptide 32 with the structure shown in formula (32) Following the method of Example 1, pyridine-3-carboxaldehyde was replaced with an equimolar amount of pyridine-4-carboxaldehyde, and 3-(isocyanomethyl)pyridine was replaced with an equimolar amount of 4-(isocyanomethyl)-1-methoxybenzene to obtain peptide 32. HRMS (ESI): m / z [M+H] + =1164.6359.

[0061] Example 33: Preparation of peptide 33 with the structure shown in formula (33) Referring to the method in Example 2, N,N-dimethylglycine was replaced with an equimolar amount of isonicotinic acid to obtain peptide 33. HRMS (ESI): m / z [M+H] + =861.4313.

[0062] Example 34: Preparation of peptide 34 with the structure shown in formula (34) Following the method of Example 1, N,N-dimethylglycine was replaced with an equimolar amount of (4-methylpiperazin-1-yl)acetic acid, pyridine-3-carboxaldehyde was replaced with an equimolar amount of pyridine-4-carboxaldehyde, and 3-(isocyanomethyl)pyridine was replaced with an equimolar amount of tert-butylisocyanate to obtain peptide 34. HRMS (ESI): m / z [M+H] + =1137.7772.

[0063] Example 35: Preparation of peptide 35 with the structure shown in formula (35) Referring to the method of Example 1, N,N-dimethylglycine was replaced with an equimolar amount of isonicotinic acid, and pyridine-3-carboxaldehyde was replaced with an equimolar amount of naphthalene-2-carboxaldehyde to obtain peptide 35. HRMS (ESI): m / z [M+H] + =1284.5572.

[0064] Example 36: Preparation of peptide 36 with the structure shown in formula (36) Following the method of Example 1, N,N-dimethylglycine was replaced with an equimolar amount of (4-methylpiperazin-1-yl)acetic acid, and pyridine-3-carboxaldehyde was replaced with an equimolar amount of pyridine-2-carboxaldehyde, yielding peptide 36. HRMS (ESI): m / z [M+H] + =1242.7165.

[0065] Example 37: Preparation of peptide 37 with the structure shown in formula (37) Referring to the method of Example 2, 3-[4-(3-aminopropyl)piperazin-1-yl]propyl-1-amine was replaced with an equimolar amount of 3-[(3-aminopropyl)(methyl)amino]propyl-1-amine, pyridine-4-carboxaldehyde was replaced with an equimolar amount of naphth-2-carboxaldehyde, and 3-(isocyanomethyl)pyridine was replaced with an equimolar amount of 4-(isocyanomethyl)-1-methoxybenzene, to obtain peptide 37. HRMS(ESI): m / z [M+H] + =922.5231.

[0066] Example 38: Preparation of peptide 38 with the structure shown in formula (38) Following the method of Example 1, N,N-dimethylglycine was replaced with an equimolar amount of (4-methylpiperazin-1-yl)acetic acid, and pyridine-3-carboxaldehyde was replaced with an equimolar amount of benzaldehyde, yielding peptide 38. HRMS (ESI): m / z [M+H] + =1239.7308.

[0067] Example 39: Preparation of peptide 39 with the structure shown in formula (39) Following the method of Example 1, N,N-dimethylglycine was replaced with an equimolar amount of 1,4-oxazacyclohexyl-4-ylacetic acid, and pyridine-3-carboxaldehyde was replaced with an equimolar amount of pyridine-2-carboxaldehyde, yielding peptide 39. HRMS (ESI): m / z [M+H] + =1203.6216.

[0068] Experimental Example 1: Quantitative Determination of Competitive Binding of Peptide-like Substances and Azurite A to Heparin Sodium Experimental materials included: heparin sodium, sourced from Yuanye Biotechnology (product number S59654-100KU); Azure A, sourced from Shanghai Maclean Biotechnology Co., Ltd. (product number A800820); and peptides prepared in Examples 1-39.

[0069] A 0.4 mg / mL azurite A stock solution, a 0.2 mg / mL heparin stock solution, and a 1 mg / mL peptide stock solution prepared in Examples 1-39 were prepared using physiological saline. Pure physiological saline wells were used as blank solvent controls, heparin-azrite A complex wells as 0% negative controls, and pure azurite A wells as 100% positive controls. The peptides prepared in Examples 1-39 were used as the test group.

