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 anticoagulants such as an Xa factor inhibitor, and has the characteristics of broad spectrum and high efficiency.

Description

[0001] This application claims priority to Chinese Patent Application No. 202411218249.X, filed on August 30, 2024, entitled “Method for treating bleeding caused by anticoagulants using an anticoagulant reversal agent”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of pharmaceutical chemistry, and more particularly to a method for preventing, treating, or reducing bleeding caused by anticoagulants using an anticoagulant reversal agent. Background Technology

[0003] Currently, there are approximately 423 million people worldwide suffering from cardiovascular diseases, a significant number of whom require anticoagulant medications for treatment. Commonly used anticoagulants include: 1. Vitamin K antagonists, which inhibit the synthesis of prothrombin and factors VII, IX, and X in the liver by antagonizing vitamin K, thus reducing coagulation. Warfarin is a representative drug. 2. Indirect thrombin inhibitors, which indirectly inhibit the activity of factors Xa and IIa through interaction with antithrombin, thus exerting an anticoagulant effect. Heparin and low molecular weight heparin are representative drugs. 3. Direct thrombin inhibitors, which inhibit thrombin, preventing fibrinogen from breaking down into fibrin, thus blocking the final step of the coagulation cascade and thrombus formation. Among them, monovalent thrombin inhibitors (dabigatran etexilate, argatroban) can directly inhibit thrombin, while divalent thrombin inhibitors (bivalirudin, recombinant hirudin) can directly inhibit thrombin and also separate thrombin and fibrin to achieve an anticoagulant effect. 4. Factor Xa inhibitors such as apixaban, rivaroxaban, edoxaban, omexaban, and fondaparinux sodium.

[0004] However, anticoagulation therapy is a double-edged sword. On the one hand, it is the foundation of treatment for the prevention and treatment of thrombotic diseases, significantly reducing the probability of thrombotic events. On the other hand, it can lead to bleeding complications, which can be life-threatening in severe cases. Therefore, there is a clinical need for an antagonist that can rapidly and effectively reverse anticoagulation to provide greater safety for patients undergoing anticoagulation therapy. Currently, there are few commercially available and investigational anticoagulation reversal drugs. Those already on the market include vitamin K (for warfarin), protamine sulfate (for heparin), idarucizumab (for dabigatran etexilate), and Andexxa (for the anticoagulation activity reversal of factor Xa inhibitors rivaroxaban / apixaban). Patent WO2013082210A1 discloses an anticoagulation reversal agent, diarginine piperazine (DAP, PER977), which can reverse the anticoagulation effects of heparin, heparin fragments, fondaparinux, and factor Xa or factor IIa inhibitors (e.g., oral factor Xa or factor IIa inhibitors), and is currently in phase III clinical trials.

[0005] Currently, there is a lack of effective methods in clinical practice to prevent, treat, or reduce bleeding caused by anticoagulants. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a method for preventing, treating or reducing bleeding caused by anticoagulants using an anticoagulant reversal agent.

[0007] This invention provides a method for preventing, treating, or reducing bleeding caused by anticoagulants, by administering, taking orally, or using a compound having the structure shown in Formula I, or a salt thereof, or a deuterated thereof; wherein the anticoagulant is one or more of a direct thrombin inhibitor, a factor Xa inhibitor, and an antiplatelet drug;

[0008]

[0009] Wherein, X, X', and X' are independently selected from substituted or unsubstituted alkyl, alkenyl, or heterocyclic groups;

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

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

[0012] Preferably, the alkyl group has 1 to 10 carbon atoms, more preferably 1 to 6, and even more preferably 2, 3, 4 or 5.

[0013] Preferably, the alkenyl group has 2 to 10 carbon atoms, more preferably 2 to 6, and even more preferably 2, 3, 4 or 5.

[0014] Preferably, the heterocyclic group has 2 to 12 carbon atoms, and more preferably, it is a monocyclic heterocyclic group, and even more preferably, it is a five- or six-membered heterocyclic group. The heteroatoms of the heterocyclic group are preferably one or more of N, O, and S.

[0015] Preferably, the substituents of the alkyl, alkenyl, or heteroaryl groups are independently selected from one or more of amino, nitro, and halogen groups, more preferably amino.

