Preparation and application of 6-site polysubstituted aminoethoxy phenyl pyridinium salt-gamma-cyclodextrin

Abstract

The invention provides 6-site polysubstituted aminoethoxy phenyl pyridinium salt-gamma-cyclodextrin with a structure as shown in a formula (I). The preparation method of the 6-site poly-substituted aminoethoxy phenyl pyridinium salt-gamma-cyclodextrin is simple and convenient, and the prepared 6-site poly-substituted aminoethoxy phenyl pyridinium salt-gamma-cyclodextrin has good biocompatibility, and can be used for preparing the 6-site poly-substituted aminoethoxy phenyl pyridinium salt-gamma-cyclodextrin. In in-vitro pig plasma experiments and in-vivo mouse experiments, the 6-site polysubstituted aminoethoxy phenyl pyridinium-gamma-cyclodextrin can well neutralize heparin and shows neutralizing activity better than that of protamine, and the cyclodextrin derivative has wide application prospects in the fields of biology and medicine.

Description

Technical Field

[0001] The invention relates to the field of biology and medical technology, in particular to the preparation and application of 6-polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin. Background Art

[0002] Heparin is a naturally occurring anionic polysaccharide widely used as an anticoagulant in clinical surgical procedures. In order to quickly restore coagulation function after surgery involving heparin, protamine is currently the only FDA-approved antagonist. However, the clinical application of protamine is limited because it can only partially neutralize low molecular weight heparin and has little effect on fondaparinux sodium, and its production and quality are also affected by factors such as source, climate and environmental conditions. In addition, protamine may also cause side effects, including hypotension, bradycardia, thrombocytopenia and allergic reactions. Therefore, the search for anti-heparin coagulants that have a good neutralization effect on heparin, are synthesized by chemical methods and have good biocompatibility has attracted widespread attention. See: 1)F.Lin,S.-B.Yu,Y.-Y.Liu,C.-Z.Liu,S.Lu,J.Cao,Q.-Y.Qi,W.Zhou,X.Li,Y.Liu,J.Tian,Z.-T.Li,Adv.Mater.2022,34,2200549; 2)Q .Huang,H.Zhao,M.Shui,D.-S.Guo,R.Wang,Chem.Sci.2020,11,9623; 3)P.-Y.Li,Y.Chen,C.-H.Chen,Y.Liu,Chem.Commun.2019,55,11790.

[0003] Cyclodextrins are widely used in the biomedical field due to their low cost, easy modification, good biocompatibility, etc. Multi-charged cyclodextrins have been successfully used to mitigate the adverse effects associated with drug overdose and toxicity, thereby promoting the restoration of normal metabolic levels.

[0004] Therefore, it is very necessary to provide a cyclodextrin derivative host molecule as a heparin antagonist. Summary of the invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a cyclodextrin derivative host molecule as a heparin antagonist. The product prepared by the present invention exhibits stronger neutralizing activity and a wider therapeutic window than protamine in antagonizing heparin, and has good biocompatibility.

[0006] The inventors can obtain cyclodextrin derivatives with strong neutralizing activity against heparin by modifying specific functional groups on cyclodextrin. The product of the present invention not only exhibits strong neutralizing activity against ordinary heparin and low molecular weight heparin, but also has good biocompatibility, which provides an effective strategy for developing cyclodextrin host molecules as drug antagonists.

[0007] The present invention provides a 6-position polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin of formula (I):

[0008]

[0009] Wherein, m is an integer from 1 to 6, specifically 1, 2, 3, 4, 5 or 6;

[0010] n is 1 or 2.

[0011] The degree of substitution of 6-position polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin is 6, and the chemical formula is C 126 H 158 I6N 12 O 40 .

[0012] Since it exhibits stronger neutralizing activity and a wider therapeutic window than protamine in antagonizing heparin and has good biocompatibility, this molecule has potential clinical application as an anti-heparin coagulant.

