Citicoline dextrophan derivatives, process for their preparation and use

Abstract

The present application relates to a kind of cytidine choline dextrohydrotalcid derivatives and its preparation method and application, belong to biological medicine technical field.The present application is aimed at solving the technical problems of low blood-brain barrier penetration rate and limited treatment effect of existing edaravone dextrohydrotalcid compound preparation.The present application prepares a dextrohydrotalcid derivative (C-B) with boronic acid structure, then it is compounded with cytidine choline in specific pH buffer system, and forms cytidine choline dextrohydrotalcid derivative (C-D-B).The present application can effectively improve the efficiency of drug crossing blood-brain barrier by using the dynamic covalent characteristics of boronic ester bond and free radical responsiveness.The experimental results show that, compared with existing combination drug, the derivative of the present application can significantly reduce the infarction area of cerebral ischemia model animal, reduce cell damage and active oxygen level, and promote vascular regeneration, and show better treatment effect on ischemic brain injury.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to citicoline dexborneol derivatives, their preparation methods, and applications. Background Technology

[0002] Stroke, commonly known as apoplexy, is a common clinical disease caused by various factors that damage cerebral blood vessels, resulting in focal or systemic damage to brain tissue. It is characterized by high incidence, high disability rate, and high recurrence rate. Ischemic stroke, also known as ischemic stroke (IS), refers to a type of cerebrovascular disease caused by narrowing or occlusion of arteries supplying the brain, leading to impaired blood supply and resulting in ischemic necrosis or cerebral softening of localized brain tissue. Currently, commonly used clinical treatments include surgical and drug therapy. Surgical treatment involves craniotomy to stop the bleeding; the commonly used drug is an injection of edaravone and dextroborneol (mass ratio 4:1).

[0003] Edaravone dexborneol, used clinically for the treatment of stroke, is a compound preparation composed of edaravone and dexborneol in a mass ratio of 4:1. It has the effects of scavenging oxygen free radicals and anti-inflammation.

[0004] The combination of daravone and dexborneol has shortcomings such as low blood-brain barrier penetration and unsatisfactory therapeutic effects. Summary of the Invention

[0005] In view of this, in order to increase the efficiency of citicoline in penetrating the blood-brain barrier and achieve a better stroke treatment effect compared with clinical drugs, the present invention aims to provide a citicoline dexborneol derivative, its preparation method, and its application.

