Application of active substance in preparation of VKOR inhibitor and / or vitamin K antagonist and anticoagulant drug

Abstract

The invention belongs to the technical field of biological medicine, and discloses application of an active substance in preparation of a VKOR inhibitor and / or a vitamin K antagonist and an anticoagulant drug. The invention provides application of an active substance shown in a formula (1) or pharmaceutically acceptable salt thereof in preparation of a vitamin K epoxide reductase inhibitor. The active substance has VKOR inhibitory activity, can prevent liver from synthesizing blood coagulation factors and activating anticoagulant factors by blocking reduction of oxidized vitamin K and inhibiting circulation of vitamin K, has potential anticoagulant activity, and has the advantages of mild anticoagulant effect, low probability of occurrence of adverse hemorrhage events, good anticoagulant effect, high safety and the like. The compound has a good application prospect in preparation of anticoagulants and / or medicines for treating thromboembolic diseases.

Description

Technical Field

[0001] This invention belongs to the technical field of biomedicine, and particularly relates to the application of active substances in the preparation of VKOR inhibitors and / or vitamin K antagonists and anticoagulant drugs. Background Technology

[0002] The vitamin K cycle refers to the metabolic process by which vitamin K is repeatedly utilized in the body through oxidation-reduction reactions. The resulting active vitamin K is an essential cofactor for the activation of a series of downstream vitamin K-dependent clotting factors, and is crucial for the post-translational modification of glutamate residues in clotting factors to form γ-carboxyglutamate. If the vitamin K cycle is blocked, the activation of clotting factors will be inhibited, thereby inhibiting the clotting process.

[0003] Therefore, obtaining an active substance that can target and interfere with the vitamin K cycle, effectively inhibit the synthesis of clotting factors, and exert an anticoagulant effect is of great significance for the development of novel anticoagulants with good anticoagulant effect and high safety. Summary of the Invention

[0004] With the aim of obtaining an active substance capable of targeting and interfering with the vitamin K cycle, the inventors of this invention, through extensive and in-depth research and numerous experiments, creatively discovered that an active molecule with the general structural formula shown in formula (1) exhibits excellent vitamin K cycle inhibitory effects in in vitro experiments. More specifically, this active substance possesses vitamin K epoxide reductase (VKOR) inhibitory activity, which can block the vitamin K cycle by preventing the reduction of oxidized vitamin K, thereby inhibiting the activation of hepatic synthesis of clotting factors and anticoagulant factors, and thus possessing potential anticoagulant activity.

[0005] Further in vivo experiments revealed that the active substance also exhibits excellent anticoagulant activity in vivo, effectively prolonging prothrombin time. Furthermore, compared to the vitamin K antagonist Warfarin, the two substances mentioned above show a more gradual trend in their inhibitory effect on vitamin K circulation with concentration changes, exhibiting a milder anticoagulant effect and a lower risk of adverse bleeding events. They combine the advantages of good anticoagulant efficacy and high safety, demonstrating promising application prospects in the preparation of anticoagulants. Based on this, the technical solution of this invention was obtained.

[0006] In a first aspect, the present invention provides the use of the active substance of formula (1) or a pharmaceutically acceptable salt thereof in the preparation of vitamin K epoxide reductase inhibitors.

[0007]

[0008] In equation (1), R1 is H or -CH3, and R2 is -(CH2)4CH3 or -(CH2)8CH3.

[0009] Furthermore, the structure of the active substance is shown in formula (2).

[0010]

[0011] Furthermore, the structure of the active substance is shown in formula (3).

[0012]

[0013] In this invention, the pharmaceutically acceptable salt refers to a salt compound formed by the active substance and a suitable acid or base through ionic bonds, and the formed salt compound retains at least the activity equivalent to that of the active substance. Specific examples include, but are not limited to, one or more of the following: potassium salts, sodium salts, bicarbonates, bisulfates, nitrates, borates, calcium salts, chlorides, iodides, acetates, propionates, citrates, benzoates, maleates, tartrates, and oxalates.

[0014] In this invention, the inhibitor refers to an inhibitor that, compared to the absence of application or the substitution with an equal amount of blank solvent, can directly or indirectly alter, interfere with, or inhibit the activity of vitamin K epoxide reductase, and the alteration, interference, or inhibition effects are statistically significantly different.

[0015] Secondly, the present invention provides the use of the above-mentioned active substance or a pharmaceutically acceptable salt thereof in the preparation of vitamin K antagonists.

