Preparation and application of positive ion type tetrahedral molecule

Abstract

The invention relates to preparation and application of a positive ion type tetrahedral molecule, in particular to preparation and application of an efficient and rapid heparin anticoagulant antagonist. According to the invention, a high-water-solubility positive ion type tetrahedral compound is designed and constructed through a chemical synthesis strategy. The novel tetrahedral compound has high water solubility, high biological safety and efficient antagonism efficiency, and has remarkable potential clinical practicability.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and specifically relates to a type of cationic tetrahedral molecule capable of realizing broad-spectrum, high-efficiency and rapid antagonism to heparin anticoagulants, as well as a preparation method and use thereof. Background Art

[0002]

[0003] Heparin drugs are a class of highly sulfated anionic linear mucopolysaccharides. In clinical applications, they are used as first-line anticoagulant and antithrombotic drugs and are widely used in the prevention and treatment of thrombosis or embolic diseases, hemodialysis, extracorporeal circulation, etc. Heparin is composed of repeating units of α-1,4-linked uronic acid and D-glucosamine, and has the highest negative charge density among biological macromolecules. Heparin can highly bind to antithrombin and significantly enhance the ability of antithrombin to inhibit the activity of serine protease coagulation factors (mainly coagulation factor Xa and thrombin) during the coagulation cascade.

[0004] Heparin drugs currently used in clinical practice include unfractionated heparin (UFH, extracted from porcine intestinal mucosa or bovine lungs, with an average molecular weight of approximately 15 kDa), low molecular weight heparins (LMWHs, fragments with an average molecular weight of approximately 3.6–6.5 kDa obtained by depolymerization of unfractionated heparin), and Fondaparinux (molecular weight 1.7 kDa), which is a modified synthesis of the natural pentose structure contained in both UFH and LMWHs.

[0005] According to publicly available statistics, 170,000 cardiovascular surgeries under extracorporeal circulation were performed nationwide in 2018, and more than 632,000 patients under hemodialysis were required. However, in the process of using heparin for treatment, especially when using low molecular weight heparin and unfractionated heparin, it is necessary to reverse the excess heparin to reduce or eliminate side effects. Protamine is currently the only heparin antagonist approved by the FDA for clinical use. It is a class of proteins containing a large number of arginine structures, which mainly achieves the reversal effect through electrostatic interactions between anions and cations. However, the use of protamine can cause many serious adverse reactions, including anaphylactic shock, systemic hypotension, severe pulmonary vasoconstriction, and idiopathic fatal cardiac arrest. In addition, protamine cannot distinguish between active and inactive heparin sequences, and in some cases, the antagonism may fail and cause anticoagulation problems again. Despite the many shortcomings of protamine, there is currently no commercial antagonist that can replace protamine, and the development of new heparin antagonists has become an urgent need.

[0006] In recent years, antagonists of heparin anticoagulants have mainly focused on macromolecular antagonists such as proteins, peptides, cationic polymers, and supramolecular assemblies. Macromolecular antagonists are usually difficult to control in quality, and are usually effective against unfractionated heparin but less effective or even ineffective against low molecular weight heparin. In addition, large-sized particles that may be formed after macromolecular antagonists bind to macromolecular heparin molecules will produce immunotoxicity. From the perspective of drug development, small molecule drugs have the advantages of a single and clear chemical structure, controllable synthesis methods and quality, predictable dose-activity relationship, stable storage performance, easier metabolism and distribution analysis, and the development of small molecule drugs has accumulated rich experience and a reliable research and development platform. However, there are currently few small molecule antagonists for heparin, and no small molecule drugs have been clinically approved for heparin antagonism.

[0007] In summary, from the perspective of drug development and drugability, based on the current status of the above small molecule heparin antagonists, developing new heparin antagonism strategies and constructing a new small molecule heparin antagonist system have important clinical significance and value for promoting research in the field of heparin antagonism. Summary of the invention

[0008] The object of the present invention is to provide a small molecule heparin antagonist having a broad spectrum of rapid antagonistic activity against anticoagulants such as unfractionated heparin and low molecular weight heparin.

[0009] In a first aspect of the present invention, there is provided a use of a cationic tetrahedral compound, or a pharmaceutically acceptable salt, hydrate, solvate, or crystal form, as shown in Formula I, for preparing an antagonist of heparin anticoagulants;

[0010]

[0011] in,

[0012] A is A cationic salt of, wherein X is a pyridinium salt, an imidazolium salt or a pyrimidinium salt;

[0013] Wherein, R1, R2, R3, R4 and R5 are each independently H, -OC 1-30 Alkylene -NH2, -SC 1-30 Alkylene -NH2, -NH-CO-C 1-30 Alkylene -NH2, C 1-30 Alkylene -NH2;

[0014] Y is C or N + ;

[0015] Z n-The anion or anion combination whose total anion valence n is 0, 4, 8 or 12 is composed of anions of pharmaceutically acceptable inorganic or organic acids selected from the following groups: chloride ion, bromide ion, iodide ion, bisulfate ion, sulfate ion, phosphate ion, maleate ion, fumarate ion, tartrate ion, palmitate ion, oxalate ion, citrate ion, succinate ion, methanesulfonate ion, benzenesulfonate ion, p-toluenesulfonate ion, or a combination thereof.

[0016] In another preferred embodiment, Z n- It is an anion or anion combination whose total anion valence is 4.

[0017] In another preferred embodiment, the compound has a structure shown in Formula II:

[0018]

[0019] Wherein, A is as described in the first aspect of the present invention,

[0020] W- is a pharmaceutically acceptable monovalent anion of an inorganic or organic acid selected from the group consisting of chloride, bromide, iodide, bisulfate, maleate, fumarate, tartrate, palmitate, oxalate, citrate, succinate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, or a combination thereof.

[0021] In another preferred embodiment, X is

[0022] In another preferred embodiment, R1, R2, R3, R4 and R5 are each independently H, -OC 1-15 Alkylene -NH2, -SC 1-15 Alkylene -NH2, -NH-CO-C 1-15 Alkylene -NH2, C 1-15 Alkylene-NH2.

[0023] In another preferred embodiment, R1, R2, R3, R4 and R5 are each independently H, -OC 1-6 Alkylene -NH2, -SC 1-6 Alkylene -NH2, -NH-CO-C 1-6 Alkylene -NH2, -C 1-6 Alkylene-NH2.

[0024] In another preferred embodiment, R1, R2, R3, R4 and R5 are each independently H, -O-(CH 2)2 -NH2、-O-(CH 2)3 -NH2、-S-(CH 2)2-NH2, -NH-CO-CH2-NH2.

[0025] In another preferred embodiment, in the structure of Formula I, at least one substituent in each A (such as R1, R2, R3, R4 and R5) is not H, preferably, at least two substituents in each A are not hydrogen, and more preferably, at least three substituents in each A are not hydrogen.

[0026] In another preferred embodiment, Z n- 4Cl - .

[0027] In another preferred embodiment, R3 is independently selected from the following group: -OC 1-6 Alkylene -NH2, -SC 1-6 Alkylene -NH2, -NH-CO-C 1-6 Alkylene-NH2; R1, R2, R4 and R5 are each independently H, -OC 1-6 Alkylene -NH2, -SC 1-6 Alkylene -NH2, -NH-CO-C 1-6 Alkylene-NH2.

[0028] In another preferred embodiment, R3 is independently selected from the following group: -OC 1-6 Alkylene -NH2, -SC 1-6 Alkylene -NH2, -NH-CO-C 1-6 Alkylene -NH2; R2, R4 are each independently -OC 1-6 Alkylene -NH2, -SC 1-6 Alkylene -NH2, -NH-CO-C 1-6 Alkylene-NH2; R1, and R5 are each independently H.

[0029] In another preferred embodiment, the compound is selected from the following group:

[0030]

[0031]

[0032] In another preferred embodiment, the compound is T2.

[0033] In another preferred embodiment, the heparin anticoagulant is selected from the following group: unfractionated heparin, low molecular weight heparin, or a combination thereof.

[0034] In another preferred embodiment, the average molecular weight of the unfractionated heparin is about 15 kDa.

[0035] In another preferred embodiment, the average molecular weight of the low molecular weight heparin is about 3.6-6.5 kDa.

[0036] In another preferred embodiment, the low molecular weight heparin is selected from the following group: dalteparin sodium, enoxaparin sodium, nadroparin calcium, or a combination thereof.

[0037] In a second aspect of the present invention, a pharmaceutical composition or preparation for antagonizing heparin anticoagulant drugs is provided, the pharmaceutical composition or preparation comprising:

[0038] (a) a tetrahedral compound of formula I as an active ingredient, or a pharmaceutically acceptable salt, hydrate, solvate, or crystal form thereof; and

[0039]

[0040] (b) a pharmaceutically acceptable carrier;

[0041] Among them, A, Z n- As described in the first aspect of the present invention.

[0042] In another preferred embodiment, the pharmaceutical composition or preparation is used to antagonize heparin anticoagulant drugs.

[0043] In another preferred embodiment, component (a) accounts for 0.001-99.99 wt % of the total weight of the preparation; preferably 0.01-99.9 wt %; more preferably 0.05-90 wt %.

[0044] In another preferred embodiment, the pharmaceutically acceptable carrier is a physiological saline solution.

[0045] In another preferred embodiment, the dosage form of the pharmaceutical composition or preparation is an injection, tablet, capsule, pill, suspension or emulsion.

[0046] In another preferred embodiment, the dosage form of the pharmaceutical composition is injection, tablet, capsule, pill, suspension or emulsion; preferably injection.

[0047] In another preferred embodiment, the injection is an aqueous solution, a sodium chloride aqueous solution or a glucose aqueous solution, wherein the concentration of component (a) as the first active ingredient is 0.001-500 mg / mL; preferably, it is a physiological saline solution.