[0006] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a citicoline dexborneol derivative, the structural formula of which is as follows: ; Furthermore, the preparation method of the aforementioned citicoline dexborneol derivative includes the following steps: S1: 4-(hydroxymethyl)phenylboronic acid pinacol ester and N,N'-carbonyldiimidazole were placed in a round-bottom flask, dissolved in acetonitrile, and reacted at 50°C under a N2 atmosphere for 1 h. After the reaction was completed, the solvent was removed and the mixture was purified to obtain compound III. S2: Dissolve compound III obtained in step S1 in toluene and place it in a round-bottom flask. Then dissolve diethylenetriamine in toluene and add it dropwise to react. React at 60°C for 8-12 hours. After the reaction is complete, remove the solvent and purify to obtain compound V. S3: p-Nitrophenyl chloroformate was dissolved in anhydrous tetrahydrofuran, and then triethylamine was added to the mixture in a round-bottom flask. The mixture was stirred at 0°C for 15 min. Then, dextrophenol was dissolved in anhydrous tetrahydrofuran and slowly added dropwise through a dropping funnel. The reaction was carried out at room temperature for 8-12 h. After the reaction was completed, the solvent was removed and the mixture was purified to obtain compound VI. S4: Dissolve compound VI obtained in step S3, compound V obtained in step S2, and 4-dimethylaminopyridine in N,N-dimethylformamide, then add triethylamine and place in a round-bottom flask. React at 60°C for 8-12 hours. After the reaction is complete, remove the solvent and purify to obtain compound VII. S5: Dissolve compound VII obtained in step S4 in dichloromethane and place it in a round-bottom flask. Then add methylboric acid and react. After reacting for 4 hours, remove the solvent and purify to obtain compound CB. S6: Dissolve the compound CB prepared in step S5 in DMSO, then add PBS buffer solution with pH=8.0, then add cytidine diphosphate choline, stir at room temperature for 30 min until the solution becomes clear, and obtain the cytidine diphosphate choline dextroborneol derivative. Preferably, in step S1, the molar ratio of 4-(hydroxymethyl)phenylboronic acid pinacol ester to N,N'-carbonyldiimidazole is 1:1.2; the purification is performed by column chromatography, and the eluent is a mixture of petroleum ether and ethyl acetate in a volume ratio of 5:1. Preferably, in step S2, the molar ratio of compound III to diethylenetriamine is 2.2:1; the purification is performed by column chromatography, and the eluent is a mixture of dichloromethane and methanol in a volume ratio of 50:1. Preferably, in step S3, the molar ratio of p-nitrophenyl chloroformate, triethylamine and dextroborneol is 1.1:2.2:1, and the purification is performed by column chromatography with the eluent being a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 20:1. Preferably, in step S4, the molar ratio of compound VI, compound V, 4-dimethylaminopyridine, and triethylamine is 1.2:1.1:0.2:2.2; the purification is performed by column chromatography, and the eluent is a mixture of dichloromethane and methanol in a volume ratio of 50:1. Preferably, in step S5, the ratio of compound VII, trifluoroacetic acid, and methylboric acid is 1 mol: 200 μL: 10 mol; the purification is performed by column chromatography, and the eluent is a mixture of dichloromethane and methanol in a volume ratio of 20:1. Preferably, in step S6, the ratio of compound CB, DMSO, and cytidine diphosphate choline is 1 mg: 40 μL: 19 mg.

[0007] The application of the citicoline dexborneol derivative prepared by the aforementioned method in the preparation of drugs for ischemic stroke.

[0008] Furthermore, the present invention provides a combination drug for the prevention and / or treatment of ischemic stroke, comprising the aforementioned citicoline dexborneol derivative.

[0009] The beneficial effects of this invention are as follows: The derivatives of this invention achieve significantly enhanced therapeutic effects. Their beneficial effects have been verified through systematic animal experiments, mainly manifested in the following aspects: 1. Significantly improves the therapeutic efficacy of cerebral ischemia-reperfusion injury. TTC staining of brain slices showed that, compared with the PBS control group and the simple citicoline-dextrin (CD) group, after 24 hours of treatment with the derivative of the present invention (CDB), the area of ​​white infarcts in the ischemic brain region of model mice was significantly reduced, which could more effectively limit the expansion of ischemic damage and had a stronger protective effect on brain tissue.

[0010] 2. Effectively reduces nerve cell damage and death. HE staining histological analysis showed that the brain tissue cells in the CDB treatment group had intact structure and the degree of damage was significantly lower than that in the control group and CD group. The derivative of this invention (CDB) can not only reduce the infarct area, but also deeply protect the nerve cells in the ischemic penumbra, reduce pathological changes such as cell edema and necrosis, and lay the foundation for the recovery of nerve function.

[0011] 3. Effectively removes excess reactive oxygen species (ROS) from the lesion area. ROS staining results showed that the ROS level in the CDB treatment group was significantly lower than that in the other groups. This confirms that the "free radical responsive" characteristic of the derivative design of this invention has been achieved, enabling it to target and efficiently clear oxygen free radicals in ischemic areas, thus breaking the damage cascade at the molecular level.

[0012] 4. Promotes nerve function repair and angiogenesis Nissl staining showed that the CDB group exhibited more significant Nissl body recovery in neurons in the ischemic brain region, suggesting that it can better promote the metabolic and functional repair of damaged neurons. CD31 immunostaining (a vascular endothelial marker) confirmed that CDB treatment significantly increased the number of new blood vessels in the ischemic hemisphere, promoting angiogenesis and recanalization. This "pro-angiogenic" effect far exceeds existing controls and is of crucial significance for improving cerebral blood flow, establishing collateral circulation, and achieving long-term functional recovery.