[0070] Add 200 μL of physiological saline to the blank solvent control wells of a 96-well white plate; add 100 μL of physiological saline, 50 μL of azure A stock solution, and 50 μL of heparin stock solution sequentially to the heparin-azure A complex wells of a 96-well white plate; add 150 μL of physiological saline and 50 μL of azure A stock solution sequentially to the azure A wells of a 96-well white plate; add 80 μL of physiological saline, 20 μL of any peptide stock solution prepared in Examples 1-39, 50 μL of azure A stock solution, and 50 μL of heparin stock solution sequentially to the test wells of a 96-well white plate; add 80 μL of physiological saline, 20 μL of any peptide stock solution prepared in Examples 1-39, 50 μL of azure A stock solution, and 50 μL of heparin stock solution sequentially to the test wells of a 96-well white plate. All 96-well white plates were incubated at 37°C for 10 minutes using a shaker, followed by absorbance measurements at λ = 600 nm using a microplate reader. Each peptide was measured three times. After subtracting background from the blank solvent control, the heparin reversal rate was calculated using the following formula: Reversal rate (%) = (OD) 测试孔 -OD 阴性对照孔 ) / (OD 阳性对照孔 -OD 阴性对照孔 )×100% Among them, OD 测试孔 The absorbance of the test well at 600 nm after background subtraction; OD 阳性对照孔 The absorbance of the Azure A aperture after background subtraction at 600 nm; OD 阴性对照孔 The absorbance of the heparin-azure A complex pore at 600 nm after background subtraction is given.

[0071] The results are shown in Table 2. All the tested peptides could reverse the binding of azurite A to heparin to varying degrees in vitro, demonstrating the ability to reverse heparin activity.

[0072] Table 2. Quantitative results of competitive binding of peptides and azurite A to heparin sodium.

[0073] Experimental Example 2: In vitro cytotoxicity assay of peptide-like substances The peptides and protamine sulfate (TICA Chemical Industry, product number P0675) prepared in Examples 1-39 were respectively prepared into stock solutions with a concentration of 5 mg / mL using PBS buffer.

[0074] HUVEC cells (ATCC CRL-1730) in the logarithmic growth phase were seeded at 4000 cells / well in 96-well plates with 100 μL of culture medium and cultured for 24 h to allow them to adhere. The stock solutions of peptide-like substances or protamine sulfate prepared in Examples 1-39 were diluted to 50 μg / mL with complete culture medium. After discarding the original culture medium, 100 μL of the solution was added to each well for 72 h. A blank control group was set up with only fresh complete culture medium, and a control group was set up with both fresh complete culture medium and HUVEC cells. Each treatment was performed in triplicate.

[0075] After culturing, 20 μL of 5 mg / mL MTT reagent was added to each well, and after incubation for 4 h, 150 μL of LDMSO was added to each well and the mixture was shaken for 5 min. The absorbance at 580 nm was measured using a microplate reader, and the cell growth inhibition efficiency was calculated using the following formula: Cell viability (%) = (OD) 待测药 -OD 培养液对照 ) / (OD 细胞对照 -OD 培养液对照 )×100%; Cell growth inhibition efficiency (%) = 1 - cell viability (%) Among them, OD 待测药 The absorbance of the peptide or protamine sulfate at 580 nm; OD 培养液对照 The absorbance at 580 nm for the blank control group with only culture medium added; OD 细胞对照 The absorbance at 580 nm is the absorbance of the control group with added culture medium and cells.

[0076] The results are shown in Table 3. Most of the peptides showed good safety (inhibition rate of HUVEC cells ≤30%).

[0077] Table 3. In vitro toxicity test results of the peptide-like HUVECs

[0078] Experimental Example 3: Hemolytic Toxicity Experiment of Peptide Mimic The fresh blood used in this experiment came from human volunteers. After venous blood collection, the blood was immediately placed in a blue sodium citrate coagulation test tube and stored at 4°C.

[0079] The peptides and protamine sulfate obtained in Examples 1-39 were prepared into solutions with a concentration of 2.5 mg / mL using PBS buffer. The solutions were then added to centrifuge tubes with fresh blood samples at a volume ratio of 9:1. Ultrapure water was used as a positive control and PBS buffer as a negative control. The mixtures were inverted and mixed thoroughly. Each treatment was performed in triplicate.