[0048] In another preferred embodiment, the concentration of the injection is 0.001-2000 mg / kg, based on the body weight of the mouse.

[0049] In the third aspect of the present invention, there is provided a use of a tetrahedral compound represented by formula I and a protamine composition for preparing an antagonist drug of heparin anticoagulant drugs;

[0050] Wherein, the tetrahedral compound represented by formula I is as follows:

[0051]

[0052] Among them, A, Z n- As described in the first aspect of the present invention.

[0053] In another preferred embodiment, in the composition, the tetrahedral compound represented by formula I accounts for 0.001-99.99wt% of the total weight of the composition; preferably 0.01-99.9wt%; more preferably 0.05-90wt%.

[0054] In the fourth aspect of the present invention, a method for antagonizing heparin-type anticoagulant drugs is provided, the method comprising: administering a therapeutically effective amount of the tetrahedral compound shown in Formula I, or the pharmaceutical composition or preparation described in the second aspect of the present invention, or the composition described in the third aspect of the present invention to a subject in need.

[0055] In another preferred embodiment, the method is in vitro.

[0056] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0057] In a fifth aspect of the present invention, there is provided a cationic tetrahedral compound of formula I, or a pharmaceutically acceptable salt, hydrate, solvate, or crystal form thereof,

[0058]

[0059] Among them, A, Z n- As described in the first aspect of the present invention;

[0060] Wherein, the tetrahedral compound represented by formula I is not:

[0061]

[0062] In a sixth aspect of the present invention, a kit is provided, comprising:

[0063] (1) a first container, and a first pharmaceutical composition in the first container, wherein the first pharmaceutical composition comprises a first compound or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier; and / or

[0064] (2) a second container, and a heparin-based anticoagulant drug in the second container, wherein the heparin-based anticoagulant drug is selected from unfractionated heparin, low molecular weight heparin, or a combination thereof; and / or

[0065] (3) an nth container, and an nth pharmaceutical composition in the nth container, wherein the nth pharmaceutical composition comprises an nth compound or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier; wherein n is any positive integer from 3 to 8;

[0066] Wherein, the first compound and the nth compound are both tetrahedral compounds of formula I selected from the following group:

[0067]

[0068] Among them, A, Z n- As described in the first aspect of the present invention;

[0069] and / or (4) optional instructions for use.

[0070] In another preferred embodiment, the instructions are used to indicate the administration method and contraindications of the kit.

[0071] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 This is the hydrogen NMR spectrum (400 MHz, D2O, 298 K) of tetrahedral compound T1.

[0073] Figure 2 This is the carbon NMR spectrum (101 MHz, D2O, 298 K) of tetrahedral compound T1.

[0074] Figure 3 This is the hydrogen NMR spectrum of tetrahedral compound T2 (400 MHz, D2O, 298 K).

[0075] Figure 4 This is the carbon NMR spectrum of tetrahedral compound T2 (101 MHz, D2O, 298 K).

[0076] Figure 5 This is the hydrogen NMR spectrum of tetrahedral compound T6 (400 MHz, D2O, 298K).

[0077] Figure 6 This is the carbon NMR spectrum of tetrahedral compound T6 (101 MHz, D2O, 298 K).

[0078] Figure 7 This is the hydrogen NMR spectrum of tetrahedral compound T4 (400 MHz, D2O, 298K).

[0079] Figure 8 This is the carbon NMR spectrum of tetrahedral compound T4 (101 MHz, D2O, 298 K).

[0080] Fig. 9 This is the hydrogen NMR spectrum of tetrahedral compound T7 (400 MHz, D2O, 298 K).

[0081] Fig.10 This is the carbon NMR spectrum (101 MHz, D2O, 298 K) of tetrahedral compound T7.

[0082] Fig.11 This is the hydrogen NMR spectrum of tetrahedral compound T8 (400 MHz, D2O, 298K).

[0083] Fig.12 This is the carbon NMR spectrum (101 MHz, D2O, 298 K) of tetrahedral compound T8.

[0084] Fig.13 The isothermal thermometric titration results of compound T1 (50 μM, saline solution) and heparin molecules (saline solution). Saline solution (a) unfractionated heparin UFH (2 mM), (b) dalte (1.5 mM), (c) enoxa (1.0 mM), (d) nadroparin calcium Nadro (2.0 mM).

[0085] Fig.14 The isothermal thermometric titration results of compound T2 (50 μM, saline solution) and heparin molecules (saline solution). Saline solution (a) unfractionated heparin UFH (1 mM), (b) dalte (0.75 mM), (c) enoxa (0.5 mM), (d) nadroparin calcium Nadro (0.5 mM).

[0086] Fig.15 The isothermal thermometric titration results of compound T3 (50 μM, saline solution) and heparin molecules (saline solution). Saline solution (a) unfractionated heparin UFH (1 mM), (b) dalte (0.75 mM), (c) enoxa (0.5 mM), (d) nadroparin calcium Nadro (0.5 mM).

[0087] Fig.16The fluorescence titration results of heparin saline solution titrated compound T2 (50μM, saline solution). (a) Unfractionated heparin UFH (1mM), (b) Dalte (0.75mM), (c) Enoxa (0.5mM), (d) Nadro (0.5mM). The emission wavelength is 330nm and the absorption wavelength is 485nm.

[0088] Fig.17 Results of activated partial thromboplastin (aPTT) test in bovine plasma. Antagonistic efficiency of compounds T1-3 and protamine against (a) unfractionated heparin (2 IU / mL), (b) dalteparin sodium (2 IU / mL), (c) enoxaparin sodium (2 IU / mL), and (d) nadroparin calcium (2 IU / mL).

[0089] Fig.18 Results of activated partial thromboplastin (aPTT) test in bovine plasma. Antagonistic efficiency of compound T4 and protamine on (a) unfractionated heparin (2 IU / mL), (b) dalteparin sodium (2 IU / mL), (c) enoxaparin sodium (2 IU / mL), and (d) nadroparin calcium (2 IU / mL).

[0090] Fig.19 Results of activated partial thromboplastin (aPTT) test in bovine plasma. Antagonistic efficiency of compound T5 and protamine on (a) unfractionated heparin (2 IU / mL), (b) dalteparin sodium (2 IU / mL), (c) enoxaparin sodium (2 IU / mL), and (d) nadroparin calcium (2 IU / mL).

[0091] Fig. 20 Results of activated partial thromboplastin (aPTT) test in bovine plasma. Antagonistic efficiency of compound T6 and protamine on (a) unfractionated heparin (2 IU / mL), (b) dalteparin sodium (2 IU / mL), (c) enoxaparin sodium (2 IU / mL), and (d) nadroparin calcium (2 IU / mL).

[0092] Fig.21 Results of activated partial thromboplastin (aPTT) test in bovine plasma. Antagonistic efficiency of compound T7 and protamine on (a) unfractionated heparin (2 IU / mL), (b) dalteparin sodium (2 IU / mL), (c) enoxaparin sodium (2 IU / mL), and (d) nadroparin calcium (2 IU / mL).

[0093] Fig. 22Results of activated partial thromboplastin (aPTT) test in bovine plasma. Antagonistic efficiency of compound T8 and protamine on (a) unfractionated heparin (2 IU / mL), (b) dalteparin sodium (2 IU / mL), (c) enoxaparin sodium (2 IU / mL), and (d) nadroparin calcium (2 IU / mL).

[0094] Fig.23 The thromboelastometry results of tetrahedral compound T2 antagonizing heparin. (a) Unfractionated heparin (2 IU / mL), (b) Dalteparin sodium (2 IU / mL), (c) Enoxaparin sodium (2 IU / mL), (d) Nadroparin calcium (2 IU / mL).

[0095] Fig.24 The thromboelastometry results of tetrahedral compound T3 antagonizing heparin. (a) Unfractionated heparin (2 IU / mL), (b) Dalteparin sodium (2 IU / mL), (c) Enoxaparin sodium (2 IU / mL), (d) Nadroparin calcium (2 IU / mL).

[0096] Fig.25 Results of the coagulation inversion test of tetrahedral compound T2 against heparin. Thromboelastograms of (a) unfractionated heparin (2 IU / mL), (b) dalteparin sodium (2 IU / mL), and (c) enoxaparin sodium (2 IU / mL).

[0097] Fig.26 This is an in vivo heparin neutralization experiment conducted by the tail model of ICR mice (18-24g, 10 mice, half male and half female): (a) total bleeding time, (b) bleeding volume. All models were administered twice, first injected with saline (negative control) unfractionated heparin UFH (200IU / kg) at t=0min, then injected with saline (negative control), compound T2 (0.4mg / kg), protamine (2.6mg / kg) at t=5min, then the mice were anesthetized with isoflurane and the distal tail of the mice was cut off at t=10min, the bleeding time and bleeding volume were recorded, the average values ​​were calculated and the significant differences of the data were compared.

[0098] Fig. 27 This is an in vivo heparin neutralization experiment conducted by the tail model of ICR mice (18-24g, 10 mice, half male and half female): (a) total bleeding time, (b) bleeding volume. All models were administered twice, first injected with saline (negative control) dalteparin sodium (200 IU / kg) at t=0min, then injected with saline (negative control), compound T2 (0.4 mg / kg), protamine (2.6 mg / kg) at t=5min, then the mice were anesthetized with isoflurane and the distal tail of the mice was cut off at t=10min, the bleeding time and bleeding volume were recorded, the average values ​​were calculated and the significant differences of the data were compared.

[0099] Fig.28 The cytotoxicity test results of tetrahedral compound T1 on (a) H9C2 cell line and (b) MCF-7 cell line.