[0013] The high charge density of the pyridinium unit and the partially protonated amino group (NH3 + ) will interact with the sulfonate and carboxylate anions of heparin through electrostatic interactions. In addition, some unprotonated amino groups (NH2) can further enhance the binding affinity with heparin through hydrogen bonding interactions.

[0014] The advantages of the present invention are: the 6-position polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin is easy to prepare, and only one supramolecular host molecule can achieve strong binding to heparin; the 6-position polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin has good biocompatibility. From the perspective of antagonistic effect, the 6-position polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin can show strong neutralizing activity to four types of heparins, namely, ordinary heparin sodium (UFH), dalteparin sodium (Dalte), enoxaparin sodium (Enoxa), and nadroparin calcium (Nadro) in a wider concentration range, and the neutralizing activity does not decrease significantly when the concentration increases, which is significantly better than protamine, the only heparin antagonist currently approved in clinical practice. In addition, the mouse tail cross-section experiment further shows the neutralizing effect of the 6-position polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin on excess heparin. Therefore, the 6-position polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin has broad application prospects in the fields of biological and drug treatment.

[0015] The present invention provides a method for preparing 6-position polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin of formula (I), comprising the following steps:

[0016] A) dissolving tert-butyl (2-(4-(pyridin-4-yl)phenoxy)ethyl)carbamate and octa-(6-iodo-6-deoxy)-γ-cyclodextrin in an organic solvent, and reacting them under an inert gas environment to obtain 6-position multi-substituted Boc-ethoxyphenylpyridinium salt-γ-cyclodextrin;

[0017] B) Dissolving 6-position polysubstituted Boc-ethoxyphenylpyridinium salt-γ-cyclodextrin in a hydrogen chloride ethyl acetate solution, stirring, centrifuging and drying to obtain 6-position polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin.

[0018] The preparation method of 6-position multi-substituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin of the structure of formula (I) provided by the present invention is firstly to dissolve tert-butyl (2-(4-(pyridin-4-yl)phenoxy)ethyl)carbamate and octa-(6-iodo-6-deoxy)-γ-cyclodextrin in an organic solvent; the organic solvent includes but is not limited to DMF.

[0019] According to the present invention, the molar ratio of tert-butyl (2-(4-(pyridin-4-yl)phenoxy)ethyl)carbamate and octa-(6-iodo-6-deoxy)-γ-cyclodextrin is 13 to 18:1; specifically, it can be 13:1, 14:1, 15:1, 16:1, 17:1, 18:1; or a range value between any two of the above.

[0020] In some preferred embodiments of the present invention, the reaction temperature is 90-110° C. and the reaction time is 40-50 h.

[0021] In some preferred embodiments of the present invention, the reaction temperature is 100° C. and the reaction time is 45 to 50 h.

[0022] In one preferred embodiment of the present invention, the reaction temperature is 100° C. and the reaction time is 48 h.

[0023] After the reaction is completed, the reaction vessel is cooled to room temperature. Next, most of the DMF is removed by spin drying, and the residue is dropped into an acetone solution, and then the solid obtained by centrifugation is washed with acetone and vacuum dried to obtain 6-position multi-substituted Boc-ethoxyphenylpyridinium salt-γ-cyclodextrin with a protective group.

[0024] The 6-position multi-substituted Boc-ethoxyphenylpyridinium salt-γ-cyclodextrin is dissolved in a hydrogen chloride ethyl acetate solution.

[0025] In the present invention, the concentration of the ethyl hydrogen chloride solution is 1.5-2.5M; preferably, the concentration of the ethyl hydrogen chloride solution is 2M.

[0026] The concentration of the 6-position multi-substituted Boc-ethoxyphenylpyridinium salt-γ-cyclodextrin dissolved in the hydrogen chloride ethyl acetate solution is 4 mg·mL -1 .

[0027] According to the present invention, the stirring is performed at room temperature for 1.5 to 2.5 hours; preferably, the stirring is performed at room temperature for 2 hours; the room temperature in the present invention is preferably 15 to 35°C.

