Methods of treating anticoagulant-induced bleeding using anticoagulant reversal agents

Abstract

The present invention provides a method for preventing, treating or alleviating bleeding caused by an anticoagulant using an anticoagulant reversal agent. The anticoagulant reversal agent has a structure as shown in formula I, can well inhibit bleeding complications caused by anticoagulants, for example, bleeding complications caused by antiplatelet drugs and other anticoagulants, and has the characteristics of broad spectrum and high efficiency.

Description

[0001] This application claims priority to the Chinese patent application No. 202411218249.X, filed on August 30, 2024, and entitled “Method of treating anticoagulant-induced bleeding using an anticoagulation reversal agent”, the entire content of which is incorporated herein by reference. TECHNICAL FIELD

[0002] The present application relates to the technical field of pharmaceutical chemistry, and in particular to a method of preventing, treating or alleviating anticoagulant-induced bleeding using an anticoagulation reversal agent. BACKGROUND

[0003] At present, the number of patients with cardiovascular diseases worldwide is about 423 million, and a considerable number of patients need to use anticoagulant drugs for treatment. Commonly used anticoagulants include: 1. Vitamin K antagonists, which can reduce the synthesis of prothrombin and factors VIIIX and X in the liver by antagonizing vitamin K to achieve anticoagulation, and the representative drug is warfarin; 2. Thrombin indirect inhibitors, which can indirectly inhibit the activity of Xa and IIa factors by interacting with antithrombin to achieve anticoagulation, and the representative drugs include heparin, low molecular weight heparin, etc.; 3. Thrombin direct inhibitors, which can inhibit thrombin to prevent fibrinogen from being cleaved into fibrin, thereby blocking the last step of the coagulation cascade and thrombus formation; among them, monovalent thrombin inhibitors (dabigatran, argatroban) can directly inhibit thrombin, and bivalent thrombin inhibitors (bivalirudin, recombinant hirudin) can directly inhibit thrombin and separate thrombin and fibrin to achieve anticoagulation; 4. Xa factor inhibitors such as apixaban, rivaroxaban, edoxaban, omacaban, and fondaparinux.

[0004] However, anticoagulant therapy is a double-edged sword. On the one hand, it is the basis for the prevention and treatment of thrombotic diseases and can significantly reduce the probability of thrombotic events, but on the other hand, it can also lead to bleeding complications, which can be life-threatening in severe cases. Therefore, there is a clinical need for an antagonist that can quickly and effectively reverse the anticoagulant effect to provide safer protection for the majority of patients undergoing anticoagulant therapy. There are few anticoagulant reversal agents on the market and in research. The ones that have been marketed include vitamin K for warfarin reversal, protamine for heparin, monoclonal antibody Idarucizumab for dabigatran, and Andexxa for Xa factor inhibitor rivaroxaban / apixaban. Patent WO2013082210A1 discloses an anticoagulant reversal agent, diarginine piperazine (DAP, PER977), which can reverse the anticoagulant effect of heparin, heparin fragments, fondaparinux, and factor Xa or factor IIa inhibitors (e.g., oral factor Xa or factor IIa inhibitors). It is currently in clinical phase III.

[0005] At present, there is a lack of efficient methods for preventing, treating or alleviating the bleeding caused by anticoagulants in the clinic. SUMMARY

[0006] Therefore, the present application aims to provide a method for preventing, treating or alleviating the bleeding caused by anticoagulants using an anticoagulant reversal agent.

[0007] The present application provides a method for preventing, treating or alleviating the bleeding caused by anticoagulants by administering, taking or using a compound having the structure shown in Formula I or a salt thereof or a deuterium compound thereof; the anticoagulant is one or more of a direct thrombin inhibitor, a factor Xa inhibitor and an anti-platelet drug.

[0008]

[0009] wherein X, X', X" are independently selected from substituted or unsubstituted alkyl, alkenyl or heterocyclic group;

[0010] Y, Y', Y" are independently selected from substituted or unsubstituted alkyl, alkenyl or heterocyclic group;

[0011] Z, Z', Z" are independently selected from a heteroatom-containing molecular fragment that can be protonated under physiological conditions.

[0012] Preferably, the number of carbon atoms in the alkyl group is 1-10, more preferably 1-6, and further preferably 2, 3, 4 or 5.

[0013] Preferably, the number of carbon atoms in the alkenyl group is 2-10, more preferably 2-6, and further preferably 2, 3, 4 or 5.