[0100] Fig.29 The cytotoxicity test results of tetrahedral compound T2 on (a) H9C2 cell line and (b) MCF-7 cell line.

[0101] Fig.30 The cytotoxicity test results of tetrahedral compound T3 on (a) H9C2 cell line and (b) MCF-7 cell line.

[0102] Fig.31 These are the results of the hemolytic toxicity experiment of tetrahedral compound T1-3 on human and rat erythrocytes.

[0103] Fig.32 The figure is a curve of the weight changes of mice after administration of different doses of T2 molecules (0.4 and 25 mg / kg).

[0104] Fig.33 These are the H&E tissue section results of the main organs of mice in the acute toxicity experiment of tetrahedral compound T2 (0.4 and 25 mg / kg) on ​​ICR mice. DETAILED DESCRIPTION

[0105] After extensive, in-depth and systematic research, the inventors have found that a cationic tetrahedral compound and a pharmaceutically acceptable salt thereof have a broad spectrum of activity in rapidly antagonizing anticoagulants such as unfractionated heparin and low molecular weight heparin. Plasma aPTT test and whole blood elastogram test confirm that such compounds can achieve rapid binding to heparin molecules in the blood environment. Animal experiments support that such compounds can achieve efficient and rapid antagonism of unfractionated heparin and low molecular weight heparin (dalteparin sodium, enoxaparin sodium, nadroparin calcium) anticoagulants in vivo. Further test results support that such compounds have high water solubility and high biosafety, and the antagonistic activity is significantly better than commercial protamine at the same dose, and has a wider antagonistic window. On this basis, the present invention has been completed.

[0106] The in vitro activity experiments in the present invention are carried out in plasma and whole blood, and the animal activity tests and maximum tolerated dose tests are carried out using ICR mice. The unique three-dimensional molecular structure can effectively avoid the precipitation of the complex and the toxicity. In vitro experiments on plasma and whole blood and in vivo experiments on mice confirm that rapid in vivo antagonism of long-chain heparin and short heparin can be achieved at a lower dose. Further biosafety test results confirm the high blood compatibility and high biosafety of the tetrahedral compound, and the therapeutic index is as high as 62.5.

[0107] the term

[0108] Heparin anticoagulants

[0109] Heparin is a linear sulfated glycosaminoglycan composed of repeating units of α-1,4-linked uronic acid and D-glucosamine, with the highest negative charge density among biological macromolecules.

[0110] Heparin can highly bind to antithrombin and significantly enhance the ability of antithrombin to inhibit the activity of serine protease coagulation factors (mainly coagulation factor Xa and thrombin) in the blood coagulation cascade.

[0111] Heparin drugs used clinically include unfractionated heparin (UFH, extracted from porcine intestinal mucosa or bovine lung, with an average molecular weight of approximately 15 kDa), low molecular weight heparins (LMWHs, small molecule fragments with an average molecular weight of approximately 3.6–6.5 kDa obtained by depolymerization of unfractionated heparin), and fondaparinux (molecular weight 1.7 kDa), which is a modified synthesis of the natural pentose structure contained in both UFH and LMWHs.

[0112] Active ingredients

[0113] As used herein, the terms "compound of the present invention" and "active ingredient of the present invention" are used interchangeably and refer to a tetrahedral compound of Formula I, wherein each substituent is as described above. It should be understood that the term also includes mixtures of the above components.

[0114]

[0115] In the present invention, pharmaceutically acceptable salts of the tetrahedral compound shown in Formula I are also included. The term "pharmaceutically acceptable salt" refers to a salt suitable for use as a drug formed by the compound of the present invention and an acid or a base. Pharmaceutically acceptable salts include inorganic salts and organic salts. A preferred class of salts is a salt formed by the polymer of the present invention and an acid. Acids suitable for forming salts include but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenesulfonic acid, benzenesulfonic acid, and acidic amino acids such as aspartic acid and glutamic acid.

[0116] Among them, Z n-It means that the compound of formula I may contain multiple anions (which may be anions of the same valence or anions of different valences), for example, the sum of the valences of the multiple anions is 0 (containing no anions), 4 (4 monovalent anions, or 2 divalent anions, or other combinations), 8 (8 monovalent anions, 4 divalent anions, or other combinations), or 12 (12 monovalent anions, 6 divalent anions, or other combinations), and is composed of anions of a pharmaceutically acceptable inorganic acid or organic acid selected from the group consisting of chloride ion, bromide ion, iodide ion, hydrogen sulfate ion, sulfate ion, phosphate ion, maleate ion, fumarate ion, tartrate ion, palmitate ion, oxalate ion, citrate ion, succinate ion, methanesulfonate ion, benzenesulfonate ion, p-toluenesulfonate ion, or a combination thereof.

[0117] The tetrahedral compound represented by Formula I of the present invention can be prepared by methods well known to those skilled in the art in the prior art, and there is no particular restriction on the reaction parameters of each step. In addition, the typical compound of the present invention can also be obtained by commercial means.

[0118] As used herein, in the tetrahedral compound represented by Formula I, if a chiral carbon atom exists, the chiral carbon atom may be in R configuration, S configuration, or a mixture of the two.

[0119] The tetrahedral compound represented by formula I of the present invention is composed of a tetraphenylmethane skeleton, a water-soluble nitrogen-containing heterocyclic ring (a pyridinium salt, an imidazole salt, a pyrimidine salt or ), a water-soluble side chain that modifies the aromatic ring structure; wherein the water-soluble side chain is selected from -OC 1-30 Alkylene -NH2, -SC 1-30 Alkylene -NH2, -NH-CO-C 1-30 Alkylene -NH2, -C 1-30 Alkylene-NH2; for example, aminoethoxy, aminopropoxy, aminoethylthio.

[0120] In a preferred embodiment, A is selected from the following group: Where Y is N + or C.

[0121] The tetrahedral compound represented by formula I of the present invention has a three-dimensional spatial structure, and different water-soluble modifying groups can be further introduced through chemical reactions, so that it can achieve broad-spectrum, efficient and rapid antagonism of unfractionated heparin and low molecular weight heparin anticoagulants through electrostatic interaction and hydrophobic interaction in various environments such as aqueous phase, physiological saline or biological medium (such as plasma, whole blood).

[0122] In the present invention, the tetrahedral compound shown in Formula I is used as a broad-spectrum antagonist for anticoagulants such as long-chain unfractionated heparin and short-chain low molecular weight heparin (dalteparin sodium, enoxaparin sodium, nadroparin calcium). In vitro experiments on plasma and whole blood and in vivo experiments on mice have confirmed that this application can achieve rapid antagonism of long-chain heparin and short-chain heparin. This class of compounds has high water solubility and high biosafety, and has significant clinical practicality.

[0123] The present invention confirms through isothermal calorimetric titration experiments and fluorescence titration experiments that the tetrahedral compound shown in formula I can achieve efficient binding to heparin molecules, showing its potential to antagonize heparin anticoagulants.

[0124] The invention confirms the in vitro high-efficiency antagonism of the tetrahedral compound to the heparin molecule through tests such as aPTT activated partial thromboplastin and thromboelastogram.

[0125] The invention confirms the in vitro high-efficiency antagonistic efficiency of the tetrahedral compound on heparin molecules through an in vivo mouse tail cross-section test.

[0126] The invention confirms the high biological safety of the tetrahedral compound through tests such as cytotoxicity, hemolysis of red blood cells and maximum tolerated dose of mice.

[0127] In the present invention, control experiments support that this type of novel tetrahedral compound can achieve faster antagonistic activity and a wider antagonistic window than the clinically used drug protamine in antagonizing unfractionated heparin.

[0128] In the present invention, control experiments support that this type of novel tetrahedral compound can achieve faster antagonistic activity against low molecular weight heparin than protamine, a clinically used drug.

[0129] In the present invention, control experiments with the clinically used drug protamine support that this type of tetrahedral compound has a significantly improved high-efficiency antagonistic window against unfractionated heparin and short-chain heparin.

[0130] In the present invention, this type of novel tetrahedral compound has a three-dimensional pre-organized structure of diamond configuration, which can be "stuck" on the linear heparin molecular chain through electrostatic interaction, thereby exerting an antagonistic effect.

[0131] The heparin antagonistic activity of the tetrahedral multivalent cationic molecule of the present invention is confirmed by in vitro plasma and whole blood antagonism tests.

[0132] The heparin antagonistic activity of the tetrahedral multivalent cationic molecule of the present invention was confirmed by an in vivo antagonism test in mice.

[0133] The present invention also provides a preparation method of the tetrahedral compound shown in formula I, which mainly involves a one-step coupling reaction and a two-step nucleophilic substitution reaction, specifically: (1) covalently coupling a nitrogen-containing heterocyclic molecule with a benzene ring or an aromatic heterocyclic ring with a modifiable site to obtain a precursor arm molecule; (2) introducing a water-soluble side chain, such as aminoethoxy, aminopropoxy, etc., at the modifiable site of the benzene ring or the aromatic heterocyclic ring through a nucleophilic substitution reaction to obtain a highly water-soluble arm molecule; (3) tetraphenylmethane tetrabenzyl bromide undergoes a nucleophilic substitution reaction with the water-soluble arm molecule, and then undergoes ion exchange to form a water-soluble tetrahedral compound shown in formula I with different counter anions.

[0134] Pharmaceutical compositions and methods of administration

[0135] The pharmaceutical composition of the present invention comprises a safe and effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable excipients or carriers. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Usually, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, and more preferably, contains 5-100 mg of the compound of the present invention per dose. Preferably, the "one dose" is a capsule or tablet.

[0136] "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatible" here means that the components in the composition can be mixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0137] There is no particular limitation on the administration of the compound or pharmaceutical composition of the present invention. Representative administration methods include (but are not limited to): oral administration, parenteral administration (intravenous administration, intramuscular administration or subcutaneous administration).