[0013] This invention provides a citicoline dexborneol derivative (CDB) that exhibits significantly superior therapeutic effects compared to existing controls (PBS and CD) in several key pathological processes, including reducing infarct area, alleviating cell damage, scavenging reactive oxygen species, promoting nerve repair, and inducing angiogenesis. These effects collectively demonstrate that this formulation has a clear positive effect and clinical application potential in the treatment of ischemic brain injury.

[0014] Other advantages, objectives, and features of the invention will be set forth in part in the description which follows, and in part will be apparent to those skilled in the art from the following examination, or may be learned from practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description

[0015] To make the objectives, technical solutions, and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, wherein: Figure 1 TTC staining images of brain slices from different treatment groups; Figure 2 HE staining images of brain sections from different treatment groups; Figure 3 ROS staining images of brain slices from different treatment groups; Figure 4 Nissl staining images of brain sections from different treatment groups; Figure 5 CD31 staining images of brain slices from different treatment groups. Detailed Implementation

[0016] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of the present invention. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0017] The accompanying drawings are for illustrative purposes only and are schematic diagrams, not actual pictures. They should not be construed as limiting the invention. To better illustrate the embodiments of the invention, some parts in the drawings may be omitted, enlarged, or reduced, and do not represent the actual product dimensions. It is understandable to those skilled in the art that some well-known structures and their descriptions may be omitted in the drawings.

[0018] In the accompanying drawings of the embodiments of the present invention, the same or similar reference numerals correspond to the same or similar components. In the description of the present invention, it should be understood that if terms such as "upper," "lower," "left," "right," "front," and "rear" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, they are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, the terms used to describe positional relationships in the drawings are only for illustrative purposes and should not be construed as limiting the present invention. For those skilled in the art, the specific meaning of the above terms can be understood according to the specific circumstances.

[0019] Example 1: Preparation of dextroborneol derivatives 1. Preparation of boric acid compound (CB) of dextroborneol: (1) Weigh 1 mol (500 mg) of 4-(hydroxymethyl)phenylboronic acid pinacol ester and 1.2 mol (416 mg) of N,N'-carbonyldiimidazole into a 100 mL round-bottom flask, add acetonitrile to dissolve, and react for 1 h under a N2 atmosphere at 50 °C. After the reaction is complete, remove the organic solvent under reduced pressure, and purify the crude product by column chromatography (the eluent is obtained by mixing petroleum ether and ethyl acetate in a volume ratio of 5:1). The crude product is purified to obtain compound III. The specific reaction formula is as follows:

[0020] (2) Weigh out compound III (2.2 mol, 200 mg), dissolve it in toluene, and place it in a 100 mL round-bottom flask. Dissolve diethylenetriamine (1 mol, 28.5 mg) in toluene and add it dropwise to the round-bottom flask. React overnight at 60 °C. After the reaction is complete, remove the organic solvent under reduced pressure. Purify the crude product by column chromatography (the eluent is obtained by mixing dichloromethane and methanol in a volume ratio of 50:1) to obtain compound V. The specific reaction formula is as follows:

[0021] (3) Weigh p-nitrophenyl chloroformate (1.1 mol, 144 mg), dissolve it in anhydrous tetrahydrofuran, and then add triethylamine (2.2 mol, 144 mg) to a 100 mL round-bottom flask. Stir the mixture at 0 °C for 15 min. Dissolve dextroborneol (1 mol, 100 mg) in anhydrous tetrahydrofuran and slowly add it dropwise through a dropping funnel. After the addition is complete, transfer the mixture to room temperature and stir overnight. After the reaction is complete, remove the organic solvent under reduced pressure and purify the crude product by column chromatography (the eluent is obtained by mixing petroleum ether and ethyl acetate in a volume ratio of 20:1). Compound VI can then be obtained. The specific reaction formula is as follows:

[0022] (4) Weigh compound VI (1.2 mol, 139 mg), compound V (1.1 mol, 300 mg), and 4-dimethylaminopyridine (0.2 mol, 11 mg) and use N... ， After dissolving N-dimethylformamide, triethylamine (2.2 mol, 97 mg) was added to a 100 mL round-bottom flask and reacted overnight at 60 °C. After the reaction was complete, the organic solvent was removed under reduced pressure, and the crude product was purified by column chromatography (the eluent was a mixture of dichloromethane and methanol in a 50:1 volume ratio) to obtain compound VII. The specific reaction formula is as follows:

[0023] (5) Weigh compound VII (1 mol, 48 mg) and trifluoroacetic acid (200 μL, 162 mg), dissolve them in dichloromethane, place the solution in a 50 mL round-bottom flask, add methylboric acid (10 mol, 35 mg), and react for 4 hours. After the reaction is complete, remove the organic solvent under reduced pressure, and purify the crude product by column chromatography (the eluent is a mixture of dichloromethane and methanol in a 20:1 volume ratio) to obtain compound (CB). The specific reaction formula is as follows:

[0024] 2. Preparation of citicoline dexborneol derivatives: 1 mg of the boric acid structure compound of dextroborneol prepared above was dissolved in 40 μL DMSO (less than 2% of the final total solution volume), and then PBS buffer solution at pH 8.0 (adjusted to pH with saturated NaHCO3) was added to a final volume of 1 mL. 19 mg of citicoline was then added to the solution, and after stirring at room temperature for 30 min until the solution became clear, it was diluted to 7 mL to obtain a new formulation of citicoline dextroborneol derivative (CDB) with a concentration of 3.3 mg / mL. The structural formula of the citicoline dextroborneol derivative formulation is as follows: .

[0025] Example 2 (1) After injecting PBS, citicoline-dextrin (CD), and citicoline-dextrin derivative (CDB) into MCAO model mice for 24 hours, the (CDB) group showed a significantly improved therapeutic effect compared with the control group. The TTC staining images of brain slices from different treatment groups are shown in the figure. Figure 1 As shown.

[0026] TTC can turn normal tissue red and ischemic tissue white. The results showed that after 24 hours of treatment with citicoline dexborneol derivative (CDB), the white area in the ischemic region was significantly smaller than that in the PBS control group and the citicoline-dexborneol (CD) group, demonstrating a good therapeutic effect.

[0027] (2) HE staining images of brain sections from different treatment groups are shown below. Figure 2 As shown, the results indicate that compared with the PBS control group and the citicoline-dextrin (CD) group, the citicoline-dextrin derivative (CDB) group showed significantly reduced cell damage.

[0028] (3) ROS staining images of brain slices from different treatment groups are shown below. Figure 3 As shown in the figure. The results indicate that compared with the PBS control group and the citicoline-dextrin (CD) group, the citicoline-dextrin derivative (CDB) group showed a significant decrease in ROS levels and a significant improvement in treatment efficacy.

[0029] (4) Nissl staining images of brain slices from different treatment groups are shown in Figure 4. The results show that the left half of the brain in the PBS group was severely ischemic and the blood flow obstruction was very obvious, while the (CDB) group showed significant recovery.

[0030] (5) CD31 staining images of brain slices from different treatment groups are shown below. Figure 5 As shown, the results indicate that the CD31-positive cell population in the ischemic hemisphere was much larger in the citicoline-dexborneol derivative (CDB) group than in the PBS and citicoline-dexborneol (CD) groups, demonstrating that citicoline-dexborneol derivative (CDB) treatment can significantly promote angiogenesis and improve vascular recanalization.