[0016] In this invention, the vitamin K antagonist refers to a class of substances that delay or inhibit the blood clotting process by blocking the vitamin K cycle and thus interfering with the physiological function of microbial K.

[0017] In this invention, the active substance blocks the reduction reaction of vitamin K epoxide by targeting and inhibiting vitamin K epoxide reductase, thereby disrupting the vitamin K cycle, and has the potential to be used as a vitamin K antagonist.

[0018] Thirdly, the present invention provides the use of the above-mentioned active substance or a pharmaceutically acceptable salt thereof in the preparation of anticoagulants.

[0019] In this invention, the anticoagulant refers to a class of substances that can inhibit the blood clotting process and prevent thrombus formation.

[0020] In this invention, the active substance acts on the initial stage of coagulation, inhibiting the vitamin K cycle and thereby hindering the synthesis of vitamin K-dependent coagulation factors and the activation of anticoagulant proteins, thus achieving an anticoagulant effect and having the potential to be used as an anticoagulant.

[0021] Fourthly, the present invention provides an anticoagulant. The anticoagulant comprises the above-mentioned active substance or a pharmaceutically acceptable salt thereof.

[0022] In this invention, the anticoagulant preferably also includes other active substances that affect the coagulation process, specific examples of which include, but are not limited to: procoagulant substances and / or anticoagulant substances.

[0023] In some specific embodiments, anticoagulants are combined with the aforementioned active substances to achieve a better anticoagulant effect through simultaneous action on the same or multiple targets. More specifically, examples of the anticoagulants include, but are not limited to, one or more of the following: anticoagulant protein, heparin, sodium citrate, argatroban, and nafamostat mesylate.

[0024] In some specific embodiments, a procoagulant substance is combined with the aforementioned active substance. Through the mutual restraint between the procoagulant substance and the active substance, the dynamic balance of the coagulation system is maintained, thereby improving the safety of the anticoagulant drug. More specifically, examples of the procoagulant substance include, but are not limited to, one or more of vitamin K, ethamsylate, tranexamic acid, and coagulation factors.

[0025] Furthermore, the anticoagulant preferably includes pharmaceutically acceptable carriers and / or excipients. Pharmaceutically acceptable carriers and / or excipients refer to substances in a pharmaceutical formulation, other than the active ingredient, used to assist in formulation formation, stabilize drug properties, improve administration routes, enhance bioavailability, or ensure drug safety. More specifically, specific examples of pharmaceutically acceptable carriers and / or excipients include, but are not limited to, one or more of: water for injection (WFI), bacteriostatic water for injection (BWFI), physiological saline, glucose solution, pH buffer solution, and Ringer's solution.

[0026] Fifthly, the present invention provides the application of the above-mentioned anticoagulant in the preparation of a medicament for the treatment of thromboembolic diseases.

[0027] In this invention, the treatment refers to the improvement or eradication of thromboembolic symptoms, which may be, but is not limited to, one or more of the following: (1) preventing the occurrence of thromboembolic symptoms; (2) preventing the development of thromboembolic symptoms; (3) alleviating or resolving thromboembolic symptoms.

[0028] In this invention, the anticoagulant drug exerts an anticoagulant effect in the body by interfering with the vitamin K cycle, preventing the synthesis of clotting factors and the activation of clotting proteins, thereby treating thromboembolic diseases.

[0029] In this invention, the inducing factors of the thromboembolic condition may be, but are not limited to, one or more of the following: (1) unstable angina; (2) acute coronary syndrome; (3) atrial fibrillation; (4) primary myocardial infarction; (5) recurrent myocardial infarction; (6) ischemic sudden death; (7) transient ischemic attack; (8) stroke; (9) atherosclerosis; (10) peripheral occlusive artery disease; (11) venous thrombosis; (12) deep vein thrombosis; (13) thrombophlebitis; (14) arterial embolism; (15) coronary artery thrombosis; (16) cerebral artery thrombosis; (17) cerebral embolism; (18) renal embolism; (19) pulmonary embolism; (20) exposure of blood to a medical implant, device, or process that may promote thrombosis. Attached Figure Description

[0030] Figure 1 The pHBLV-FIXgla-PC lentiviral vector spectrum provided in Example 1 of this invention;

[0031] Figure 2 This is a graph showing the experimental results of the test on the inhibitory effect of 6-Paradol on the vitamin K cycle provided in Example 1 of this invention;

[0032] Figure 3 This is a graph showing the experimental results of the test on the inhibitory effect of 10-Gingerol on the vitamin K cycle provided in Example 1 of this invention;