[0138] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

[0139] Solid dosage forms such as tablets, pills, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifiers, and the release of the active compound or compounds in such compositions can be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active compound can also be formed into microencapsulated form with one or more of the above-mentioned excipients.

[0140] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage form may contain inert diluents conventionally used in the art, water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.

[0141] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0142] Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances.

[0143] Compositions for parenteral injection may include physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0144] The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.

[0145] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage during administration is a pharmaceutically effective dosage, and for a person weighing 60 kg, the daily dosage is usually 1 to 2000 mg, preferably 5 to 100 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the health status of the patient, which are all within the skill range of a skilled physician.

[0146] Compared with the prior art, the main advantages of the present invention include:

[0147] (1) The cationic tetrahedral small molecule compound of the present invention can achieve a broad-spectrum, rapid and efficient antagonism of heparin anticoagulants, and has high antagonistic activity, high biocompatibility and high water solubility;

[0148] (2) The present invention designs a heparin antagonist molecule with multiple binding sites, which for the first time separates the binding sites of electrostatic interaction and hydrogen bonding. By dispersing the inherent positive ion skeleton on the tetraphenylmethane skeleton and connecting the amino-containing hydrogen bonding side chain to the end of the positive ion skeleton, this has a completely different structural feature from the antagonist developed based on arginine fragments. This positive charge structural feature not only makes it highly efficient in antagonistic activity, but the inherent charges dispersed on the rigid skeleton will also effectively avoid the toxicity of the usual ionic compounds after charge aggregation.

[0149] (3) The present invention adopts a two-level synergistic strategy to enhance the interaction between the tetrahedral compound and the heparin molecule. The synergistic electrostatic interaction of the four cationic side arms and the multivalent electrostatic interaction of the flexible amino side chain exert a synergistic effect, thereby achieving efficient antagonism of the heparin molecule.

[0150] (4) The tetrahedral cationic molecule of the present invention is a small chemical molecule with a clear molecular structure, simple synthesis, easy modification, and convenient mass preparation;

[0151] (5) The tetrahedral cationic molecule of the present invention has a wider antagonistic window than the commercial protamine antagonist, has a distinct antagonistic platform, and has a more efficient antagonistic efficiency against low molecular weight heparin;

[0152] (6) The tetrahedral cationic molecule of the present invention has high water solubility and can effectively avoid the toxicity caused by the precipitation of the complex after binding with the heparin molecule; the positive charge of the four side arms can not only maintain the water solubility of the complex, but also avoid the aggregation and precipitation of the complex.

[0153] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are generally performed under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0154] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0155] Example 1

[0156]

[0157] Weigh 3,4,5-trimethoxyphenylboronic acid (5.00 g, 23.6 mmol), 4-bromopyridine hydrochloride (5.50 g, 28.3 mmol) and potassium carbonate (9.80 g, 70.9 mmol), add (DMF) 50 mL and water (25 mL) to dissolve, add tetrakistriphenylphosphine palladium (28 mg, 24.2 μmol) under N2 protection, stir and react at 110 ° C for 16 h. TLC monitors the reaction progress (DCM:EA=1:1, R f =0.7). After the reaction, water (100 mL) and ethyl acetate (100 mL) were added for extraction, and water was reversely extracted (50 mL × 2). The organic phases were combined, washed with water (100 mL × 3), washed with saturated brine (100 mL × 2), and dried over anhydrous sodium sulfate. The solid obtained by removing the solvent was washed with n-hexane reflux, and dried to obtain compound 1-3 as a white powdery solid (5.0 g, 87%). 1 H NMR (400MHz, CDCl3): δ8.66 (d, J = 6.2Hz, 2H), 7.49 (d, J = 6.3Hz, 2H), 6.84 (s, 2H), 3.94 (d, J = 14.4Hz, 9H).

[0158] Weigh compound 1-3 (0.62 g, 2.53 mmol) and dissolve it in dichloromethane (10 mL). Cool it to 0°C in an ice bath, and slowly drop a dichloromethane (10 mL) solution of boron tribromide (1.5 mL, 15.6 mmol) under stirring. After the addition is complete, continue the reaction for 19 h. Monitor the reaction progress (EA, R f =0.1). After the reaction was completed, the reaction solution was added dropwise to ice water (100 mL), and a large amount of yellow precipitate was generated. The filter cake was collected by suction filtration and washed with a small amount of water to obtain compound 1-4 as a yellow solid (0.46 g, 90%). 1 H NMR (400MHz, CD3OD): δ8.67 (d, J = 7.1Hz, 2H), 8.14 (d, J = 7.1Hz, 2H), 6.99 (s, 2H).

[0159] Compound 1-4 (0.30 g, 1.47 mmol), Boc-bromoethylamine (1.98 g, 8.84 mmol) and cesium carbonate (2.88 g, 8.84 mmol) were weighed, and DMF (10 mL) was added and stirred at room temperature for 36 h. The reaction progress was monitored by TLC (DCM / MeOH=20:1, R f =0.6). After the reaction, the mixture was filtered, and water (100 mL) and ethyl acetate (50 mL) were added for extraction. The aqueous phase was reversely extracted (50 mL×2). The organic phases were combined, washed with water (50 mL×3), washed with saturated brine (50 mL), and dried over anhydrous sodium sulfate. After the solvent was removed, column chromatography was performed (polarity was gradually changed from DCM to DCM / MeOH=20:1). After drying, Boc-protected compound 1-6 was obtained as a white solid powder (0.79 g, 85%). 1 H NMR (400MHz, CDCl3): δ8.63(d,J=6.2Hz,2H),7.44(d,J=6.2Hz,2H),6.85(s,2H),5.77(s,1H),5.28(s,2H) ,4.13(dt,J1=11.4,J2=5.7Hz,6H),3.58(q,J=5.3Hz,4H),3.44(q,J=5.3Hz,2H),1.45(d,J=15.9Hz,27H). 13 C NMR (101MHz, CDCl3): δ156.12,155.97,153.16,150.23,148.17,138.07,1 34.61,121.70,106.31,79.99,79.83,72.79,68.96,40.79,40.29,28.53.

[0160] Weigh compound 1-6 (1.85 g, 2.93 mmol) and tetrakis(4-(bromomethyl)phenyl)methane (0.29 g, 0.42 mmol) in a 25 mL eggplant-shaped bottle, add dry DMF (12 mL) and react at 80 ° C for 16 h under nitrogen protection. After the reaction is completed, remove DMF by rotary evaporation, add a small amount of dichloromethane (5 mL) to dissolve the oil, and drop it into n-hexane (50 mL) to precipitate a large amount of yellow precipitate. Centrifuge, recrystallize the solid using dichloromethane and n-hexane, and disperse the obtained light yellow solid into 1.0 M HCl ethyl acetate solution (15 mL) and react at room temperature for 12 h. After the reaction is completed, filter with suction, wash the solid with ethyl acetate (20 mL × 3), and dry to obtain a light yellow solid. The solid was dissolved in water, and saturated ammonium hexafluorophosphate solution was added until no precipitation was generated. After stirring at room temperature for 1 hour, the mixture was filtered off with suction, washed with water (20 mL×3), and dried. The obtained solid was dissolved in acetonitrile, and saturated tetrabutylammonium chloride was added dropwise until no precipitation was generated. After stirring at room temperature for 1 hour, the mixture was filtered off with suction, washed with acetonitrile (50 mL×3), and dried to obtain tetrahedral compound T1 as a light yellow solid powder (211 mg, 74%). 1 H NMR (400MHz, D2O): δ8.71(d,J=6.9Hz,8H),8.16(d,J=7.2Hz,8H),7.40–7.24(m,16H),7.16(s ,8H),5.61(s,8H),4.34(s,16H),4.20(t,J=5.1Hz,8H),3.39(s,16H),3.25(t,J=5.0Hz,8H). 13 C NMR(101MHz,D2O): δ156.04,152.15,147.79,144.08,138.62,131.57,131 .21,130.86,129.00,125.46,107.44,69.69,65.47,63.16,39.51,38.93.

[0161] Example 2

[0162] According to the method in Example 1, the Boc amine ethoxy pyridine compound 2-6 with two side chains was used to replace the Boc amine ethoxy pyridine compound 1-6 with three side chains.

[0163]

[0164] Weigh 3,4-dimethoxyphenylboronic acid (3.30 g, 18.1 mmol), 4-bromopyridine hydrochloride (4.20 g, 21.6 mmol) and potassium carbonate (4.20 g, 30.4 mmol), add DMF (40 mL) and water (20 mL) to dissolve, add tetrakistriphenylphosphine palladium (22.0 mg, 19.2 μmol) under N2 protection, stir and react at 110 ° C for 16 h. TLC monitors the reaction progress (EtOAc, R f =0.7). After the reaction, water (200 mL) and ethyl acetate (200 mL) were added for extraction, and aqueous phase was reversely extracted (50 mL × 2). The organic phases were combined, washed with water (200 mL × 3), washed with saturated brine (100 mL × 2), and dried over anhydrous sodium sulfate. The solid obtained by removing the solvent was recrystallized with EtOAc / Hexane, and compound 2-3 was obtained after drying as transparent block crystals (3.3 g, 85%). 1 H NMR (400MHz, CDCl3): δ8.64 (d, J=6.1Hz, 2H), 7.50 (d, J=6.2Hz, 2H), 7.25 (dd, J1= 8.5Hz, J2=2.3Hz, 1H), 7.16 (d, J=2.1Hz, 1H), 6.99 (d, J=8.3Hz, 1H), 3.98 (s, 6H).