[0080] The mixed sample was incubated at 37℃ for 1 h, then centrifuged at 3500 rpm for 15 min. The supernatant was collected from the centrifuge tube, and the absorbance was measured at 575 nm using an ELISA reader. The hemolysis rate was calculated using the following formula: Hemolysis rate (%) = (OD) 待测药 -OD PBS对照 ) / (OD 超纯水对照 -OD PBS对照 )×100% Among them, OD 待测药 The absorbance value at 575 nm of blood samples treated with peptides or protamine sulfate; OD PBS对照 The absorbance value at 575 nm is the negative control; OD 超纯水对照 The absorbance value at 575 nm is the positive control.

[0081] The results, as shown in Table 4, indicate that most of the peptides of this invention exhibit no hemolytic toxicity (hemolysis rate ≤5%) at extremely high concentrations of 2.5 mg / mL, demonstrating good safety. In contrast, the positive control, protamine sulfate, showed significant hematologic toxicity at high concentrations. The hemolytic toxicity of protamine sulfate is one of its important toxic characteristics, and the experimental data are consistent with literature reports and clinical data. The peptides of this invention are non-hemolytic and meet the requirements for intravenous injection materials.

[0082] Table 4 Results of hemolytic toxicity assays for peptide-like substances

[0083] Experimental Example 4: Coagulation Factor Activity Assay of Peptides (Anti-Factor Xa Assay) According to BiopHen TM In accordance with the instructions for the Heparin Anti-Xa Kit (product number 221005), dilute the sample to be tested and reconstitute the kit using Tris-NaCl buffer at pH 7.4.

[0084] Peptides 2, 3, 6, 9, 12, 13, 14, 16, 17 and protamine sulfate were diluted to 2 × 10⁻⁶. -4 mg / mL, enoxaparin sodium (Biode Pharmaceuticals, product number: BD01148943) diluted to 1×10 -4mg / mL, the two were mixed in equal volumes to prepare the test sample, with ultrapure water as the heparin-free reference standard, and enoxaparin sodium was diluted with ultrapure water to obtain 5 × 10 mg / mL. -5 Enoxaparin solution at mg / mL is used as a heparin reference standard.

[0085] Add 40 μL of the test sample to each well of a 96-well plate, with three replicates for each compound. First, add 40 μL of antithrombin solution to each well, vortex to mix, and incubate at 37°C for 2 min. Then, add 40 μL of coagulation factor Xa solution to each well, vortex to mix, and incubate at 37°C for 2 min. Next, add 40 μL of the specific chromogenic substrate solution for coagulation factor Xa to each well, vortex to mix, and incubate precisely at 37°C for 2 min. Finally, add 80 μL of 2% citric acid solution to each well, vortex to mix, and immediately read the absorbance at 405 nm using a microplate reader. The neutralization activity of the peptide-like compound for enoxaparin is expressed as the neutralization rate, calculated using the following formula: Neutralization rate (%) = (OD) 待测药 -OD 无肝素对照 ) / (OD 肝素对照 -OD 无肝素对照 )×100%, Among them, OD 待测药 The absorbance value of the sample at 405 nm; OD 肝素对照 The absorbance value of the heparin control well at 405 nm; OD 无肝素对照 The absorbance value at 405 nm is the absorbance value of the heparin-free control well.

[0086] The results are as follows Figure 1 As shown, all the peptides of this invention possess anti-Xa activity and can reverse the anticoagulant activity of enoxaparin. The positive control, protamine sulfate, can only partially reverse the activity of coagulation factor Xa under the action of low molecular weight heparin, which is one of the factors affecting its clinical application. In contrast, peptides 2, 3, 6, 9, 12, 13, 14, 16, and 17 of this invention exhibit anti-Xa neutralization activity exceeding 70%, demonstrating potent heparin reversal activity.