[0016] Preferably, YZ, Y'-Z', and Y”-Z” are independently selected from residues of basic amino acids; more preferably, the residues of basic amino acids are histidine, arginine, or lysine residues.

[0017] In some specific embodiments, the residue refers to the group remaining after an amino acid loses its carboxyl group.

[0018] Preferably, the heteroatom-containing molecular fragment is selected from amino, guanidinyl, or imidazole groups.

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

[0020]

[0021] Wherein, R1, R2, R3, R4, R5, and R6 are independently selected from substituted or unsubstituted C1-C6 alkyl, C2-C6 alkenyl, or monocyclic heterocyclic groups, more preferably C2-C5 alkyl, C2-C5 alkenyl, or monocyclic heterocyclic groups, and even more preferably methylene, ethylene, n-propylene, isopropylene, n-butylene, isobutylene, tert-butylene, n-pentylene, isopentylene, vinylene, propenylene, butenylene, pyrrolylene, pyridylene, thiophenylene, or furanylene.

[0022] Preferably, the compound has the following structure:

[0023]

[0024] Preferably, the compound has the following structure:

[0025]

[0026] Preferably, the anticoagulant is one or more of a direct thrombin inhibitor, a factor Xa inhibitor, and an antiplatelet drug; more preferably, the direct thrombin inhibitor is dabigatran etexilate, bivalirudin, argatroban, or recombinant hirudin; the factor Xa inhibitor is apixaban, rivaroxaban, edoxaban, omexaban, or fondaparinux sodium; and the antiplatelet drug is aspirin.

[0027] The present invention also provides the use of a compound having the structure shown in Formula I, or a salt thereof, or a deuterated product thereof, in the preparation of a medicament for the prevention, treatment, or reduction of bleeding caused by anticoagulants; wherein the anticoagulant is one or more of a direct thrombin inhibitor, a factor Xa inhibitor, and an antiplatelet drug;

[0028]

[0029] Wherein, X, X', and X' are independently selected from substituted or unsubstituted alkyl, alkenyl, or heterocyclic groups;

[0030] Y, Y', and Y' are independently selected from substituted or unsubstituted alkyl, alkenyl, or heterocyclic groups;

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

[0032] Preferably, the alkyl group has 1 to 10 carbon atoms, more preferably 1 to 6, and even more preferably 2, 3, 4 or 5.

[0033] Preferably, the alkenyl group has 2 to 10 carbon atoms, more preferably 2 to 6, and even more preferably 2, 3, 4 or 5.

[0034] Preferably, the heterocyclic group has 2 to 12 carbon atoms, and more preferably, it is a monocyclic heterocyclic group, and even more preferably, it is a five- or six-membered heterocyclic group. The heteroatoms of the heterocyclic group are preferably one or more of N, O, and S.

[0035] Preferably, the substituents of the alkyl, alkenyl, or heteroaryl groups are independently selected from one or more of amino, nitro, and halogen groups, more preferably amino.

[0036] Preferably, YZ, Y'-Z', and Y”-Z” are independently selected from residues of basic amino acids; more preferably, the residues of basic amino acids are histidine, arginine, or lysine residues.

[0037] In some specific embodiments, the residue refers to the group remaining after an amino acid loses its carboxyl group.

[0038] Preferably, the heteroatom-containing molecular fragment is selected from amino, guanidinyl, or imidazole groups.

[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 substituted or unsubstituted C1-C6 alkyl, C2-C6 alkenyl, or monocyclic heterocyclic groups, more preferably C2-C5 alkyl, C2-C5 alkenyl, or monocyclic heterocyclic groups, and even more preferably methylene, ethylene, n-propylene, isopropylene, n-butylene, isobutylene, tert-butylene, n-pentylene, isopentylene, vinylene, propenylene, butenylene, pyrrolylene, pyridylene, thiophenylene, or furanylene.

[0042] Preferably, the compound has the following structure:

[0043]

[0044] Preferably, the compound has the following structure:

[0045]

[0046] In one specific embodiment of the present invention, the direct thrombin inhibitor is dabigatran etexilate, bivalirudin, argatroban, or recombinant hirudin; the factor Xa inhibitor is apixaban, rivaroxaban, edoxaban, omexaban, or fondaparinux sodium; and the antiplatelet drug is aspirin.

[0047] The present invention also provides a kit comprising the above-described drugs for the prevention, treatment or reduction of bleeding caused by anticoagulants.