[0028] The solid obtained by centrifugation after the reaction was washed with ethyl acetate and dried in vacuo to obtain a yellow-brown product, 6-polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin.

[0029] The present invention does not limit the specific parameters of centrifugation, which are well known to those skilled in the art.

[0030] The vacuum drying parameters of the present invention are vacuum drying at 40° C. for 8 hours.

[0031] The present invention provides the use of 6-polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin of formula (I) or 6-polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin prepared by the preparation method described in any one of the above technical schemes in neutralizing heparin.

[0032] According to the present invention, the heparin includes unfractionated heparin sodium, dalteparin sodium, enoxaparin sodium or nadroparin calcium.

[0033] The present invention also provides a heparin antagonist product, the raw materials of which include 6-polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin of the structure of formula (I) described in the above technical scheme or 6-polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin prepared by the preparation method described in any one of the above technical schemes.

[0034] The heparin antagonist product of the present invention includes medicines or health products.

[0035] The dosage form of the product of the present invention is not limited, and can be tablets, pills, capsules, syrups, drop pills or granules. The product of the present invention also includes pharmaceutically acceptable excipients, which are not limited.

[0036] The present invention preferably measures the neutralizing activity by an activated partial thromboplastin time test, and the specific method is:

[0037] 1.) Preparation of a series of heparinized plasma: Heparin solution (200 IU mL -1 , dissolved in saline) was diluted with porcine plasma to obtain a concentration of 2 IU·mL-1 Heparinized plasma of different concentrations was prepared by half dilution method, with the concentration range of 0.03125-2IU·mL -1 .

[0038] 2.) Preparation of a series of antagonized plasma: dilute to a concentration of 0.6 mg mL -1 6-polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin solution or protamine solution (dissolved in physiological saline) to obtain a series of 6-polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin solution or protamine solution with a concentration gradient (0.06-0.6 mg·mL -1 ) 10 groups each. Subsequently, the above solution (10 μL) was added to heparinized plasma (90 μL) to obtain antagonized plasma to be tested, which were numbered 1 to 10.

[0039] 3.) Neutralization activity test: Place the test plasma (100μL) and aPTT ellagic acid solution (100μL) in a 37°C water bath and incubate for 5 minutes. Then, mix the two and continue to incubate at 37°C for 5 minutes. Next, add the incubated CaCl2 (100μL, 25mM) and start timing immediately. The solution can be shaken continuously during this process. Once the appearance of fibrin filaments is observed, stop timing immediately and record the coagulation time. In the experiment, the coagulation time of blank porcine plasma was first tested to determine the normal coagulation time. Then, a series of heparinized plasma (0.03125-2IU·mL -1 ) to obtain a standard curve. Then, a series of antagonized plasmas numbered 1 to 10 were tested for clotting time to determine the neutralization effect. The neutralization activity (%) of 6-position polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin or protamine on heparin was calculated by the standard curve.

[0040]

[0041] The results of the activated partial thromboplastin time test are as follows: The highest neutralization activity of 6-position polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin against UFH is 94%, and at 0.012-0.06 mg·mL -1 In the dosage range, it can still maintain more than 90% neutralization activity. In addition, the highest neutralization activity of 6-position multi-substituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin against Dalte, Enoxa, and Nadro is 91%, 99%, and 85%, respectively. When the concentration increases, its neutralization activity decreases slightly. When the dosage increases to 0.06 mg·mL -1When the concentration was increased, the neutralization activity of 6-position polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin against Dalte, Enoxa, and Nadro remained above 82%, 85%, and 80%, respectively. However, protamine had a neutralization activity of more than 90% against UFH, and the highest neutralization activity against Dalte, Enoxa, and Nadro was 87%, 89%, and 69%, respectively. When the concentration increased, its neutralization activity decreased rapidly. When the dose increased to 0.06 mg·mL -1 When the neutralization activity of protamine on Dalte, Enoxa and Nadro was reduced to 61%, 29% and 27% respectively, it indicates that high doses of protamine may have the risk of bleeding. The above results show that 6-position multi-substituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin can not only almost completely antagonize UFH, but more importantly, it has a wider therapeutic window than protamine in antagonizing Dalte, Enoxa and Nadro.