[0087] Experimental Example 5: Peptide-based mouse tail docking experiment Tail docking following high-dose heparin injection in mice leads to prolonged blood loss time and increased blood loss. Intravenous heparin injection followed by the use of a heparin reversal agent can reverse the blood loss time and reduce the blood loss. The specific experimental steps are as follows: 12-13 week old, 20-22 g female C57 mice were purchased from Shanghai Bikaico Biotechnology Co., Ltd., and randomly divided into 3 groups, including: The blank control group (Saline) was injected with normal saline via the tail vein. Five minutes later, normal saline was injected again via the tail vein, and 2.5% aphthol was immediately injected intraperitoneally for anesthesia. A total of 3 animals were included. Heparin group: 3 mg / kg of unfractionated heparin (heparin sodium, UFH), low molecular weight heparin (enoxaparin sodium, LMWH), or ultra-low molecular weight heparin (fondaparinux sodium, from Maclean, product number: F857015-5 mg, ULMWH) in saline solution was injected via the tail vein. After 5 minutes, saline was injected via the tail vein, and 2.5% aphthol was immediately injected intraperitoneally for anesthesia. Three animals were treated with each type of heparin. Experimental groups: 1) 3 mg / kg unfractionated heparin (heparin sodium) was injected via the tail vein, followed by a 4.5 mg / kg peptide mimicry as a reversal agent 5 min later (including peptide mimicry 2, 3, 6, 9, 12, 13, 14, 16, and 17, all prepared with normal saline) and immediate intraperitoneal anesthesia with 2.5% aphthol; 2) 3 mg / kg low molecular weight heparin (enoxaparin sodium) was injected via the tail vein, followed by a 4.5 mg / kg peptide mimicry as a reversal agent 5 min later (including peptide mimicry 2 and 16, all prepared with normal saline) and immediate intraperitoneal anesthesia with 2.5% aphthol; 3) 3 mg / kg ultra-low molecular weight heparin (fondaparinux sodium) was injected via the tail vein, followed by a 4.5 mg / kg peptide mimicry as a reversal agent 5 min later (including peptide mimicry 2 and 16, all prepared with normal saline) and immediate intraperitoneal anesthesia with 2.5% aphthol; each heparin and peptide mimicry combination was administered to 3 animals.

[0088] Ten minutes after the reversal agent was injected via the tail vein, the mouse tail was transversely cut at a diameter of 2.5 mm. The tail was immediately immersed in a centrifuge tube containing 15 mL of 37°C physiological saline, and blood flowing from the wound was collected. The blood loss observation window was 20 minutes. Hemostasis was performed immediately after the experiment.

[0089] The results are as follows Figure 2 As shown, the tested peptides can all significantly reverse blood loss induced by unfractionated heparin; simultaneously, as Figure 3 As shown, peptides 2 and 16 can also significantly reverse blood loss caused by low molecular weight heparin and ultra-low molecular weight heparin.

[0090] Experimental Example 6: Acute toxicity evaluation of peptide-mimicking peptides in mice ICR mice, aged 6-8 weeks and weighing 18-20g, were purchased from Jiangsu Qinglongshan Biotechnology Co., Ltd., and randomly divided into 3 groups of 6 mice each, with half males and half females, including: Saline control group: Saline was injected via tail vein; Low-dose peptide group: peptide 16 prepared with normal saline was injected via tail vein at a dose of 20 mg / kg. High-dose peptide group: peptide 16 prepared with physiological saline was injected via tail vein at a dose of 30 mg / kg.

[0091] The activity level of mice was observed regularly after injection. Seven days after a single tail vein injection, the mice were dissected, and their body weight and the size of each organ were observed and recorded. The organ coefficient was calculated using the following formula: Organ coefficient (%) = organ wet weight / mouse body weight × 100%.

[0092] The results are as follows Figure 4 , 5 As shown, the peptide of the present invention has good safety.

[0093] In summary, the small molecule peptide of this invention, after quantitative testing of competitive binding to heparin sodium by azurite A, in vitro cytotoxicity and hemolytic toxicity tests, anti-Xa factor tests, acute toxicity tests, and in vivo heparin reversal tests in mice, showed potent and low-toxicity heparin reversal activity. The anticoagulant activity of peptide 16 in reversing heparin in both in vitro and in vivo was superior to or comparable to that of the positive control protamine sulfate, and it also had good biocompatibility.

Claims

1. A peptide-like compound, characterized in that, The peptide-like substance has the following characteristics: The structure is shown in equation (I): Formula (I); Or, as shown in equation (II): Formula (II); Or, as shown in equation (III): Formula (III); R1, R2, R3, R4, R5, and R6 may be the same as or different from each other, and are independently selected from any one or more of optionally substituted alkyl, optionally substituted alkylamino, optionally substituted heterocyclic alkyl, optionally substituted aryl, and optionally substituted heterocyclic aryl. All nitrogen atoms in the pseudopeptide may optionally be quaternized or oxidized.