[0165] Weigh compound 2-3 (2.24 g, 10.4 mmol) and dissolve it in dichloromethane (20 mL). Cool it to 0°C in an ice bath, and slowly drop a dichloromethane (20 mL) solution of boron tribromide (5 mL, 51.9 mmol) under stirring. After the addition is complete, continue the reaction for 9 hours. Monitor the reaction progress (EA, R f =0.1). After the reaction was completed, the reaction solution was added dropwise to ice water (100 mL), and a large amount of yellow precipitate was generated. The filter cake was collected by suction filtration, and the filtrate was concentrated and the pH was adjusted to 1. Saturated brine was gradually added, and a large amount of yellow precipitate was generated. The solids were combined twice to obtain compound 2-4 as a yellow solid powder (1.93 g, 99%). 1 H NMR (400MHz, DMSO-d6): δ10.08(s,1H),9.45(s,1H),8.80(d,J=6.9Hz,2H),8.21(d,J=6.9Hz,2H),7.50–7.39(m,2H),6.97(d,J=8.0Hz,1H).

[0166] Compound 2-4 (0.60 g, 3.21 mmol), Boc-bromoethylamine (2.20 g, 9.82 mmol) and cesium carbonate (5.20 g, 16.0 mmol) were weighed, and DMF (20 mL) was added and stirred at room temperature for 20 h. The reaction progress was monitored by TLC (DCM / MeOH=20:1, R f =0.6). After the reaction, the mixture was filtered, and water (150 mL) and ethyl acetate (100 mL) were added for extraction. The aqueous phase was reversely extracted (50 mL × 2). The organic phases were combined, washed with water (100 mL × 3), washed with saturated brine (100 mL), and dried over anhydrous sodium sulfate. After the solvent was removed, column chromatography was performed (the polarity was gradually changed from DCM to DCM / MeOH = 20:1). After drying, compound 2-6 was obtained as a light yellow oily liquid (1.3 g, 86%). 1 H NMR (400MHz, CDCl3): δ8.60(d,J=4.7Hz,2H),7.44(d,J=4.8Hz,2H),7.30–7.14(m,2H),7.00(d,J=8.2Hz,1H),4.12(s,3H),3.54(s,3H),1.44(s,18H).

[0167]

[0168] Weigh tetrakis(4-(bromomethyl)phenyl)methane (0.32 g, 0.46 mmol) and arm molecule 2-6 (1.58 g, 3.33 mmol) and add DMF (10 mL) under nitrogen protection, and stir at 80 ° C for 12 h. After the reaction, spin-dry DMF, add a small amount of acetonitrile to dissolve the product, and then evaporate to a small amount of acetonitrile, add a large amount of low-temperature ether to disperse in 4.0M HCl ethyl acetate solution (20 mL, excess), and react at room temperature for 12 h. After the reaction is completed, filter and wash the solid with ethyl acetate (20 mL × 3), and dry to obtain a light yellow solid. The solid was dissolved in water, and saturated ammonium hexafluorophosphate solution was added until no precipitation was generated. After stirring at room temperature for 1 hour, the mixture was filtered off with suction, washed with water (20 mL×3), and the solid obtained after drying was dissolved in acetonitrile. Saturated tetrabutylammonium chloride was added dropwise until no precipitation was generated. After stirring at room temperature for 1 hour, the mixture was filtered off with suction, washed with acetonitrile (50 mL×3), and recrystallized from ethanol / water to obtain tetrahedron T2 as a light yellow solid powder (0.73 g, 94%). 1 H NMR (400MHz, D2O): δ8.63(d,J=7.2Hz,8H), 8.11(d,J=6.0Hz,8H), 7.51(d,J=8. 6Hz, 4H), 7.44 (s, 4H), 5.56 (s, 8H), 4.30 (t, J = 4.2Hz, 16H), 3.41–3.34 (m, 17H).13 C NMR (100MHz, D2O): δ155.69,150.75,147.64,143.80,131.52,128.87,124.39,123.21,113.80,113.00,65.44,65.08,62.84,38.84,38.74.

[0169] Example 3

[0170] According to the method in Example 1, the Boc amine ethoxy pyridine compound 6-4 with two side chains was used to replace the Boc amine ethoxy pyridine compound 1-6 with three side chains.

[0171]

[0172] Weigh 4-bromobenzenethiol (0.79 g, 4.18 mmol), potassium carbonate (0.87 g, 6.29 mmol) and Boc-bromoethylamine (0.92 g, 4.10 mmol) in a 100 mL eggplant-shaped bottle, add DMF (25 mL) and react at room temperature under nitrogen protection for 3 h, during which thin layer chromatography monitoring (DCM: Hexane = 1: 1) was used. After the reaction, water (100 mL) and ethyl acetate (50 mL) were added for extraction, and the aqueous phase was reversely extracted (50 mL × 2), the organic phase was combined, the organic phase was washed with water (100 mL × 2), washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and separated by column chromatography (eluent DCM) after removing the solvent to obtain compound 6-2 as a white solid (1.27 g, 91%). 1 HNMR (400MHz, CDCl3): δ7.42 (d, J = 8.4Hz, 2H), 7.25 (d, J = 8.4Hz, 2H), 3.32 (s, 2H), 3.03 (t, J = 6.4Hz, 2H), 1.45 (s, 9H).

[0173] Weigh compound 6-2 (0.5 g, 1.51 mmol), 4-pyridine boronic acid (0.21 g, 1.71 mmol) and potassium carbonate (0.63 g, 4.56 mmol) in a 25 mL eggplant-shaped bottle, add DMF (6 mL) and water (3 mL), add tetrakis triphenylphosphine palladium (8.5 mg, 0.5% eq. 7.36 μmol) under nitrogen protection, and then react at 80 ° C for 20 h under nitrogen atmosphere, during which thin layer chromatography monitoring (developing agent DCM) was used. After the reaction, water (100 mL) and ethyl acetate (50 mL) were added for extraction, and the aqueous phase was reversely extracted (50 mL × 2), the organic phase was combined, the organic phase was washed with water (100 mL × 2), washed with saturated brine (100 mL), dried over anhydrous sodium sulfate, and separated by column chromatography (eluent DCM: EA = 1: 1) after removing the solvent to obtain compound 6-4 as a light yellow solid (0.23 g, 46%). 1 H NMR (400MHz, CDCl3): δ8.67(d,J=6.1Hz,2H),7.59(d,J=8.4Hz,2H),7.52(d,J=6.1H z,2H),7.48(d,J=8.4Hz,2H),3.45–3.33(m,2H),3.13(t,J=6.5Hz,2H),1.45(s,9H). 13 C NMR (100MHz, CDCl3): δ155.81,149.83,148.14,137.64,135.59,129.44,127.64,121.48,39.78,33.50,28.47.

[0174]

[0175] Weigh compound 6-4 (0.6 g, 1.82 mmol) and tetrakis(4-(bromomethyl)phenyl)methane (0.2 g, 0.29 mmol) and dissolve in dry DMF (7 mL), and react at 80 ° C for 24 h under nitrogen protection. After the reaction is completed, cool to room temperature, and gradually add the reaction solution dropwise to ethyl acetate (60 mL), and a large amount of yellow precipitate is generated. Centrifuge, wash the solid with ethyl acetate (20 mL × 3) and ether (20 mL), disperse in 1.0M HCl ethyl acetate solution (20 mL, excess), and react at room temperature for 12 h. After the reaction is completed, filter, wash the solid with ethyl acetate (20 mL × 3), and dry to obtain a yellow solid. The solid was dissolved in water, and saturated ammonium hexafluorophosphate solution was added until no precipitation was generated. After stirring at room temperature for 1 hour, the mixture was filtered off with suction, washed with water (20 mL×3), and dried. The obtained solid was dissolved in acetonitrile, and saturated tetrabutylammonium chloride was added dropwise until no precipitation was generated. After stirring at room temperature for 1 hour, the mixture was filtered off with suction, washed with acetonitrile (50 mL×3), and dried to obtain aminethioxo tetrahedron T6 as a yellow solid powder (301 mg, 87%). 1 H NMR (400MHz, DMSO-d6): δ9.25(d,J=7.1Hz,8H),8.56(d,J=7.0Hz,8H),8.06(d,J=8.6Hz,8H),7.64(d,J=8 .6Hz,8H),7.48(d,J=8.5Hz,8H),7.21(d,J=8.5Hz,8H),5.81(s,8H),3.42(t,J=7.5Hz,8H),3.03(s,8H). 13 C NMR (100MHz, DMSO-d6): δ154.60,147.22,145.35,141.75,133.09,131.32,130.67,129.39,128.86,127.85,124.85,64.57,61.93,38.41,28.01.

[0176] Example 4

[0177] According to the method in Example 1, the Boc amine ethoxy pyridine compound 4-3 with two side chains was used to replace the Boc amine ethoxy pyridine compound 1-6 with three side chains.

[0178]

[0179] Add 4-1 (1.0 g, 5.8 mmol), anhydrous potassium carbonate (1.6 g, 8.7 mmol) and DMF (15 mL) to a 250 mL single-mouth bottle, add tert-butyloxycarbonyl protected bromopropylamine (2.1 g, 8.7 mmol) and DMF (15 mL) under uniform stirring, and react at room temperature overnight. After the reaction is completed, add ice water (30 mL), continue stirring for 20 minutes, and then filter to obtain a white solid product 4-3, which weighs 1.7 g after drying, and the yield is 89%. 1 H NMR (400MHz, DMSO-d6): δ8.58(d,J=5.2Hz,2H),7.78(d,J=8.0Hz,2H),7.67(d,J=5.2Hz,2H),7.07(d,J=8.4 Hz,2H),6.93(d,J=6.8Hz,1H),4.05(t,J=6.0Hz,2H),3.13-3.08(m,2H),1.86(t,J=6.4Hz,2H),1.38(s,9H). 13 C NMR (100MHz, DMSO-d6): δ160.08,156.08,150.55,146.91,129.52,128.47,120.94,115.51,77.94,65.85,37.32,29.59,28.68.