[0048] This invention also provides a kit comprising the above-mentioned compound or its salt or deuterated derivative, and one or more anticoagulants; preferably, the anticoagulant is one or more of a direct thrombin inhibitor, a factor Xa inhibitor, and an antiplatelet drug; more preferably, the direct thrombin inhibitor is dabigatran etexilate, bivalirudin, argatroban, or recombinant hirudin; the factor Xa inhibitor is apixaban, rivaroxaban, edoxaban, omexaban, or fondaparinux sodium; and the antiplatelet drug is aspirin. Compared with the prior art, this invention provides a method for preventing, treating, or reducing anticoagulant-induced bleeding using an anticoagulant reversal agent. This anticoagulant reversal agent has the structure shown in Formula I or its salt or deuterated derivative, and can effectively inhibit bleeding complications caused by anticoagulants. For example, it can inhibit bleeding complications caused by direct thrombin inhibitors, factor Xa inhibitors, aspirin, dabigatran etexilate, bivalirudin, argatroban, recombinant hirudin, apixaban, rivaroxaban, edoxaban, omexaban, and fondaparinux sodium, and has the characteristics of broad spectrum and high efficacy. Attached Figure Description

[0049] Figure 1 This is the mass spectrum of compound 1;

[0050] Figure 2 This is the 1H NMR spectrum of compound 1. Detailed Implementation

[0051] To further illustrate the present invention, the following describes in detail, with reference to embodiments, the method provided by the present invention for preventing, treating or reducing bleeding caused by anticoagulants using anticoagulant reversal agents.

[0052] Example 1

[0053] first step:

[0054]

[0055] Operating steps

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

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

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

[0059] 4. Bring to room temperature and stir for 1 hour;

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

[0061] 6. Stir overnight at room temperature, then stop.

[0062] Post-processing:

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

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

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

[0066] 10. The concentrated residue was purified by silica gel column chromatography (eluent: ethyl acetate / methyl tert-butyl ether = 1 / 1);

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

[0068] 12. A colorless oily substance was obtained.

[0069] Step Two:

[0070]

[0071] Operating steps:

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

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

[0074] 3. Control the temperature at 20±5℃ and stir for 16 hours;

[0075] 4. Precipitation of solid products;

[0076] Post-processing:

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

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

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

[0080] Step 3:

[0081]

[0082] Operating steps

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

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

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

[0086] 4. Bring to room temperature and react overnight (16 hours);

[0087] 5. Add water (30 mL) to the reaction solution, a solid precipitates, and filter under vacuum;

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

[0089] 7. Extract the filtrate with methyl ether (2 x 30 mL);

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

[0091] 9. The organic phase was concentrated to dryness to obtain 0.3g of oily substance.

[0092] Step 4:

[0093]

[0094] Operating steps

[0095] 1. Add CG659D / DCM (0.5g / 3mL) solution to a 100mL three-necked reaction flask;

[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, add TFA (2 mL), and continue stirring for 5 hours.

[0098] Post-processing:

[0099] 4. Concentration was used to remove DCM and most of the TFA from the system to obtain 0.7 g of crude product;

[0100] 5. While stirring, add anhydrous ethanol (10 mL), a solid will precipitate, and stir for 1 hour;

[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 marginal ear 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, physiological saline, PER977, or different doses of compound 1 were injected into the marginal ear vein. Two minutes after administration, the wound was heated with a heat lamp for 5 minutes. A 1 cm long incision was made along the marginal ear vein 3 cm from the ear tip, and the bleeding loss (BL) was recorded for 5 minutes. Ten minutes after the incision, 1 ml of blood was collected from the jugular vein without anticoagulant and injected into a preheated 37°C test tube (1 ml / tube). A stopwatch was immediately started, and the blood coagulation status in the test tube was observed every 30 seconds in a 37°C water bath until the blood coagulated. The time was recorded with a stopwatch, which is the WBCT.

[0110] The results are shown in Table 1.

[0111] Table 1: Effect of apixaban on hemorrhage volume in rabbits

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

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

[0114] Analysis of experimental results:

[0115] Compared with the Control group, the amount of hemorrhage induced by liver laceration and ear hemorrhage in rabbits was significantly increased (P<0.01, P<0.001), increasing 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 at 0.1 and 0.5 mg / kg significantly reduced the amount of bleeding induced by ear hemorrhage and liver laceration in rabbits (P<0.05, P<0.001) and significantly shortened the WBCT (P<0.01, P<0.001).