[0014] Preferably, the number of carbon atoms in the heterocyclic group is 2-12, and the heterocyclic group is more preferably a monocyclic heterocyclic group, and further preferably a five- or six-membered heterocyclic group. The heteroatom in the heterocyclic group is preferably one or more of N, O or S.

[0015] Preferably, the substituent of the alkyl group, the alkenyl group or the heteroaryl group is independently selected from one or more of amino, nitro or halogen, and more preferably amino.

[0016] Preferably, Y-Z, Y'-Z' and Y"-Z" are independently selected from the residue of a basic amino acid; more preferably, the residue of the basic amino acid is the residue of histidine, arginine or lysine.

[0017] In some embodiments, the residue refers to the group remaining after the carboxyl group of an amino acid is removed.

[0018] Preferably, the heteroatom-containing molecular fragment is selected from amino, guanidino or imidazolyl.

[0019] Preferably, the compound has the structure shown in Formula I-a:

[0020]

[0021] wherein R1, R2, R3, R4, R5, R6 are independently selected from substituted or unsubstituted C1-C6 alkyl, C2-C6 alkenyl or monocyclic heterocyclyl, more preferably C2-C5 alkyl, C2-C5 alkenyl or monocyclic heterocyclyl, further preferably methylene, ethylene, n-propylene, i-propylene, n-butylene, i-butylene, t-butylene, n-pentylene, i-pentylene, ethenylene, propenylene, butenylene, pyrrolylene, pyridylene, thienylene or furanylene.

[0022] Preferably, the compound has the structure shown in Formula I-a:

[0023]

[0024] Preferably, the compound has the structure shown in Formula I-a:

[0025]

[0026] Preferably, the anticoagulant is one or more of a direct thrombin inhibitor, a factor Xa inhibitor and an anti-platelet agent, more preferably the direct thrombin inhibitor is dabigatran, bivalirudin, argatroban, recombinant hirudin, the factor Xa inhibitor is apixaban, rivaroxaban, edoxaban, otamixaban, fondaparinux; and the anti-platelet agent is aspirin.

[0027] The present application also provides a use of a compound having the structure shown in Formula I or a salt thereof or a deuterated compound thereof in the preparation of a medicament for preventing, treating or alleviating bleeding caused by an anticoagulant, wherein the anticoagulant is one or more of a direct thrombin inhibitor, a factor Xa inhibitor and an anti-platelet agent;

[0028]

[0029] wherein X, X', X" are independently selected from substituted or unsubstituted alkyl, alkenyl or heterocyclyl;

[0030] Y, Y', Y" are independently selected from substituted or unsubstituted alkyl, alkenyl or heterocyclyl;

[0031] Z, Z', Z" are independently selected from a heteroatom-containing molecular fragment that can be protonated under physiological conditions.

[0032] Preferably, the number of carbon atoms in the alkyl group is 1-10, more preferably 1-6, further preferably 2, 3, 4 or 5.

[0033] Preferably, the number of carbon atoms of the alkenyl group is 2 to 10, more preferably 2 to 6, and further preferably 2, 3, 4, or 5.

[0034] Preferably, the number of carbon atoms of the heterocyclic group is 2 to 12, and the heterocyclic group is more preferably a monocyclic heterocyclic group, and further preferably a five- or six-membered heterocyclic group. The heteroatom of the heterocyclic group is preferably one or more of N, O, and S.

[0035] Preferably, the substituent of the alkyl group, the alkenyl group, or the heteroaryl group is independently selected from one or more of an amino group, a nitro group, and a halogen, and more preferably an amino group.

[0036] Preferably, Y-Z, Y'-Z', and Y"-Z" are independently selected from the residue of a basic amino acid; more preferably, the residue of the basic amino acid is the residue of histidine, arginine, or lysine.

[0037] In some embodiments, the residue refers to the group remaining after the carboxyl group of an amino acid is removed.

[0038] Preferably, the heteroatom-containing molecular fragment is selected from an amino group, a guanidino group, or an imidazolyl group.

[0039] Preferably, the compound has the structure shown in Formula I-a:

[0040]

[0041] wherein R1, R2, R3, R4, R5, and R6 are independently selected from a substituted or unsubstituted C1-C6 alkyl group, a C2-C6 alkenyl group, or a monocyclic heterocyclic group, and more preferably a C2-C5 alkyl group, a C2-C5 alkenyl group, or a monocyclic heterocyclic group, and further preferably a methylene group, an ethylene group, a n-propylene group, an i-propylene group, a n-butylene group, an i-butylene group, a t-butylene group, a n-pentylene group, an i-pentylene group, an ethenylene group, a propenylene group, a butenylene group, a pyrrolylene group, a pyridylene group, a thiophenylene group, or a furanylene group.