[0031] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A citicoline-dextrin derivative, characterized in that: Its structural formula is as follows: 。 2. The method for preparing the citicoline dexborneol derivative according to claim 1, characterized in that, Includes the following steps: S1: 4-(hydroxymethyl)phenylboronic acid pinacol ester and N,N'-carbonyldiimidazole were placed in a round-bottom flask, dissolved in acetonitrile, and reacted at 50°C under a N2 atmosphere for 1 h. After the reaction was completed, the solvent was removed and the mixture was purified to obtain compound III. S2: Dissolve compound III obtained in step S1 in toluene and place it in a round-bottom flask. Then dissolve diethylenetriamine in toluene and add it dropwise to react. React at 60°C for 8-12 hours. After the reaction is complete, remove the solvent and purify to obtain compound V. S3: p-Nitrophenyl chloroformate was dissolved in anhydrous tetrahydrofuran, and then triethylamine was added to the mixture in a round-bottom flask. The mixture was stirred at 0°C for 15 min. Then, dextrophenol was dissolved in anhydrous tetrahydrofuran and slowly added dropwise through a dropping funnel. The reaction was carried out at room temperature for 8-12 h. After the reaction was completed, the solvent was removed and the mixture was purified to obtain compound VI. S4: Dissolve compound VI obtained in step S3, compound V obtained in step S2, and 4-dimethylaminopyridine in N,N-dimethylformamide, then add triethylamine and place in a round-bottom flask. React at 60°C for 8-12 hours. After the reaction is complete, remove the solvent and purify to obtain compound VII. S5: Dissolve compound VII obtained in step S4 in dichloromethane and place it in a round-bottom flask. Then add methylboric acid and react. After reacting for 4 hours, remove the solvent and purify to obtain compound CB. S6: Dissolve the compound CB prepared in step S5 in DMSO, then add PBS buffer solution with pH=8.0, then add cytidine diphosphate choline, stir at room temperature for 30 min until the solution becomes clear, and obtain the cytidine diphosphate choline dextroborneol derivative.

3. The preparation method according to claim 2, characterized in that, In step S1, the molar ratio of 4-(hydroxymethyl)phenylboronic acid pinacol ester to N,N'-carbonyldiimidazole is 1:1.2; the purification is performed by column chromatography, and the eluent is a mixture of petroleum ether and ethyl acetate in a volume ratio of 5:

1.

4. The preparation method according to claim 2, characterized in that, In step S2, the molar ratio of compound III to diethylenetriamine is 2.2:1; the purification is performed by column chromatography, and the eluent is a mixture of dichloromethane and methanol in a volume ratio of 50:

1.

5. The preparation method according to claim 2, characterized in that, In step S3, the molar ratio of p-nitrophenyl chloroformate, triethylamine and dextroborneol is 1.1:2.2:

1. The purification is performed by column chromatography, and the eluent is a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 20:

1.

6. The preparation method according to claim 1, characterized in that, In step S4, the molar ratio of compound VI, compound V, 4-dimethylaminopyridine, and triethylamine is 1.2:1.1:0.2:2.2; the purification is performed by column chromatography, and the eluent is a mixture of dichloromethane and methanol in a volume ratio of 50:

1.

7. The preparation method according to claim 2, characterized in that, In step S5, the ratio of compound VII, trifluoroacetic acid, and methylboric acid is 1 mol: 200 μL: 10 mol; the purification is performed by column chromatography, and the eluent is a mixture of dichloromethane and methanol in a volume ratio of 20:

1.

8. The preparation method according to claim 2, characterized in that, In step S6, the ratio of compound CB, DMSO, and cytidine diphosphate choline is 1 mg: 40 μL: 19 mg.

9. The use of the citicoline dexborneol derivative prepared by any one of the preparation methods according to claims 2-8 in the preparation of drugs for ischemic stroke.

10. A combination drug for the prevention and / or treatment of ischemic stroke, characterized in that: Includes the cytidine diphosphate choline dexborneol derivative as described in claim 1.

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