[0117] Compared with the control group (compound 1 0.5 mg / kg + NS), no significant changes were observed in bleeding volume and WBCT, suggesting that administration of compound 1 does not cause changes in coagulation parameters and bleeding volume in normal animals.

[0118] Example 3 Antagonism of dabigatran etexilate

[0119] Rat liver laceration model: Except for the Control group and the Compound 1 10mg / kg + NS control group, dabigatran etexilate (30mg / kg) was administered by gavage. 1.5h 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℃. Jugular vein cannulation was completed within 30min. 2min later, physiological saline, PER977 or different doses of Compound 1 were injected through the jugular vein cannula. 5min after administration, the abdominal cavity was opened, and three standard incisions 1cm long and 2mm deep were made on the liver. Blood flowing from the liver was wiped with pre-weighed dry cotton, and the bleeding loss (BL) was recorded.

[0120] Rat tail transection hemorrhage model: Except for the Control group and the Compound 1 10mg / kg + NS control group, dabigatran (30mg / kg) was administered by gavage. 30 min 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℃. Jugular vein cannulation was completed within 30 min. 2 min later, physiological saline, PER977 or different doses of Compound 1 were injected through the jugular vein cannula. 5 min after administration, an incision was made 5mm from the tail tip of the rat, and BL (bleeding loss) was recorded for 15 min. 2.5 mL of blood was drawn from the inferior vena cava, and 0.5 mL was separated and injected into a preheated test tube at 37°C without anticoagulant. The stopwatch was started immediately and the test tube was tilted and observed every 30 seconds in a 37°C water bath until the blood coagulated. The time was recorded with the stopwatch and is the WBCT.

[0121] The results are shown in Table 2.

[0122] Table 2: Effects of dabigatran etexilate on hemorrhage volume in rats

[0123] Group n Blood loss at tail transection (g) Hemorrhage volume from liver laceration (g) WBCT(s) Control (NS) 8 0.0154±0.0051 0.0323±0.0191 263±42 Vehicle (Dabigatran) 9 <![CDATA[0.0856±0.0474 * ]]> <![CDATA[0.5665±0.2039 ** ]]> <![CDATA[720±212 ** ]]> PER977 20mg / kg + Dabigatran 7 <![CDATA[0.0161±0.0067 # ]]> <![CDATA[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 <![CDATA[0.0083±0.0039 # ]]> <![CDATA[0.0500±0.0299 ## ]]> 626±182 Compound 1 10 mg / kg + NS 8 0.0118±0.0074 0.0152±0.0080 259±55

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

[0125] Analysis of experimental results:

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

[0127] Compared with the Vehicle group, compound 1 at 10 mg / kg significantly reduced the increase in hemorrhage induced by tail transection and liver laceration in rats (P<0.05, P<0.01), with an inhibition rate of 91.2% on hemorrhage induced by liver laceration in rats; compound 1 also had a certain effect on shortening WBCT, but there was no statistical difference.

[0128] Compared with the control group (compound 1 10 mg / kg + NS), no significant changes were observed in bleeding volume and WBCT, suggesting that administration of compound 1 does not cause changes in coagulation parameters and bleeding volume in normal animals.

[0129] Example 4: Antagonizing Aspirin

[0130] Rat liver laceration and hemorrhage model: Except for the Control group and the Compound 1 5.0 mg / kg + NS control group, aspirin was administered by gavage. Seven days later, 0.3 mL / 100 g body weight of 10% chloral hydrate was injected intraperitoneally. The anesthetized animals were placed on a preheated electric blanket at 37°C. Jugular vein cannulation was completed within 30 min. Two min later, physiological saline, PER977 or different doses of Compound 1 were injected through the jugular vein cannula. Five min after administration, the abdominal cavity was opened, and three standard incisions of 1 cm in length and 2 mm in depth were made on the liver. The blood flowing from the liver was wiped with pre-weighed dry cotton, and the bleeding loss (BL) was recorded.