[0042] The present invention provides a 6-position polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin of the structure of formula (I). The preparation method of the 6-position polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin is simple, and the prepared 6-position polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin has good biocompatibility; in in vitro pig plasma and in vivo mouse experiments, the 6-position polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin can well neutralize heparin, showing better neutralization activity than protamine, and the cyclodextrin derivative has broad application prospects in the biological and medical fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The invention is a synthetic route of 6-polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin;

[0044] Figure 2 It is a 6-position multi-substituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin 1 H NMR spectrum;

[0045] Figure 3 2D COSY spectrum of 6-position multi-substituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin;

[0046] Figure 4 It is a high-resolution mass spectrum of 6-polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin;

[0047] Figure 5 The neutralizing activity of different concentrations of 6-position polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin in porcine plasma against (a) unfractionated heparin sodium (UFH), (b) dalteparin sodium (Dalte), (c) enoxaparin sodium (Enoxa), and (d) nadroparin calcium (Nadro);

[0048] Figure 6 The actual coagulation effects of different concentrations of 6-position polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin in porcine plasma on (a) unfractionated heparin sodium (UFH), (b) dalteparin sodium (Dalte), (c) enoxaparin sodium (Enoxa), and (d) nadroparin calcium (Nadro);

[0049] Figure 7 The hemolysis rate and cytotoxicity of 6-polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin;

[0050] Figure 8 A standard curve was established for measuring the absorbance of blood at 405 nm.

[0051] Fig. 9 The bleeding time and amount in different groups in mice included normal saline group, heparin alone group, heparin and polysubstituted aminoethoxyphenylpyridinium-γ-cyclodextrin group, and heparin and protamine group.

[0052] Fig.10 The figures are the changes in body weight, hematological parameters, liver and kidney function markers, and H&E staining results of heart, liver, spleen, lung, and kidney tissue sections of mice injected with normal saline or 6-polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin;

[0053] Fig.11 Schematic diagram of the neutralization process of heparin by 6-polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin. DETAILED DESCRIPTION

[0054] The present invention provides a preparation and application of 6-position polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they all belong to the scope of protection of the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0055] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0056] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0057] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural.

[0058] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0059] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0060] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0061] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0062] Some cases are recorded in the embodiments and comparative examples of the present invention, wherein the embodiments show certain implementation modes of the present invention. However, this does not mean that the effects of the present invention can only be achieved in these cases.

[0063] In order to further illustrate the present invention, a 6-polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin provided by the present invention is described in detail below in conjunction with examples.

[0064] Example 1

[0065] A 6-position polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin for treating excessive heparin. The building unit of the 6-position polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin is based on 6-position fully substituted γ-cyclodextrin as the main body, and tert-butyl (2-(4-(pyridin-4-yl)phenoxy)ethyl) carbamate is obtained by nucleophilic substitution reaction and deprotection group.

[0066] A method for preparing 6-polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin based on neutralization of excess heparin, comprising the following steps:

[0067] 1) Synthesis of 6-Polysubstituted Boc-ethoxyphenylpyridinium-γ-cyclodextrin

[0068] According to the literature, tert-butyl (2-(4-(pyridin-4-yl)phenoxy)ethyl)carbamate (115.45 mg) was synthesized, dissolved in DMF (500 μL) with octa-(6-iodo-6-deoxy)-γ-cyclodextrin (50 mg), and stirred at 100°C for 48 hours under nitrogen protection. After the reaction, the reaction bottle was cooled to room temperature, and then most of the DMF was removed by spin drying. Then, the residue was dropped into an acetone solution, and the solid obtained by centrifugation was washed twice with acetone and vacuum dried to obtain 6-position polysubstituted Boc-ethoxyphenylpyridinium salt-γ-cyclodextrin with protective groups.