[0180]

[0181] Weigh compound 4-3 (1.30 g, 3.96 mmol) and tetrakis(4-(bromomethyl)phenyl)methane (0.46 g, 0.66 mmol) and dissolve in dry DMF (10 mL), and react at 90 ° C for 12 h under nitrogen protection. After the reaction is completed, cool to room temperature, and gradually add the reaction solution dropwise to acetonitrile (30 mL), and a large amount of white precipitate is generated. Then disperse it in 4.0M HCl ethyl acetate solution (15 mL, excess) and react at room temperature for 12 h. After the reaction is completed, filter and wash the solid with acetonitrile (20 mL × 3), and dry to obtain a white solid. The solid was dissolved in water, and saturated ammonium hexafluorophosphate solution was added until no precipitation was generated. After stirring at room temperature for 2 h, the mixture was filtered off with suction, washed with water (20 mL × 3), and dried. The obtained solid was dissolved in acetonitrile, and saturated tetrabutylammonium chloride (about 2 drops) was added dropwise until no precipitation was generated. After stirring at room temperature for 2 h, the mixture was filtered off with suction, washed with acetonitrile (50 mL × 3), and dried to obtain pyridinamine propoxy tetrahedron T4 as a white solid powder (308 mg, 72%). 1H NMR (400MHz, DMSO-d6): δ9.19(d,J=6.4Hz,8H),8.50(d,J=6.4Hz,8H),8.15-8.10(m,20H),7.45(d,J=8.4 Hz,8H),7.21-7.17(m,16H),5.77(s,8H),4.21(t,J=6.0Hz,8H),2.99-2.94(m,8H),2.08(t,J=6.8Hz,8H). 13 CNMR (100MHz, DMSO-d6): δ162.42,154.76,147.09,145.06,133.13,131.27, 130.64,128,71,125.83,124.03,116.13,65.83,64.50,61.65,36.48,27.12.

[0182] Example 5

[0183] According to the method in Example 1, the Boc amine ethoxy pyridine compound 7-2 with two side chains was used to replace the Boc amine ethoxy pyridine compound 1-6 with three side chains.

[0184]

[0185] Weigh 4-(Imidazol-1-yl)phenol (1.0 g, 6.24 mmol), potassium carbonate (1.73 g, 12.5 mmol) and Boc-bromoethylamine (2.8 g, 12.5 mmol), add DMF (15 mL) and dissolve under stirring. React at room temperature for 24 h under nitrogen protection, during which time it is monitored by thin layer chromatography (DCM:MeOH=20:1). After the reaction, potassium carbonate was removed by filtration, and water (200 mL) and ethyl acetate (50 mL) were added for extraction, and aqueous phase was reversely extracted (50 mL × 2). The organic phases were combined, washed with water (100 mL × 3), washed with saturated brine (100 mL), and dried over anhydrous sodium sulfate. After removing the solvent, the crude product was a yellow oily liquid, which was separated by column chromatography (DCM: MeOH = 40: 1). A small amount of dichloromethane was added to the obtained oily liquid to dissolve it, and then a large amount of n-hexane was added to precipitate a white solid. After filtration and drying, compound 7-2 was obtained as a white solid (1.2 g, 63%). 1 H NMR (400MHz, CDCl3): δ7.84 (s, 1H), 7.32 (d, J = 9.0Hz, 2H), 7.22 (s, 2H), 7.00 (d, J = 8.9Hz, 2H),5.00(s,1H),4.07(t,J=5.1Hz,2H),3.57(q,J=5.1Hz,2H),2.71(s,1H),1.47(s,9H). 13C NMR (101MHz, CHLOROFORM-D): δ158.03,155.98,135.89,131.07,130.14,123.32,118.81,115.56,79.75,77.34,67.66,40.11,28.47.

[0186]

[0187] Weigh compound 7-2 (0.6 g, 1.98 mmol) and tetrakis(4-(bromomethyl)phenyl)methane (0.23 g, 0.33 mmol) and dissolve in dry DMF (10 mL), and react at 90 ° C for 12 h under nitrogen protection. After the reaction is completed, cool to room temperature, and gradually add the reaction solution dropwise to ethyl acetate (60 mL), and a large amount of white precipitate is generated. Then disperse it in 4.0M HCl ethyl acetate solution (15 mL, excess) and react at room temperature for 12 h. After the reaction is completed, filter and wash the solid with acetonitrile (20 mL × 3), and dry to obtain a white solid. The solid was dissolved in water, and saturated ammonium hexafluorophosphate solution was added until no precipitation was generated. After stirring at room temperature for 2 h, the mixture was filtered off with suction, washed with water (20 mL × 3), and dried. The obtained solid was dissolved in acetonitrile, and saturated tetrabutylammonium chloride (about 2 drops) was added dropwise until no precipitation was generated. After stirring at room temperature for 2 h, the mixture was filtered off with suction, washed with acetonitrile (50 mL × 3), and dried to obtain imidazoleamine ethoxytetrahedron T7 as a white solid powder (290 mg, 77%). 1 H NMR (400MHz, D2O): δ9.13(s,4H),7.68(t,J=1.7Hz,4H),7.49(t,J=1.8Hz,4H),7.44(d,J=9.0Hz,8H),7.38(d,J=8 .4Hz,8H),7.29(d,J=8.4Hz,8H),7.08(d,J=9.0Hz,8H),5.35(s,8H),4.24(t,J=4.8Hz,8H),3.36(t,J=4.9Hz,8H). 13 C NMR(101MHz,D2O)δ158.62,147.84,134.03,131.52,131.33,128.89,128. 20,123.82,123.06,122.20,115.83,115.78,64.52,64.35,52.66,38.92.

[0188] Example 6

[0189] According to the method in Example 1, the Boc amine ethoxy pyridine compound 8-2 with two side chains was used to replace the Boc amine ethoxy pyridine compound 1-6 with three side chains.

[0190]

[0191] Weigh 6-hydroxyisoquinoline (3.00 g, 20.7 mmol), cesium carbonate (13.4 g, 41.1 mmol) and Boc-bromoethylamine (6.90 g, 30.8 mmol) in a 250 mL eggplant-shaped bottle, add DMF (60 mL) and react at room temperature under nitrogen protection for 24 h, during which thin layer chromatography monitoring (DCM: EtOAc = 1: 2) was used. After the reaction was completed, cesium carbonate was removed by suction filtration, water (300 mL) and ethyl acetate (100 mL) were added for extraction, and aqueous back extraction (50 mL × 2) was performed, the organic phases were combined, washed with water (100 mL × 3), washed with saturated brine (100 mL), and dried over anhydrous sodium sulfate. After removing the solvent, the crude product was a yellow oily liquid, which was separated by column chromatography (DCM: EtOAc = 1: 2) to obtain compound 8-2 as a white solid (4.80 g, 80%). 1 H NMR (400MHz, DMSO-d6): δ9.15(s,1H),8.40(d,J=5.7Hz,1H),8.02(d,J=9.0Hz,1H),7.69(d,J=5.8Hz,1H),7.35(d,J=2.1Hz ,1H),7.28(dd,J1=8.9Hz,J2=2.4Hz,1H),7.08(t,J=5.1Hz,1H),4.13(t,J=5.7Hz,2H),3.38(q,J=5.7Hz,2H),1.38(s,9H). 13 C NMR (101MHz, CDCl3): δ159.89,156.00,151.67,143.61,137.68,129.48,124.60,120.34,119.82,104.84,79.70,67.47,39.99.

[0192]

[0193] Weigh compound 8-2 (1.30 g, 4.51 mmol) and tetrakis(4-(bromomethyl)phenyl)methane (00.50 g, 0.72 mmol) and dissolve in dry DMF (10 mL). React at 80 °C for 24 h under nitrogen protection. After the reaction is completed, cool to room temperature and gradually add the reaction solution dropwise to ethyl acetate (60 mL). A large amount of white precipitate is generated. Centrifuge, wash the solid with ethyl acetate (20 mL × 3), and dry to obtain compound 8-3 as a white solid (1.26 g, 95%).

[0194] Weigh compound 8-3 (0.40 g, 0.22 mmol) and disperse it in 1.0 M HCl ethyl acetate solution (20 mL, excess), and react at room temperature for 12 h. After the reaction is completed, filter with suction, wash the solid with ethyl acetate (20 mL × 3), and dry to obtain a white solid. Dissolve the solid in water, add saturated ammonium hexafluorophosphate solution until no precipitation occurs, stir at room temperature for 1 h, filter with suction, wash with water (20 mL × 3), and dry the obtained solid, dissolve it in acetonitrile, dropwise add saturated tetrabutylammonium chloride until no precipitation occurs, stir at room temperature for 1 h, filter with suction, wash with acetonitrile (50 mL × 3), and dry to obtain aminoethoxyisoquinoline tetrahedron T6 as a white solid powder (0.91 g, 91%). 1 H NMR (400MHz, D2O): δ9.40 (s, 4H), 8.23 ​​(d, J = 6.9Hz, 4H), 8.16 (d, J = 9.2Hz, 4H), 8.06 (d, J = 6.9Hz, 4H), 7.52 (dd, J1 = 9.2Hz, J2 = 2.2Hz ,4H),7.48(d,J=2.0Hz,4H),7.32(d,J=8.4Hz,8H),7.25(d,J=8.4Hz,8H),5.66(s,8H),4.45(t,J=4.8Hz,8H),3.46(t,J=4.8Hz,8H). 13 C NMR (101MHz, D2O): δ164.34,147.59,147.18,140.25,134.18,132.21,131.75, 131.55,128.63,124.64,124.29,123.20,106.43,65.17,64.49,62.95,38.71.