[0131] Rat tail transection hemorrhage model: Except for the Control group and the Compound 1 5.0 mg / kg + NS control group, aspirin was administered by gavage. Seven days later, 0.3 ml / 100 g 10% chloral hydrate was injected intraperitoneally. The anesthetized animals were placed on a preheated electric blanket at 37°C, and the jugular vein was separated within 30 minutes. Saline, PER977, or different doses of Compound 1 were administered. Five minutes after administration, a new incision was made 5 mm from the tail tip, and bleeding loss (BL) was recorded for 15 minutes. 2.5 mL of blood was collected from the inferior vena cava, and 0.5 mL was separated. The blood without anticoagulant was injected into a preheated 37°C test tube (0.5 mL / tube). A stopwatch was immediately started, and the blood coagulation status in the test tube was observed every 30 seconds in a 37°C water bath until coagulation occurred. The time was recorded using a stopwatch.

[0132] The results are shown in Table 3.

[0133] Table 3: Effects of aspirin on hemorrhage volume in rats

[0134] Group n Blood loss from tail severance WBCT n Liver laceration bleeding volume Control (NS) 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 20mg / 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 <![CDATA[0.0186±0.0079 ### ]]> <![CDATA[262.5±49.7 # ]]> 8 <![CDATA[0.0536±0.0180 # ]]> Compound 1 5.0 mg / kg + NS 12 0.0263±0.0233 265.0±38.0 8 0.0500±0.0162

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

[0136] Analysis of experimental results:

[0137] Compared with the Control group, the amount of hemorrhage from tail transection and liver laceration was significantly increased in the Vehicle group (P<0.001, P<0.05). The amount of hemorrhage in the rat tail transection hemorrhage model increased by 5.5 times, and the amount of hemorrhage in the rat liver laceration model increased by 6 times. WBCT was also prolonged to some extent.

[0138] Compared with the Vehicle group, compound 1 at 5.0 mg / kg significantly reduced hemorrhage in rat tail transection and liver laceration models (P<0.001, P<0.05) and significantly shortened WBCT (P<0.05).

[0139] Compared with the control group (compound 1 5.0 mg / kg + NS), no significant changes were observed in bleeding volume and WBCT, suggesting that administration of compound 1 does not cause changes in bleeding volume and coagulation parameters in normal animals.

[0140] Example 5: Antagonizing Aspirin

[0141] Rabbit liver laceration and hemorrhage model: Except for the control group and the compound 1 2.1 mg / kg + NS control group, aspirin was administered by gavage. Seven days later, 30 minutes after the last gavage, 1 ml / kg chloral hydrate was injected intravenously for anesthesia. The anesthetized animals were placed on a preheated electric blanket at 37°C. Within 2 minutes, physiological saline and different doses of compound 1 were injected into the ear vein. Five minutes after administration, the abdominal cavity was opened, and three parallel, 0.5 cm long, fully penetrating standard incisions were made at the lower edge of the left lobe of the liver. Blood flowing from the liver was wiped away with pre-weighed dry cotton balls. The weight of the cotton balls was weighed again after 30 minutes, and the difference between the two weights was the bleeding loss (BL). 1 ml of blood was collected from the inferior vena cava and injected into a preheated test tube at 37°C. A stopwatch was immediately started, and the coagulation state of the blood in the test tube was observed every 30 seconds in a 37°C water bath until the blood coagulated. The time was recorded using a stopwatch (WBCT).

[0142] The results are shown in Table 4.

[0143] Table 4. Effects of aspirin-induced bleeding volume and coagulation function in rabbits.

[0144] Group n WBCT Liver laceration bleeding volume Control (NS) 8 577.5±31.1 0.7745±0.1795 Vehicle (Asp.) 8 <![CDATA[675.0±62.1 *** ]]> <![CDATA[2.0315±0.1407 *** <!-- 11 -->]]> 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 <![CDATA[592.5±50.1 ## ]]> <![CDATA[1.0021±0.5710 # ]]> Compound 1 2.1 mg / kg + NS 8 570.0±35.9 0.5362±0.2735

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

[0146] Analysis of experimental results:

[0147] Compared with the control group, the WBCT of animals in the vehicle group was significantly prolonged (P<0.001), and the amount of bleeding in the vehicle group was also significantly increased (P<0.001). The amount of bleeding in the rabbit liver laceration model increased by 1.6 times.