[0042] Preferably, the compound has the structure shown in Formula I-a:

[0043]

[0044] Preferably, the compound has the structure shown in Formula I-a:

[0045]

[0046] In one embodiment, the direct thrombin inhibitor is dabigatran, bivalirudin, argatroban, or recombinant hirudin; the factor Xa inhibitor is apixaban, rivaroxaban, edoxaban, otamixaban, or fondaparinux; and the anti-platelet agent is aspirin.

[0047] The present application also provides a kit comprising the above-mentioned medicament for preventing, treating or alleviating bleeding caused by an anticoagulant.

[0048] The present application also provides a kit comprising the above-mentioned compound or salt thereof or deuterated compound thereof and one or more anticoagulants; preferably, the anticoagulant is one or more of a direct thrombin inhibitor, a factor Xa inhibitor and an anti-platelet drug; more preferably, the direct thrombin inhibitor is dabigatran, bivalirudin, argatroban, recombinant hirudin, the factor Xa inhibitor is apixaban, rivaroxaban, edoxaban, otamixaban, fondaparinux; and the anti-platelet drug is aspirin. Compared with the prior art, the present application provides a method for preventing, treating or alleviating bleeding caused by an anticoagulant using an anticoagulant reversal agent. The anticoagulant reversal agent has the structure shown in Formula I or a salt thereof or a deuterated compound thereof, and can effectively inhibit bleeding complications caused by an anticoagulant, such as bleeding complications caused by a direct thrombin inhibitor, a factor Xa inhibitor, aspirin, dabigatran, bivalirudin, argatroban, recombinant hirudin, apixaban, rivaroxaban, edoxaban, otamixaban, fondaparinux, and has the characteristics of broad spectrum and high efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 is a mass spectrum of compound 1;

[0050] Figure 2 is a nuclear magnetic hydrogen spectrum of compound 1. DETAILED DESCRIPTION

[0051] In order to further illustrate the present application, the method for preventing, treating or alleviating bleeding caused by an anticoagulant using an anticoagulant reversal agent provided by the present application is described in detail below in conjunction with examples.

[0052] Example 1

[0053] First step:

[0054]

[0055] Operation steps

[0056] 1. Add DMF (500 mL) to a 1000 mL three-necked flask and start stirring;

[0057] 2. Control the temperature at 0-10°C and slowly add NaH (27.6 g);

[0058] 3. Control the temperature at 0-10°C and slowly add xanthine (21 g);

[0059] 4. Stir at room temperature for 1 hour;

[0060] 5. Slowly add N-Boc-bromopropylamine (115 g);

[0061] 6. Stop stirring at room temperature overnight;

[0062] Work-up:

[0063] 7. Add the reaction solution to 1000 mL of ice water and extract with 1000 mL of dichloromethane (1000 mL*2 twice);

[0064] 8. Wash the organic phase with 500 mL of saturated brine twice (500 mL*2);

[0065] 9. Concentrate the organic phase to dryness;

[0066] 10. Purify the concentrated residue with silica gel column chromatography (eluent: ethyl acetate / methyl tert-butyl ether = 1 / 1);

[0067] 11. Concentrate the product-containing fraction to dryness;

[0068] 12. Obtain a colorless oil.

[0069] Second step:

[0070]

[0071] Operation steps:

[0072] 1. Add dioxane to the bottle containing CG659A oil and stir to dissolve;

[0073] 2. Control the temperature at 10±5°C and slowly add dioxane hydrochloric acid solution;

[0074] 3. Control the temperature at 20±5°C and stir for 16 h;

[0075] 4. Precipitate the solid product;

[0076] Work-up:

[0077] 5. Filter under nitrogen protection, and wash the filter cake with dioxane (25 mL*2);

[0078] 6. Add the above filter cake to methyl tert-butyl ether (150 mL) and stir at room temperature for 1 h;

[0079] 7. Filter under nitrogen protection, and wash the filter cake with methyl tert-butyl ether (25 mL).