[0069] 2) Synthesis of 6-Polysubstituted Aminoethoxyphenylpyridinium Salt-γ-Cyclodextrin

[0070] 6-position polysubstituted Boc-ethoxyphenylpyridinium salt-γ-cyclodextrin (20 mg) was dissolved in 2M hydrogen chloride ethyl acetate solution (5 mL) and stirred at room temperature for 2 hours. The solid obtained by centrifugation was washed twice with ethyl acetate and vacuum dried to obtain yellow-brown solid 6-position polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin.

[0071] The compound is characterized as follows: 1 H NMR(400MHz,D2O): δ8.86(s,2H),8.72-8.37(m,10H),8.35-8.03(m,12H),7 .99(s,5H),7.85(s,2H),7.71(d,J=7.0Hz,2H),7.61(d,J=8.2Hz,3H),7.23 (s,2H),7.01(d,J=6.8Hz,4H),6.89(s,6H),5.11(d,J=8.2Hz,8H),4.51-4. 20(m,12H),4.18-3.42(m,48H),3.28(d,J=30.0Hz,12H).HRMS(ESI):Calcd for[M-3I] 3+:953.60,Found:953.63.

[0072] Verification Example

[0073] Forty ICR mice (female, 4-6 weeks old, 20-25 g) were randomly divided into eight groups for the tail transection experiment. In the normal saline control group, both the first and second injections were saline; in the heparin negative control group, the first injection was heparin solution (UFH or Enoxa solution, 200 IU kg -1 ), and the second injection of normal saline; in the experimental group, the first injection of heparin solution (UFH or Enoxa solution, 200 IU kg -1 ), and the second injection of 6-polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin solution (2.6 mg kg -1 ); in the protamine positive control group, the first injection of heparin solution (UFH or Enoxa solution, 200 IU kg -1 ), the second injection of protamine solution (2.6 mg kg -1 ). Heparin solution (20 IU·mL -1 ), 6-polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin solution (0.52 mg mL -1 ) and protamine solution (0.52 mg mL -1 ) were prepared with normal saline, and the administration volume was controlled within the range of 0.1-0.15 mL according to the body weight of each mouse. The specific experimental process is as follows: ICR mice are fixed in a mouse fixator, but normal blood circulation must be ensured. First, the first injection is made through the tail vein of the mouse, and the second injection is made 7 minutes later. After another 7 minutes, cut it off with scissors at 0.25 cm from the tail end, record the bleeding time, and collect blood drops with filter paper strips until no new blood drops appear. In addition, the standard curve of blood is established by measuring the absorbance of different volumes of blood (2.5-100 mL) collected from the heart of normal mice and dissolved in 3 mL of 10wt% NaOH solution. Finally, according to the standard curve method, the volume of blood loss is calculated by measuring the absorbance of the filter paper strips with blood drops collected after dissolving in the NaOH solution of the same concentration.

[0074] The experimental results are as follows: The average bleeding time and blood loss of the saline control group were 10.03±2.95 minutes and 5.22±3.40μL, respectively. For the heparin group, the average bleeding time and blood loss of the UFH and Enoxa groups were extended to 52.02±12.85 minutes and 47.62±27.78μL and 47.34±12.82 minutes and 42.74±31.99μL, respectively. When the mice injected with heparin, UFH and Enoxa were further injected with 6-polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin, the average bleeding time and blood loss were shortened to 10.17±2.42 minutes and 7.98±2.16μL and 9.49±6.22 minutes and 4.38±2.59μL, respectively. The average bleeding volume and blood loss time of the 6-position polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin experimental group were close to those of the saline group, indicating that 6-position polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin can completely neutralize heparin UFH and Enoxa. As for the protamine group, when protamine was continued to be injected into mice injected with heparin UFH and Enoxa, the average bleeding time and blood loss were shortened to 14.21±8.39 minutes and 12.70±12.12μL and 20.60±12.18 minutes and 5.90±2.98μL, respectively. Compared with the saline control group, the average bleeding time and blood loss of the protamine group did not fully recover to the level of the saline group, indicating that 6-position polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin showed a better in vivo neutralization effect than protamine.