[0033] Figure 4 The figure shows the experimental results of the test on the inhibitory effect of Warfarin on the vitamin K cycle provided in Example 1 of this invention;

[0034] Figure 5 This is a diagram showing the experimental results of the target testing of active substances in the vitamin K cycle provided in Example 1 of this invention;

[0035] Figure 6 The figure shows the experimental results of the in vivo anticoagulant effect test of the active substance provided in Example 2 of this invention (Note: ***P<0.001, ****P<0.0001). Detailed Implementation

[0036] The embodiments of the present invention are described in detail below. These embodiments are intended to explain the present invention and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0037] It should be noted that any steps, reagents, and conditions not mentioned in the experiments described in the following embodiments are all prior art and can be referred to existing publicly available patent or non-patent literature. In addition, in different groups under the same test, all other conditions are kept consistent except for the differences mentioned.

[0038] Example 1

[0039] This embodiment illustrates the inhibitory effect of two active substances having the structure shown in formula (1) on the vitamin K cycle, specifically including:

[0040] 1. Construction of FP-D7 cells [1]

[0041] References [1] , construct Figure 1 The pHBLV-FIXgla-PC lentiviral vector shown was introduced into HEK-293T cells, followed by resistance selection, green fluorescence sorting, and cloning to obtain the FIXgla-PC-D7-HEK-293T(FP-D7) cell line.

[0042] [1]A cell-based high-throughput screen identifies drugs that cause bleeding disorders by off-targeting the vitamin K cycle.Xuejie Chen, CaihongLi, Da-Yun Jin, Brian Ingram, Zhenyu Hao,

[0043] 2. The inhibitory effect of active substances on the vitamin K cycle

[0044] (1) According to 2×10 4For the seeding amount of cells / well, the above-mentioned FP-D7 cells were added to a 96-well plate, and vitamin K1 2,3-epoxide (KO) was added at a final concentration of 5 μM. At the same time, 6-Paradol (structure shown in formula (2)) or 10-Gingerol (structure shown in formula (3)) was added at final concentrations of 0.01 μM, 0.1 μM, 1 μM, 5 μM and 10 μM, respectively. Warfarin (structure shown in formula (4)) of the same concentration was used as a positive control instead of the above-mentioned active substances, and DMSO of the same concentration was used as a blank control instead of the above-mentioned active substances. After co-incubation at 37℃ and 5% CO2 for 48 h, the supernatant was collected.

[0045]

[0046]

[0047] (2) GMA-001 (Green Mountain Antibodies, catalog number GMA-001) was used as the capture antibody and Anti-PC (Affinity Biologicals, catalog number SAPC-HRP) was used as the detection antibody. The absorbance of each supernatant was detected by ELISA sandwich method, and the carboxylation efficiency (in %) was calculated according to the following formula:

[0048] Carboxylation efficiency = A x / A0×100%

[0049] Among them, A x A1 represents the absorbance value measured in the supernatant of the experimental group or positive control group; A2 represents the absorbance measured in the supernatant of the corresponding concentration of the blank control group.

[0050] (3) The cell viability of FP-D7 cells co-incubated with 6-Paradol, 10-Gingerol, Warfarin, or DMSO was tested using the CCK-8 cell viability assay kit (MedChemExpress, catalog number HY-K0301) and in accordance with the instructions. The cell viability (in %) was calculated using the following formula:

[0051] Cell activity = OD x / OD0×100%

[0052] Among them, OD x OD of the culture medium in the experimental group or positive control group 450 Value, OD0 is the OD of the culture medium in the corresponding concentration of the blank control group. 450 Value. Result as follows Figures 2-4 As shown.

[0053] Depend on Figures 2-4 The results show that 6-Paradol and 10-Gingerol can effectively inhibit the vitamin K cycle without affecting cell activity.

[0054] 3. Target sites of active substances in the vitamin K cycle

[0055] (1) According to 2×10 4 FP-D7 cells were seeded into 96-well plates. 6-Paradol and 10-Gingerol were then added to a final concentration of 2.5 μM. Vitamin K (VK) was added at final concentrations of 0.001 nM, 0.05 nM, 0.2 nM, 0.5 nM, 2.5 nM, 10 nM, 50 nM, 150 nM, 650 nM, 2500 nM, or 10000 nM. Warfarin at the corresponding concentration was used as a positive control, and DMSO at the corresponding concentration was used as a blank control. The plates were co-incubated at 37 °C and 5% CO2 for 48 h, and the supernatant was collected.