[0080] Third step:

[0081]

[0082] Operation steps

[0083] 1. In a 100 mL three-necked flask, add Boc-L-Arg(Pbf)-OH (CG659C, 2.0 g, 3.3 eq), DMF (30 mL), and DIPEA (1.5 g, 10 eq) under stirring;

[0084] 2. Cool to 0±5°C, and add HATU (1.5 g, 3.3 eq) and keep for 30 min;

[0085] 3. Control the temperature at 0±5°C, and add CG659-B (0.5 g, 1.0 eq);

[0086] 4. Raise to room temperature, and react overnight (16 h);

[0087] 5. Add water (30 mL) to the reaction solution, and extract the solid by suction filtration;

[0088] 6. Dissolve in ethyl acetate, wash with water, and concentrate to dryness to obtain 1.9 g of a thick substance

[0089] 7. Extract the filtrate with methyl tert-butyl ether (2*30 mL);

[0090] 8. Combine the organic phases, and wash with water (25 mL);

[0091] 9. Concentrate the organic phase to dryness to obtain 0.3 g of an oily substance.

[0092] Fourth Step:

[0093]

[0094] Operation Steps

[0095] 1. In a 100 mL three-necked flask, add a CG659D / DCM (0.5 g / 3 mL) solution;

[0096] 2. Add TFA (1 mL, 50 eq), water (29.2 mg, 6.0 eq), and TIS (141 mg, 3.3 eq) at room temperature;

[0097] 3. Stir at room temperature overnight, and supplement with TFA (2 mL) and continue stirring for 5 h.

[0098] Post-treatment:

[0099] 4. Concentrate to remove DCM and most of the TFA in the system to obtain 0.7 g of a crude product;

[0100] 5. Add anhydrous ethanol (10 mL) under stirring, and extract the solid by stirring for 1 h;

[0101] 6. Filter by suction, wash the filter cake with MTBE (2×2mL) to obtain 0.3g of off-white solid product, add 3mL of water, adjust the pH to 8-9 with 5% NaOH aqueous solution, extract with DCM (2×10mL) to obtain 0.06g of oily substance, remove water in batches with 100mL of toluene, add 50mL of isopropanol to dissolve and filter, evaporate the filtrate to dryness to obtain about 0.2g of oily liquid, the product is designated as compound 1.

[0102] Structural characterization:

[0103] The structure of the product was characterized by mass spectrometry and nuclear magnetic resonance, and the results are shown in the figure. Figure 1 , Figure 2 As shown.

[0104] Mass spectrometry results: [M+H] + 792.5.

[0105] Depend on Figure 1 Based on the molecular ion peaks obtained from medium- and low-resolution mass spectrometry, the molecular weight of the free base of this product is 791.5. Furthermore, according to the nitrogen rule, the accurate molecular weight of the product is an odd number, consistent with the chemical formula of the target compound.

[0106] The structure of the product was verified by NMR results.

[0107] Example 2 Antagonism of Apixaban

[0108] Rabbit liver laceration and hemorrhage model: Except for the control group and the compound 10.5 mg / kg + NS control group, apixaban was administered by gavage. One hour later, chloral hydrate 1 ml / kg was injected intravenously for anesthesia. The anesthetized animals were placed on a preheated electric blanket at 37°C. Physiological saline, PER977 or different doses of compound 1 were injected into the ear vein. Five minutes after administration, the abdominal cavity was opened, and three parallel standard incisions of 0.5 cm in length and 2 mm in depth were made at the lower edge of the left lobe of the liver. Blood flowing from the liver incisions was wiped with pre-weighed dry cotton balls. The weight of the cotton balls was weighed again after 5 minutes. The difference between the two weights was the bleeding loss (BL) at 5 minutes.

[0109] Rabbit ear margin bleeding model: Except for the Control group and the Compound 10.5 mg / kg + N.S. control group, apixaban was administered by gavage, and 1 h later, normal saline, PER977 or different doses of Compound 1 were injected intravenously at the ear margin. Two minutes after administration, a 1 cm long incision was made along the ear margin 3 cm from the ear tip under the heating of a lamp for 5 min, and the BL (Bleeding loss) was recorded for 5 min. Ten minutes after the surgical incision, 1 ml of blood was collected from the jugular vein without anticoagulant and injected into a 37°C preheated test tube at 1 ml per tube. The stopwatch was immediately started, and the coagulation state of the blood in the test tube was observed every 30 s in a 37°C water bath until the blood coagulated. The time recorded by the stopwatch was the WBCT.

[0110] The results are shown in Table 1.