[0075] Table 1 Activated partial thromboplastin time assay to determine 6-polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin or protamine (0.006-0.06 mg mL -1 ) for four heparin UFH (2IU·mL -1 )、Dalte(2IU·mL -1 )、Enoxa(2IU·mL -1 ) and Nadro (2 IU·mL -1 ) Neutralization activity (%)

[0076]

[0077]

[0078] Table 2 Mouse tail cross-section experiment to determine the saline group, heparin UFH (200IU·kg -1 ) or Enoxa (200 IU kg -1 ) Negative control group, UFH (200 IU·kg -1 ) or Enoxa (200 IU kg -1)+6-position polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin (2.6 mg kg -1 ) experimental group, heparin UFH (200 IU kg -1 ) or Enoxa (200 IU kg -1 )+ protamine (2.6 mg kg -1 ) Bleeding time (minutes) and blood loss (μL) of the positive control group

[0079]

[0080]

[0081] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. 6-position polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin of formula (I): in, m is an integer of 1 to 6, and n is 1 or 2.

2. A method for preparing 6-position polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin of formula (I), characterized in that: The steps include: A) dissolving tert-butyl (2-(4-(pyridin-4-yl)phenoxy)ethyl)carbamate and octa-(6-iodo-6-deoxy)-γ-cyclodextrin in an organic solvent, and reacting them under an inert gas environment to obtain 6-position multi-substituted Boc-ethoxyphenylpyridinium salt-γ-cyclodextrin; B) Dissolving 6-position polysubstituted Boc-ethoxyphenylpyridinium salt-γ-cyclodextrin in a hydrogen chloride ethyl acetate solution, stirring, centrifuging and drying to obtain 6-position polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin.

3. The preparation method according to claim 2, characterized in that: Step A) The molar ratio of tert-butyl (2-(4-(pyridin-4-yl)phenoxy)ethyl)carbamate to octa-(6-iodo-6-deoxy)-γ-cyclodextrin is 13-18:

1.

4. The preparation method according to claim 2, characterized in that: Step A) The reaction temperature is 90-110° C. and the reaction time is 40-50 h.

5. The preparation method according to claim 2, characterized in that: Step A) After the reaction, the reaction further comprises cooling, drying the solvent by spin drying, and then dissolving the solid with acetone, centrifuging, washing, and drying.

6. The preparation method according to claim 2, characterized in that: Step B) the concentration of ethyl hydrochloride is 1.5-2.5M; The concentration of the 6-position multi-substituted Boc-ethoxyphenylpyridinium salt-γ-cyclodextrin dissolved in the hydrogen chloride ethyl acetate solution is 4 mg·mL -1 .

7. The preparation method according to claim 2, characterized in that: In step B), the stirring is carried out at room temperature for 1.5 to 2.5 hours; after the reaction, centrifugation and vacuum drying are also carried out; the parameters of the vacuum drying are vacuum drying at 40° C. for 8 hours.

8. Use of 6-polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin of formula (I) according to claim 1 or 6-polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin prepared by the preparation method according to any one of claims 2 to 7 in neutralizing heparin.

9. The use according to claim 8, characterized in that: The heparin includes unfractionated heparin sodium, dalteparin sodium, enoxaparin sodium or nadroparin calcium.

10. A heparin antagonist product, characterized in that: The raw materials include 6-polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin of the formula (I) according to claim 1 or 6-polysubstituted aminoethoxyphenylpyridinium salt-γ-cyclodextrin prepared by the preparation method according to any one of claims 2 to 7.

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