2. The peptide-like substance according to claim 1, characterized in that, The peptides also include pharmaceutically acceptable salts, esters, stereoisomers, solvates, hydrates, metabolites, metabolic precursors, or prodrugs of compounds with structures as shown in Formula (I), Formula (II), or Formula (III).

3. The peptide analogue according to claim 1, characterized in that, The alkyl group is C1-C. 10 The terms are: straight-chain or branched alkyl groups; the alkylamino group refers to an alkyl group containing 1 to 6 carbon atoms connected to the remainder of the molecule via an amino group, wherein the nitrogen atom of the amino group may optionally be quaternized; the heterocyclic alkyl group is a 6-membered monocyclic non-aromatic ring structure connected to the remainder of the molecule via a heteroatom, a carbon atom, or an alkyl group; the heterocyclic aryl group is a saturated 6-membered monocyclic or 6-membered heterocyclic aryl group containing one heteroatom selected from nitrogen on the ring, connected to the remainder of the molecule via a heteroatom, a carbon atom, or an alkyl group; the heteroatom may occupy an intermolecular linking position, and the heteroatom is selected from nitrogen or oxygen; the optional substitutions are selected from one or more of halogens, C1-C5 alkyl groups, hydroxyl groups, cycloalkyl groups, heterocyclic alkyl groups, alkoxy groups, carbonyl groups, heterocyclic aryl groups, and aryl groups.

4. The peptide analogue according to claim 1, characterized in that, R1 is selected from: , , , , , , , Any one of the following; the R2 is selected from: , , , , , , , Any one of the following; the R3 is selected from: , , , , , Any one of the following; the R4 is selected from: , , , Any one of the following; the R5 is selected from: , , , , , Any one of the following; the R6 is selected from: , Any one of them.

5. The peptide according to any one of claims 1 to 4, characterized in that, The peptide has a structure as shown in any one of formulas (1) to (39): , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , .

6. A method for preparing the peptide according to any one of claims 1 to 5, characterized in that, The steps are as follows: ， Using methanol as the reaction solvent, the structure is as shown in formula (B) m Compounds and structures as shown in formula (C) p The compound reacts with the addition of a structure as shown in formula (A) n Compounds and structures as shown in formula (D) q The compound was further reacted to give a peptide with the structure shown in formula (I), wherein A n B m : C p :D q The molar ratio is (0.8~1.2): (0.8~1.2): (0.8~1.2): (0.8~1.2); or, , Using methanol as the reaction solvent, the structure is as shown in formula (E x Compounds and structures as shown in formula (C) p The compound reacts with the addition of a structure as shown in formula (A) n Compounds and structures as shown in formula (D) q The compound was further reacted to give a peptide with the structure shown in formula (II), wherein A n E x : C p :D q The molar ratio is (1.8~2.2): (0.8~1.2): (1.8~2.2): (1.8~2.2); or, , Using methanol as the reaction solvent, the structure is as shown in formula (F y Compounds and structures as shown in formula (C) p The compound reacts with the addition of a structure as shown in formula (A) n Compounds and structures as shown in formula (D) q The compound was further reacted to give a peptide with the structure shown in formula (III), wherein A n : F y : C p :D q The molar ratio is (2.8~3.2): (0.8~1.2): (2.8~3.2): (2.8~3.2); The definitions of R1, R2, R3, R4, R5, and R6 are the same as those previously defined.

7. A composition, characterized in that, The composition contains the peptide as described in claim 1 or a pharmaceutically acceptable salt, ester, stereoisomer, solvate, hydrate, metabolite, metabolic precursor or prodrug as the active ingredient.

8. A reagent for reversing the anticoagulant activity of an anticoagulant, characterized in that, The reagent contains the peptide according to any one of claims 1 to 5 or the composition according to claim 7.

9. The use of the peptide according to any one of claims 1 to 5, the composition according to claim 7, or the reagent according to claim 8 in the preparation of a medicament for reversing the anticoagulant activity of an anticoagulant.

10. The application according to claim 9, characterized in that, The anticoagulant is unfractionated heparin, low molecular weight heparin, or ultra-low molecular weight heparin.

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