[0111] Table 1: Effect of Compound 1 on the bleeding volume induced by apixaban in rabbits

[0112] Group n Amount of liver laceration bleeding (g) Amount of ear bleeding (g) WBCT (s) Control (N.S.) 8 0.7333±0.2785 2.30±1.48 243.75±38.06 Vehicle (Apixaban) 8 2.6514 ± 0.8091 ** ]] 7.07 ± 1.00 *** ]] 468.75±39.51*** PER977 20 mg / kg + Apixaban 8 0.6487 ± 0.3366 ## ]] 2.83 ± 1.85 ### ]] 318.75 ± 44.84 ### ]] Compound 1 0.02 mg / kg + Apixaban 8 3.3005±3.0503 5.63±1.23 435.00±51.96 Compound 1 0.1 mg / kg + Apixaban 8 1.2462±0.7622 4.00 ± 1.63 # ]] 378.75 ± 44.84 ## ]] Compound 1 0.5 mg / kg + Apixaban 8 0.7991 ± 0.4202 ## ]] 1.80 ± 1.76 ### ]] 315.00 ± 30.00 ### ]] Compound 1 0.5 mg / kg + N.S. 8 0.6496±0.2403 1.97±1.30 243.75±34.98

[0113] **P<0.01, ***P<0.001 vs. Control; #P<0.05, ##P<0.01, ###P<0.001 vs. Vehicle

[0114] Analysis of experimental results:

[0115] Compared with Control, the liver tearing and ear bleeding induced bleeding volume of the animals in the Vehicle group were significantly increased (P<0.01, P<0.001), increased by 2.6 times and 2.1 times, respectively, and the WBCT was also significantly prolonged (P<0.001).

[0116] Compared with the Vehicle group, Compound 1 0.1, 0.5 mg / kg significantly reduced the ear bleeding and liver tearing induced bleeding volume in rabbits (P<0.05, P<0.001), and also significantly shortened the WBCT (P<0.01, P<0.001).

[0117] Compared with Control, the bleeding volume and WBCT of the Compound 1 control group (Compound 1 0.5 mg / kg + N.S.) showed no significant change, indicating that the administration of Compound 1 did not cause changes in the coagulation indicators and bleeding volume of normal animals.

[0118] Example 3 Antagonism of dabigatran etexilate

[0119] Rat liver laceration model: Except for the Control group and the Compound 1 10 mg / kg + N.S control group, Dabigatran (30 mg / kg) was administered by gavage, 1.5 h later, 0.3 mL / 100 g body weight of 10% chloral hydrate was injected intraperitoneally, and the anesthetized animals were placed on a preheated electric blanket at 37°C. The jugular vein cannulation was completed within 30 min, 2 min later, normal saline, PER977 or different doses of Compound 1 were injected through the jugular vein cannula, 5 min after administration, three standard incisions of 1 cm long and 2 mm deep were made on the liver, and the blood flowing out of the liver was wiped with pre-weighed dry cotton, and the bleeding loss (BL) was recorded.

[0120] Rat tail transection bleeding model: Except for the Control group and the Compound 1 10 mg / kg + N.S control group, Dabigatran (30 mg / kg) was administered by gavage, 30 min later, 0.3 mL / 100 g body weight of 10% chloral hydrate was injected intraperitoneally, and the anesthetized animals were placed on a preheated electric blanket at 37°C. The jugular vein cannulation was completed within 30 min, 2 min later, normal saline, PER977 or different doses of Compound 1 were injected through the jugular vein cannula, 5 min after administration, an incision was made on the rat tail 5 mm from the tail end, and the BL (Bleeding loss) was recorded for 15 min. 2.5 mL of blood was taken from the inferior vena cava, 0.5 mL was separated, and no anticoagulant was added. It was injected into a 37°C preheated test tube, 0.5 mL per tube, and the stopwatch was immediately started. The coagulation state of the blood in the test tube was observed every 30 s in a 37°C water bath until the blood coagulated. The time recorded by the stopwatch was the WBCT.

[0121] The results are shown in Table 2.

[0122] Table 2: Effect on rat bleeding volume induced by Dabigatran

[0123] Group n Amount of tail transection bleeding (g) Amount of liver laceration bleeding (g) WBCT (s) Control (N.S.) 8 0.0154±0.0051 0.0323±0.0191 263±42 Vehicle (Dabigatran) 9 0.0856 ± 0.0474 * ]] 0.5665 ± 0.2039 ** ]] 720 ± 212 ** ]] PER977 20 mg / kg + Dabigatran 7 0.0161 ± 0.0067 # ]] 0.0635 ± 0.0469 ## ]] 583±169 Compound 1 2.5 mg / kg + Dabigatran 8 0.0663±0.0707 0.6368±0.1505 724±204 Compound 1 5 mg / kg + Dabigatran 8 0.0257±0.0178 0.3441±0.0998 664±165 Compound 1 10 mg / kg + Dabigatran 8 0.0083 ± 0.0039 # ]] 0.0500 ± 0.0299 ## ]] 626±182 Compound 1 10 mg / kg + N.S. 8 0.0118±0.0074 0.0152±0.0080 259±55