[0056] (2) The absorbance of the supernatant in each group was tested using the method provided in "2. Inhibitory effect of active substances on vitamin K cycle". The carboxylation efficiency of cells (in %) and the EC50 of vitamin K were calculated according to the following formula. 50 (Unit: nM):

[0057] Carboxylation efficiency = A x / A max ×100%

[0058] Among them, A x A represents the absorbance value measured in the supernatant of the experimental group, positive control group, or blank control group. max The maximum absorbance measured in the supernatant of the blank control group is shown below. Figure 5 As shown in Table 1.

[0059] Table 1.

[0060] active substance <![CDATA[EC 50 (nM)]]> Increase multiple DMSO 8.185 1 Warfarin 3186.000 393 6-Paradol 576.200 70 10-Gingerol 1113.000 137

[0061] Depend on Figure 5 As shown in Table 1, the inhibitory effect of 6-Paradol and 10-Gingerol on the vitamin K cycle can be restored by vitamin K. Furthermore, 6-Paradol and 10-Gingerol exhibit concentration-dependent rescue characteristics similar to those of Warfarin, indicating that the target of 6-Paradol and 10-Gingerol in the vitamin K cycle is VKOR. 6-Paradol and 10-Gingerol interfere with the vitamin K cycle by inhibiting VKOR.

[0062] In addition, the introduction of 6-Paradol and 10-Gingerol respectively increased the EC of vitamin K. 50 The values ​​increased by 70 times and 137 times, which had a strong inhibitory effect on VKOR.

[0063] Example 2

[0064] This embodiment illustrates the in vivo anticoagulant effects of two active substances having the structure shown in formula (1), specifically including:

[0065] Twenty-four eight-week-old female BALB / c mice were randomly divided into four groups of six mice each (n=6). After acclimatization under the same feeding conditions for seven days, the following procedures were performed:

[0066] (1) 6-Paradol group: 6-Paradol was administered to mice by gavage at a dose of 100 mg / (kgd) (injection volume of 300 μL) for 7 days.

[0067] (2) 10-Gingerol group: 10-Gingerol was administered to mice by gavage at a dose of 150 mg / (kgd) (injection volume of 300 μL) for 7 days.

[0068] (3) Warfarin group: Warfarin was administered to mice by gavage at a dose of 3 mg / (kgd) (injection volume of 300 μL) for 7 days.

[0069] (4) DMSO group: DMSO was administered to mice by gavage at a dose of 300 μL / d for 7 days.

[0070] After drug administration, blood was collected from the retro-orbital vascular plexus of mice, and the prothrombin time (PT, in seconds) was measured using a micro-point coagulation analyzer. The results are as follows: Figure 6 As shown.

[0071] Depend on Figure 6 The results show that, compared with the DMSO group, mice treated with 6-Paradol or 10-Gingerol by gavage had prolonged PT, indicating that 6-Paradol and 10-Gingerol have excellent in vivo anticoagulant effects.

[0072] comprehensive Figures 1-6 As shown in Table 1, compared to Warfarin, the inhibitory effects of 6-Paradol and 10-Gingerol on the vitamin K cycle exhibit a more gradual trend with concentration changes, and the EC50 of vitamin K in exerting its inhibitory and restorative effects on the vitamin K cycle is also higher.50 The lower concentrations of 6-Paradol and 10-Gingerol result in milder anticoagulant effects, a lower probability of bleeding adverse events, and better safety profiles.

[0073] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. The use of the active substance represented by formula (1) or a pharmaceutically acceptable salt thereof in the preparation of vitamin K epoxide reductase inhibitors, In equation (1), R1 is H or -CH3, and R2 is -(CH2)4CH3 or -(CH2)8CH3.

2. The use of the active substance according to claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a vitamin K epoxide reductase inhibitor, characterized in that, The structure of the active substance is shown in formula (2).

3. The use of the active substance according to claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a vitamin K epoxide reductase inhibitor, characterized in that, The structure of the active substance is shown in formula (3).

4. The use of the active substance or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 3 in the preparation of a vitamin K antagonist.

5. The use of the active substance or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 3 in the preparation of an anticoagulant.

6. An anticoagulant drug, characterized in that, The anticoagulant includes the active substance as described in any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof.

7. The anticoagulant drug according to claim 6, characterized in that, The anticoagulant includes a pharmaceutically acceptable carrier and / or excipient.

8. The use of the anticoagulant drug according to claim 6 or 7 in the preparation of a medicament for the treatment of thromboembolic diseases.

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