[0195] Example 7

[0196] Binding capacity testing: isothermal calorimetric titration and fluorescence titration experiments

[0197] Compound T2 is used as an example to illustrate the isothermal calorimetric titration experiment and fluorescence titration experiment of tetrahedral multivalent cationic molecules and heparin in physiological saline, in which the disaccharide repeating unit of the heparin molecule is used as the molar concentration unit to replace the polymer heparin.

[0198] Isothermal calorimetric titration experiment: First, tetrahedral compounds and heparin were prepared into physiological saline solution. The specific titration process was as follows: 50 μM compound T2 molecules (200 μL) were placed in the sample pool, and the titration needle contained unfractionated long-chain heparin UFH (1 mM), or dalte sodium Dalte (0.75 mM), or enoxa (0.5 mM), or nadroparin calcium Nadro (0.5 mM). The total number of titrations was 20 times, of which the first drop was 0.5 μL, and the subsequent 19 drops were 2 μL. The drop interval was 2 minutes. The heat released by each needle was recorded at T = 298.15 K, and the stirring speed was set to 750 rpm. After obtaining the titration binding diagram, the equilibrium binding constant was calculated and determined by the 1:1 binding model in the Malvern MicroCal ITC200 analysis software after data fitting correction.

[0199] Fluorescence titration experiment: First, tetrahedral compounds and heparin are prepared into physiological saline solution. The specific titration process is as follows: 5μM compound T2 molecules (2mL) are placed in a cuvette, and unfractionated long-chain heparin UFH or low molecular weight heparin (1.0mM) is added dropwise until the fluorescence intensity no longer changes. At a wavelength of 330nm, heparin itself has no fluorescence. It can be seen that with the addition of heparin molecules, the fluorescence gradually increases, and the fluorescence is enhanced by about 2.5 times when equilibrium is finally reached.

[0200] like Figure 13-15 The ITC titration fitting results showed that the binding constant of compound T2 with UFH was (2.15±0.86)×10 7 M -1 The binding constant with Dalte is (1.51±0.67)×10 7 M -1 The binding constant with Enoxa is (2.31±0.89)×10 7 M -1 The binding constant with Nadro is (2.46±0.74)×10 7 M -1 The binding constants of compounds T1 and T3 to heparin are ~10 6 M -1 .like Fig.16 Fluorescence titration results showed that one tetrahedral molecule could bind 1.8-3.0 heparin disaccharide repeating units.

[0201] The above results indicate that tetrahedral molecules represented by compound T2 have strong binding ability to both unfractionated heparin and low molecular weight heparin anticoagulant drug heparin.

[0202] Example 8

[0203] In vitro antagonist test (1): activated partial thromboplastin test (aPTT)

[0204] The in vitro aPTT test in the present invention uses 3.8% sodium citrate anticoagulated poor platelet plasma (PPP) for related experiments. This experiment can also be performed using animal plasma, such as cattle and sheep plasma. Among them, human poor platelet plasma (PPP) comes from scientific research blood approved by Shanghai Blood Center.

[0205] The present invention uses compound T2 as an example to illustrate the experimental process of the activated partial thromboplastin test of tetrahedral multivalent positive ion molecules and heparin in plasma. The present invention uses a fully automatic coagulation analyzer (UP3000, Shanghai Sun Biotechnology) to perform aPTT experiment, and automatically tests the "PPP-heparin-antagonist system" according to the manufacturer's instructions, using low platelet plasma as the experimental medium.

[0206] First, heparin or saline was mixed with low platelet plasma at a ratio of 1:99, and the obtained heparinized plasma was incubated in a 37°C water bath for 5 minutes. Then 18 μL of tetrahedral molecule solution or saline solution of protamine in a certain gradient concentration was added to 182 μL of heparinized plasma (10%, v / v), and the final concentration of unfractionated heparin and low molecular weight heparin was 2 IU / mL of heparin solution. Among them, "PPP-saline" was a blank control, "PPP-heparin-saline" was a negative control, and "PPP-heparin-protamine" was a positive control.

[0207] The first step is to establish a standard curve. First, heparinized plasma (4 IU / mL) was prepared, and then doubled to obtain heparin samples with gradient concentrations (0.078-4.0 IU / mL) for the activated partial thromboplastin test. . Test procedure: The fully automatic coagulation analyzer was preheated to 37°C for half an hour. Before the test, a quality control test was performed to calibrate the analyzer. After starting the test, the sample (0.2 mL) was manually added to the sample cup and the automatic program was started. 50 μL of sample was automatically drawn from the sample cup and added to the test cup through the sampling probe. Then, the sample was incubated at 37°C for 50 seconds. Subsequently, 50 μL of preheated (37°C) activated partial thromboplastin test reagent was added and incubated for 190 seconds. Finally, 50 μL of 25 mM pre-incubated calcium chloride solution was added and the clotting time was recorded. Finally, the antagonistic effect of tetrahedral multivalent positive ion molecules on drug-like heparin was calculated with the standard curve.

[0208] like Figure 17-22The results of aPTT experiments showed that at the same dose, the highest antagonistic efficiency of protamine against unfractionated heparin (UFH) reached more than 95%, while the antagonistic efficiency against low molecular weight heparin (Dalte, Enoxa, Nadro) could only reach 60% to 80%. Moreover, the "peak-shaped" antagonistic efficiency caused the antagonistic efficiency of protamine to drop sharply after deviating from the maximum efficiency, which is consistent with the fact that the dosage of protamine is difficult to control in clinical practice.

[0209] In contrast, tetrahedral compounds T1-T6 showed a wider antagonistic window against unfractionated heparin and low molecular weight heparin, and showed a clear antagonistic platform within a certain concentration range, indicating that tetrahedral compounds are easier to set and control the antagonistic dosage; among them, T2 molecule showed the highest antagonistic efficiency against low molecular weight heparin (Dalte, Enoxa, Nadro), reaching 100%. The above results show that tetrahedral multivalent cationic molecules have better antagonistic activity and dosage window than commercial protamine.

[0210] Example 9

[0211] In vitro antagonist test (2): thromboelastography (TEG)

[0212] The in vitro TEG test in the present invention uses 3.8% sodium citrate anticoagulated whole blood for relevant experiments. This experiment can also be performed using animal plasma, such as cattle and sheep plasma. Among them, human whole blood comes from scientific research blood approved by Shanghai Blood Center.

[0213] The present invention uses compound T2 as an example to illustrate the thromboelastogram results of tetrahedral multivalent positive ion molecules in human whole blood. The experiment used cryopreserved blood from healthy volunteers provided by Shanghai Blood Center, which was sealed and collected in a 200mL whole blood collection bag, and anticoagulated with 3.8% sodium citrate, with a blood / anticoagulant ratio of 9:1. The thromboelastogram was performed on the Hamma T4 instrument of Shenzhen McLean, and the final concentration of heparinized whole blood was 2IU / mL.

[0214] The thromboelastometry test method of the antagonist neutralizing heparin in the present invention is as follows: 1) First, mix whole blood (1 mL) with 10.1 μL of heparin (200 IU / mL heparin sodium; 200 IU / mL dalteparin sodium; 200 IU / mL enoxaparin sodium; 200 IU / mL nadroparin calcium) or saline, and incubate at 37°C for 5 minutes. 2) Then add 112.2 μL of antagonist solution or saline, mix, and incubate for another 5 minutes. The test concentration of the tetrahedral compound is 3-20 μg / mL. 3) Pipette 1 mL of the above mixed whole blood into a plastic tube containing kaolin activator, mix it upside down five times, and then transfer 340 μL of the mixed solution into a reaction cup preheated to 37°C (20 μL of 0.2 mol / L calcium chloride solution needs to be added to the reaction cup in advance). 4) Immediately start the thromboelastometry measurement for not less than 60 minutes. Then the test was started, and the thrombus strength curves that changed over time were collected and plotted. "Whole blood-normal saline" was the blank control, "whole blood-heparin-normal saline" was the negative control, and "whole blood-heparin-protamine" was the positive control.

[0215] like Figure 23-24 As shown, the experimental results show that the windows of tetrahedral compound T3 for antagonizing unfractionated heparin and low molecular weight heparin are 12-29μg / mL and 10-25μg / mL, respectively. The antagonistic window of tetrahedral compound T2 for antagonizing unfractionated heparin to restore whole blood to normal is 4-16ug / mL, the window for antagonizing dalteparin sodium is 4-16ug / mL, the window for antagonizing enoxaparin sodium is 4ug / mL-14ug / mL, and the therapeutic window for antagonizing nadroparin calcium is 4ug / mL-14ug / mL. In contrast, T2 has a lower antagonistic dose and has obvious advantages. In addition, compared with protamine, tetrahedral compounds have higher antagonistic efficiency and a wider antagonistic window, which is consistent with the results of aPTT experiments.

[0216] Example 10

[0217] In vitro antagonist activity test: coagulation inversion test

[0218] Compound T2 and unfractionated heparin are used as examples to further illustrate the in vitro antagonistic activity of tetrahedral compounds. Fresh whole blood from rats was collected and anticoagulated with 3.8%wt sodium citrate, then activated with 0.02mM calcium chloride, and the activated whole blood was divided into four portions and transferred to 2.0mL centrifuge tubes. An equal volume (50μL) of normal saline, unfractionated heparin (200IU / mL), unfractionated heparin (200IU / mL) + T2 (10μg / mL), and unfractionated heparin (200IU / mL) + T2 (15μg / mL) were added respectively. Gently shake the centrifuge tube 5 times and let it stand. After 15 minutes, invert the centrifuge tube to observe whether the whole blood is agglutinated.