[0124] *P <0.05, **P <0.01 vs. Control; #P <0.05, ##P <0.01 vs. Vehicle

[0125] Analysis of experimental results:

[0126] Compared with Control, the rat bleeding volume induced by tail transection and liver laceration in the Vehicle group was significantly increased (P <0.05, P <0.01), increased by 4.6 times and 16.5 times, respectively, and the WBCT was also significantly prolonged (P <0.01).

[0127] Compound 110mg / kg could significantly reduce the increase of bleeding volume induced by tail transection and liver laceration in rats (P<0.05, P<0.01) compared with Vehicle group, and the inhibition rate of bleeding volume induced by liver laceration reached 91.2%; Compound 1 also had a certain effect on shortening WBCT, but there was no statistical difference.

[0128] Compared with Control, the bleeding volume and WBCT of Compound 1 control group (Compound 110mg / kg + N.S.) did not change significantly, indicating that the administration of Compound 1 would not cause changes in coagulation indicators and bleeding volume of normal animals.

[0129] Example 4 Antagonizing Aspirin

[0130] Rat liver laceration bleeding model: Except for the Control group and the Compound 15.0mg / kg + N.S. control group, aspirin (Asp.) was administered by gavage, and 7 days later, 0.3mL / 100g body weight of 10% chloral hydrate was injected intraperitoneally. The anesthetized animals were placed on a preheated electric blanket at 37°C, and the jugular vein was cannulated within 30min. After 2min, normal saline, PER977 or different doses of Compound 1 were injected through the jugular vein cannula. Five minutes after administration, three standard incisions of 1cm in length and 2mm in depth were made on the liver, and the blood flowing out of the liver was wiped with pre-weighed dry cotton. The bleeding volume BL (Bleeding loss, BL) was recorded.

[0131] Rat tail transection bleeding model: Except for the Control group and the Compound 15.0mg / kg + N.S. control group, aspirin (Asp.) was administered by gavage, and 7 days later, 0.3ml / 100g 10% chloral hydrate was injected intraperitoneally. The anesthetized animals were placed on a preheated electric blanket at 37°C, and the jugular vein was cannulated within 30min. Normal saline, PER977 or different doses of Compound 1 were administered, and 5min after administration, an incision was made on the rat tail 5mm from the tail end. The BL (Bleeding loss) was recorded for 15min. 2.5mL of blood was taken from the inferior vena cava, and 0.5mL was separated. The blood without anticoagulant was injected into a 37°C preheated test tube at 0.5mL per tube. The stopwatch was started immediately, and the coagulation state of the blood in the test tube was observed every 30s in the 37°C water bath until the blood coagulated. The time was recorded with the stopwatch.

[0132] The results are shown in Table 3.

[0133] Table 3: Effect on bleeding volume of rats induced by aspirin

[0134] Group n Amount of tail transection bleeding WBCT n Amount of liver laceration bleeding Control (N.S.) 12 0.0165±0.0115 277.5±42.7 8 0.0562±0.0186 Vehicle (Asp.) 12 0.1068±0.0316*** 317.5±69.4 8 0.3930±0.1791* PER977 20 mg / kg + Asp. 12 0.1310±0.1136 285.0±62.0 8 0.3809±0.1275 Compound 1 0.2 mg / kg + Asp. 12 0.1030±0.0619 300.0±51.2 8 0.3726±0.1110 Compound 1 1.0 mg / kg + Asp. 12 0.0651±0.0730 292.5±29.0 8 0.1482±0.0578 Compound 1 5.0 mg / kg + Asp. 12 0.0186 ± 0.0079 ### ]] 262.5 ± 49.7 # ]] 8 0.0536 ± 0.0180 # ]] Compound 1 5.0 mg / kg + N.S. 12 0.0263±0.0233 265.0±38.0 8 0.0500±0.0162

[0135] *P<0.05, ***P<0.001 vs. Control; #P<0.05, ###P<0.001 vs. Vehicle

[0136] Analysis of experimental results:

[0137] Compared with Control, the bleeding volume of tail transection and liver laceration in Vehicle group was significantly increased (P<0.001, P<0.05), the bleeding volume of tail transection model was increased by 5.5 times, the bleeding volume of liver laceration model was increased by 6 times, and WBCT was prolonged to a certain extent.