[0148] Compared with the Vehicle group, compound 1 at 2.1 mg / kg significantly shortened WBCT (P<0.01); compound 1 at 2.1 mg / kg also significantly reduced the amount of bleeding caused by liver laceration (P<0.05), with a reduction of up to 50.6%.

[0149] Compared with the control group (compound 1 2.1 mg / kg + NS), no significant changes were observed in WBCT and bleeding volume, suggesting that administration of compound 1 does not cause changes in coagulation parameters and bleeding volume in normal animals.

[0150] Example 6 Antagonizing Rivaroxaban

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

[0152] Rat tail transection hemorrhage model: Except for the Control group and the compound 1 0.13 mg / kg + NS control group, rivaroxaban was administered by gavage. Anesthetized animals were placed on a preheated electric blanket at 37°C. Within 30 minutes, the jugular vein was isolated and cannulated. Rivaroxaban was administered, and 30 minutes later, physiological saline, PER977, or different doses of compound 1 were injected into the jugular vein. Five minutes after administration, an incision was made 5 mm from the tail tip of the rat, and the bleeding loss (BL) was recorded over 15 minutes. Temperature was strictly controlled throughout the experiment, including room temperature and water bath temperature. 3 mL of blood was collected from the abdominal aorta and divided into two portions: 2 mL and 1 mL. The 2 mL portion was rapidly injected into an EP tube containing 0.2 mL of anticoagulant (3.8% sodium citrate solution), gently inverted to mix, and centrifuged at 3500 rpm for 10 minutes to obtain platelet-poor plasma (PPP). A fully automated coagulation analyzer was used to measure PT, TT, and APTT, and the results were recorded upon completion of the test. Another blood sample was used to determine whole blood clotting time (WBCT).

[0153] The results are shown in Table 5.

[0154] Table 5: Effect of rivaroxaban on hemorrhage volume in rats

[0155]

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

[0157] Analysis of experimental results:

[0158] Compared with the control group, the APTT, PT, TT and WBCT of the animals in the Vehicle group were significantly prolonged (P<0.01, P<0.001), with APTT being prolonged by 0.99 times. The amount of hemorrhage from tail transection in the Vehicle group was also significantly increased (P<0.05), with a 4.25-fold increase in hemorrhage. The amount of hepatic laceration hemorrhage in the Vehicle group was also significantly increased (P<0.01), with a 10.35-fold increase in hemorrhage.

[0159] Compared with the vehicle group, compound 1 at 0.005, 0.026, and 0.13 mg / kg dose-dependently shortened APTT (P<0.01, P<0.001), dose-dependently shortened WBCT (P<0.05, P<0.001), and dose-dependently reduced hemorrhage. The effects were most significant at 0.026 and 0.13 mg / kg (P<0.05, P<0.01), with reductions of 79.9% and 80.8% in tail transection hemorrhage and 81.3% and 89.0% in liver laceration hemorrhage, respectively.

[0160] Compared with the control group (administered only compound 1 0.13 mg / kg), no significant changes were observed in APTT, PT, TT, WBCT, and bleeding volume, suggesting that administration of compound 1 does not cause changes in coagulation parameters and bleeding volume in normal animals.

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

[0162] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

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 a factor Xa inhibitor; wherein X, X', X" are independently selected from substituted or unsubstituted alkyl, alkenyl or heterocyclyl; Y, Y', Y" are independently selected from substituted or unsubstituted alkyl, alkenyl or heterocyclyl; Z, Z', Z" are independently selected from a heteroatom-containing molecular fragment that can be protonated under physiological conditions.

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

3. The method of claim 1, wherein, Y-Z, Y'-Z', Y"-Z" are independently selected from the residue of a basic amino acid; preferably, the residue of the 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, guanidino or imidazolyl.

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

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 of any one of claims 1-7, wherein, The factor Xa inhibitor is one or more of apixaban, rivaroxaban, edoxaban, otamixaban and fondaparinux.

9. The method according to any one of claims 1 to 7, wherein, The factor Xa inhibitor is rivaroxaban.

10. A kit comprising a compound used in any one 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 a factor Xa inhibitor; more preferably, the factor Xa inhibitor is one or more of apixaban, rivaroxaban, edoxaban, otamixaban and fondaparinux; more preferably, the factor Xa inhibitor is rivaroxaban.

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