[0219] like Fig.25 As shown, at a dose of 10-15 μg / mL, T2 was able to achieve complete antagonism of unfractionated heparin and low molecular weight heparin.

[0220] Embodiment 11

[0221] In vivo antagonistic activity test: mouse tail cutting test

[0222] Compound T2 is used as an example to illustrate the mouse tail cutting experiment process of tetrahedral compounds. A group of mouse tail cutting experiments used 40 ICR mice (half male and half female, weighing 18-24g), which were randomly divided into four groups, namely, a "normal saline" control group, a "heparin" (unfractionated heparin UFH, low molecular weight dalteparin Dalte) negative control group, a "heparin-protamine" positive control group, and a "heparin-tetrahedral compound" test group.

[0223] Each group had 10 ICR mice, half of them were male and half were female. The injection dose of heparin was 200 IU / kg, the injection dose of tetrahedral compound T2 was 0.4 mg / kg, and the injection dose of protamine was 2.6 mg / kg, all of which were prepared with physiological saline, and the injection volume was 0.1-0.2 mL.

[0224] The specific experimental process is as follows: 1) At 0 minutes, saline or heparin was injected through the tail vein, and the injection concentration of unfractionated heparin and dalteparin sodium was 30 IU / mL, 0.1-0.2 mL); 2) At 5 minutes, a second injection (saline or heparin antagonist, 0.1-0.2 mL) was performed through the tail vein; 3) At 7 minutes, the mice were anesthetized by inhaling a 5% mixed isoflurane-air mixture, and then the proportion of mixed isoflurane was reduced to 1.5% at 8 minutes to maintain the anesthesia of the mice ; 4) At the 10th minute of the experiment, the scalpel was cleaned and disinfected, and then the tail of each mouse was cut off 2.5 mm above the tail with the scalpel, and the blood was collected with a circular filter paper with a radius of 1 cm. The bleeding time of the mouse was recorded until there was no new bleeding mark on the filter paper within 1 minute; the filter paper was placed in 2 mL of 10 wt % NaOH aqueous solution to lyse the red blood cells, and the amount of bleeding in each test tube was calculated by the microplate reader according to its absorbance at (405 nm), and the total amount of bleeding and coagulation time of the mouse were calculated together.

[0225] like Fig.26 and 27 The experimental results showed that at a lower therapeutic dose (0.4 mg / kg), tetrahedral compounds represented by compound T2, that is, mice with heparin overdose, completely recovered to normal in terms of bleeding time and bleeding volume, with no significant difference from the normal saline group (bleeding time 20.9 min, bleeding volume 20.5 μL).

[0226] Compared with the negative control group of "heparin + saline" (bleeding time 87min, bleeding volume 324.4μL), the coagulation time and bleeding volume of the "heparin + T2" experimental group (bleeding time 22min, bleeding volume 26.1μL) were significantly different; compared with the positive control group of protamine antagonizing heparin (bleeding time 44min, bleeding volume 116.0μL), there were significant differences in coagulation time and bleeding volume. This verifies the excellent antagonistic activity of tetrahedral compounds in vivo.

[0227] Example 12

[0228] In vitro biosafety: cytotoxicity assay

[0229] Compound T2 is used as an example to illustrate the cytotoxicity experimental process of tetrahedral compounds. Using the Cell Counting Kit-8 (CCK-8), we used normal rat cardiomyocytes (H9C2) and human breast cancer cells (MCF-7) as models to study the in vitro cytotoxicity of compound T2. Figure 28-30 The results showed that when the sample contents of compound T1-3 were 200 μg / mL and 300 μg / mL, respectively, the survival rates of normal rat cardiomyocytes H9C2 could be maintained above 96% and 98%, respectively, and the survival rates of MCF-7 cells could be maintained above 93% and 80%, respectively. The threshold concentration of cytotoxicity was much higher than the concentration of in vitro and in vivo antagonism tests, further indicating the high biosafety of tetrahedral compounds.

[0230] Example 13

[0231] Blood compatibility test: hemolysis test

[0232] The hemolytic toxicity experiment process of tetrahedral compounds is illustrated by taking compound T2 as an example. We used rat erythrocytes and human erythrocytes as models to study the hemolytic toxicity of compound T2 through an ELISA instrument. The T2 molecule was prepared into a stock solution of physiological saline of different concentrations and added to a 5% erythrocyte solution (10%, v / v). After incubation at 37°C for 1 hour, the supernatant was collected by centrifugation at 3000rpm for 15 minutes for ELISA testing. Pure water was added to the positive control group, and physiological saline was added to the negative control group. The hemolysis rate of different concentrations of T2 was calculated accordingly.

[0233] like Fig.31 As shown, compounds T1-3 had no hemolytic toxicity below 256 μg / mL, showing good blood compatibility.

[0234] Embodiment 14

[0235] In vivo biosafety: maximum tolerated dose study in mice

[0236] The present invention takes the maximum tolerated dose of compound T2 by ICR mice as an example to investigate the in vivo biosafety of tetrahedral compounds. The specific operation process is as follows: ICR mice weighing 18-24 grams are selected as biological models to test the biological toxicity of compound T2 (three male mice and three female mice are selected for each group of experiments); weigh and record the initial weight of mice; inject the normal saline injection of compound T2 into mice through the tail vein at a time, and the doses are 0mg / kg, 15mg / kg, 20mg / kg, 25mg / kg, respectively, and the injection volume is controlled at 0.2-0.3mL, and the injection is slow (injected within 50 seconds); then weigh and record the changes in the weight of mice every 48 hours, and continue to record for 14 days. After the observation, the mice were dissected, and the tissue morphology of the organs was observed using a fluorescence microscope after the main organs were sliced ​​and stained.

[0237] like Fig.32 The test results showed that the maximum tolerable dose of the cationic tetrahedral compound T2 in ICR mice was 25 mg / kg. Under this high dose condition, there was no death in the mice; and from the perspective of body weight changes, the weight growth trend of the experimental group injected with compound T2 was consistent with that of the saline control group, further indicating the in vivo biosafety of the tetrahedral compound T2.

[0238] like Fig.33 According to the slice results, no organ damage was found in mice at the maximum tolerated dose (25 mg / kg) after 14 days, which was consistent with the tissue slice results of the blank group. The therapeutic index (TI) was calculated based on the effective dose (0.4 mg / kg) and the maximum tolerated dose (25 mg / kg). The therapeutic index of compound T2 was greater than 62.5, which means that under the condition of use far below the maximum tolerated dose, compound T2, as a heparin anticoagulant used in vivo, showed extremely high biosafety.

[0239] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A use of a cationic tetrahedral compound of formula I, or a pharmaceutically acceptable salt, hydrate, solvate, or crystal form thereof, characterized in that: Antagonists for the preparation of heparin anticoagulants; in, A is A cationic salt of, wherein X is a pyridinium salt, an imidazolium salt or a pyrimidinium salt; Wherein, R1, R2, R3, R4 and R5 are each independently H, -OC 1-30 Alkylene -NH2, -SC 1-30 Alkylene -NH2, -NH-CO-C 1-30 Alkylene -NH2, C 1-30 Alkylene -NH2; Y is C or N + ; Z n- The anion or anion combination whose total anion valence n is 0, 4, 8 or 12 is composed of anions of pharmaceutically acceptable inorganic or organic acids selected from the following groups: chloride ion, bromide ion, iodide ion, bisulfate ion, sulfate ion, phosphate ion, maleate ion, fumarate ion, tartrate ion, palmitate ion, oxalate ion, citrate ion, succinate ion, methanesulfonate ion, benzenesulfonate ion, p-toluenesulfonate ion, or a combination thereof.

2. The use according to claim 1, characterized in that X is 3. The use according to claim 1, characterized in that R1, R2, R3, R4 and R5 are each independently H, -OC 1-15 Alkylene -NH2, -SC 1-15 Alkylene -NH2, -NH-CO-C 1-15 Alkylene -NH2, C 1-15 Alkylene-NH2.

4. The use according to claim 1, characterized in that In the structure of formula I, at least one substituent in each A (such as R1, R2, R3, R4 and R5) is not H, preferably, at least two substituents in each A are not hydrogen, and more preferably, at least three substituents in each A are not hydrogen.

5. The use according to claim 1, characterized in that R3 is independently selected from the following group: -OC 1-6 Alkylene -NH2, -SC 1-6 Alkylene -NH2, -NH-CO-C 1-6 Alkylene-NH2; R1, R2, R4 and R5 are each independently H, -OC 1-6 Alkylene -NH2, -SC 1-6 Alkylene -NH2, -NH-CO-C 1-6 Alkylene-NH2.

6. The use according to claim 1, characterized in that The compound is selected from the following group:

7. The use according to claim 1, characterized in that The heparin anticoagulant is selected from the following group: unfractionated heparin, low molecular weight heparin, or a combination thereof.

8. A pharmaceutical composition or preparation for antagonizing heparin anticoagulant drugs, characterized in that: The pharmaceutical composition or preparation comprises: (a) a tetrahedral compound of formula I as an active ingredient, or a pharmaceutically acceptable salt, hydrate, solvate, or crystal form thereof; and (b) a pharmaceutically acceptable carrier; Among them, A, Z n- As claimed in claim 1.

9. Use of a tetrahedral compound represented by formula I and a protamine composition, characterized in that: Antagonists for the preparation of heparin anticoagulant drugs; Wherein, the tetrahedral compound represented by formula I is as follows: Among them, A, Z n- As described in claim 1.

10. A cationic tetrahedral compound of formula I, or a pharmaceutically acceptable salt, hydrate, solvate, or crystal form thereof, in, A. Z n- As claimed in claim 1; Wherein, the tetrahedral compound represented by formula I is not:

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