[0138] Compared with Vehicle group, Compound 1 5.0 mg / kg could significantly reduce the bleeding volume of tail transection and liver laceration models (P<0.001, P<0.05), and could also significantly shorten WBCT (P<0.05).

[0139] Compared with Control, the bleeding volume and WBCT of Compound 1 control group (Compound 1 5.0 mg / kg + N.S.) were not significantly changed, suggesting that Compound 1 would not cause changes in bleeding volume and coagulation indicators of normal animals.

[0140] Example 5 Antagonism of aspirin

[0141] Rabbit liver laceration bleeding model: Except for Control group and Compound 1 2.1 mg / kg + N.S. control group, aspirin (Asp.) was administered by gavage, and 7 days later, 1 ml / kg of chloral hydrate was injected intravenously 30 min after the last gavage to anesthetize the animals. The anesthetized animals were placed on a 37°C preheated electric blanket, and 2 min later, physiological saline and different doses of Compound 1 were injected into the marginal ear vein. Five minutes after administration, three standard incisions were made on the lower edge of the left lobe of the liver, and the bleeding volume (Bleeding loss, BL) was calculated by weighing the dry cotton balls before and after the bleeding. 1 ml of blood was taken from the inferior vena cava and injected into a 37°C preheated test tube. The coagulation state of the blood in the test tube was observed every 30 s in a 37°C water bath until the blood coagulated, and the time was recorded (WBCT) using a stopwatch.

[0142] The results are shown in Table 4.

[0143] Table 4 Effect on bleeding volume and coagulation function of rabbits induced by aspirin

[0144] Group n WBCT Amount of liver laceration bleeding Control (N.S.) 8 577.5±31.1 0.7745±0.1795 Vehicle (Asp.) 8 675.0 ± 62.1 *** ]] 2.0315 ± 0.1407 *** ]] Compound 1 0.7 mg / kg + Asp. 8 656.3±37.4 1.9115±0.1457 Compound 1 1.4 mg / kg + Asp. 8 630.0±50.7 1.7714±0.3466 Compound 1 2.1 mg / kg + Asp. 8 592.5 ± 50.1 ## ]] 1.0021 ± 0.5710 # ]] Compound 1 2.1 mg / kg + N.S. 8 570.0±35.9 0.5362±0.2735

[0145] ***P < 0.001 vs. Control; #P < 0.05, ##P < 0.01 vs. Vehicle

[0146] Analysis of experimental results:

[0147] Compared with Control, the WBCT of the Vehicle group was significantly prolonged (P < 0.001), and the bleeding volume of the Vehicle group was also significantly increased (P < 0.001), and the bleeding volume of the rabbit liver tear model was increased by 1.6 times.

[0148] Compared with the Vehicle group, Compound 1 2.1 mg / kg could significantly shorten the WBCT (P < 0.01); Compound 1 2.1 mg / kg could also significantly reduce the bleeding volume caused by liver tear (P < 0.05), and the reduction rate was 50.6%.

[0149] Compared with Control, the WBCT and bleeding volume of the Compound 1 control group (Compound 1 2.1 mg / kg + N.S.) showed no obvious change, indicating that the administration of Compound 1 would not cause changes in the coagulation indicators and bleeding volume of normal animals.

[0150] Example 6 Antagonizing rivaroxaban

[0151] Rat liver tear model: Except for the Control group and the Compound 1 0.13 mg / kg + N.S. control, rivaroxaban (Riv) was administered by gavage, 10% chloral hydrate was injected intraperitoneally, and the anesthetized animals were placed on a preheated electric blanket at 37°C. The jugular vein was isolated and cannulated within 30 min, and 30 min after the administration of rivaroxaban, normal saline, PER977 or different doses of Compound 1 were injected into the jugular vein. Five minutes after administration, the abdominal cavity was opened, three standard incisions of 1 cm long and 2 mm deep were made on the liver, and the blood flowing out of the liver was wiped with pre-weighed dry cotton. The bleeding loss (BL) was recorded for 30 min.

[0152] Rat tail transection bleeding model: except Control group, Compound 1 0.13 mg / kg + N.S. control, Rivastigmine (Riv) was given by gavage, the animals after anesthesia were placed on a 37℃ preheated electric blanket, the jugular vein was separated and cannulated within 30 min, 30 min after the administration of Rivastigmine, normal saline, PER977 or different doses of Compound 1 were injected into the jugular vein, 5 min after the administration, an incision was made at 5 mm from the tail tip of the rat tail, and the bleeding loss (BL) was recorded for 15 min. The temperature was strictly controlled during the experiment, including the room temperature and the temperature of the water bath. 3 mL of blood was collected from the abdominal aorta, which was divided into two parts, 2 mL and 1 mL. 2 mL of blood was quickly injected into an EP tube containing 0.2 mL of anticoagulant (3.8% sodium citrate solution), and mixed gently by inverting up and down, and centrifuged at 3500 rpm for 10 min to obtain platelet poor plasma (PPP). The automatic coagulation analyzer was used to detect PT, TT, APTT, and the results were recorded at the end of the test. The other part of the blood was used to determine the whole blood clotting time (WBCT).

[0153] The results are shown in Table 5.

[0154] Table 5: Effect on rivastigmine-induced bleeding in rats

[0155]

[0156] *P<0.05, **P<0.01, ***P<0.001 vs. Control; #P<0.05, ##P<0.01, ###P<0.001 vs. Vehicle

[0157] Analysis of experimental results:

[0158] Compared with Control, the APTT, PT, TT and WBCT of the animals in the Vehicle group were significantly prolonged (P<0.01, P<0.001), among which the APTT was prolonged by 0.99 times, the tail transection bleeding of the animals in the Vehicle group was also significantly increased (P<0.05), the bleeding was increased by 4.25 times, the liver tear bleeding of the animals in the Vehicle group was significantly increased (P<0.01), the bleeding was increased by 10.35 times.

[0159] Compared with the Vehicle group, compound 10.005, 0.026, 0.13 mg / kg can dose-dependently shorten APTT (P<0.01, P<0.001), dose-dependently shorten WBCT (P<0.05, P<0.001), and dose-dependently reduce the amount of bleeding, wherein 0.026, 0.13 mg / kg have the most obvious effect (P<0.05, P<0.01), and the amount of tail transection bleeding is reduced by 79.9%, 80.8%, and the amount of liver laceration bleeding is reduced by 81.3%, 89.0%.

[0160] Compared with the Control, the APTT, PT, TT, WBCT and the amount of bleeding of the compound 1 control group (only given compound 10.13 mg / kg) do not change obviously, which indicates that giving compound 1 does not cause the change of the coagulation index and the amount of bleeding of normal animals.

[0161] The above test results show that the compound prepared in the application has a better anticoagulation reversal effect than PER977.

[0162] The above examples are only used to help understand the method of the application and its core idea. It should be noted that for those skilled in the art, some improvements and modifications can be made to the application without departing from the principles of the application, and these improvements and modifications also fall within the protection scope of the claims of the application.

Claims

1. A method for preventing, treating or alleviating bleeding caused by an anticoagulant, characterized in that, administering, taking or using a compound having a structure of Formula I or a salt thereof or a deuterated compound thereof; the anticoagulant is an anti-platelet agent; X, X', X" are independently selected from substituted or unsubstituted alkyl, alkenyl or heterocyclic group; Y, Y', Y" are independently selected from substituted or unsubstituted alkyl, alkenyl or heterocyclic group; Z, Z', Z" are independently selected from heteroatom-containing molecular fragments that can be protonated under physiological conditions.

2. The method of claim 1, wherein, The number of carbon atoms of the alkyl group is 1-10, the number of carbon atoms of the alkenyl group is 2-10, and the number of carbon atoms of the heterocyclic group is 2-12.

3. The method of claim 1, wherein, Y-Z, Y'-Z', Y"-Z" are independently selected from the residue of basic amino acid; preferably, the residue of basic amino acid is the residue of histidine, arginine or lysine.

4. The method of claim 1, wherein, The heteroatom-containing molecular fragment is selected from amino group, guanidino group or imidazolyl group.

5. The method of claim 1, wherein, The compound has a structure of Formula I-a: R1, R2, R3, R4, R5, R6 are independently selected from substituted or unsubstituted C1-C6 alkyl, C2-C6 alkenyl or monocyclic heterocyclic group.

6. The method of claim 1, wherein, The compound has the following structure:

7. The method of claim 1, wherein, The compound has the following structure:

8. The method according to any one of claims 1 to 7, wherein, The anti-platelet agent is aspirin.

9. A kit comprising a compound used in any of the methods of claims 1-7 or a salt thereof or a deuterated compound thereof and one or more anticoagulants; preferably, the anticoagulant is an anti-platelet agent; more preferably, the anti-platelet agent is aspirin.

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