Eicosapentaenoic acid derivative, preparation method and application thereof, and medicine for preventing and treating nervous system diseases

Abstract

The invention provides an eicosapentaenoic acid derivative, a preparation method and application thereof and a medicine for preventing and treating nervous system diseases, and belongs to the technical field of medicines. The eicosapentaenoic acid derivative provided by the invention has a structure as shown in a formula I, is an ester derivative of eicosapentaenoic acid (EPA), is high in lipid solubility, and can improve the pharmacological effect of the EPA. Pharmacological experiments show that the EPA derivative has a better nerve cell protection effect than EPA, and the medicinal value of EPA is expected to be expanded. And the formula I of the # imgabs0 # is shown in the specification.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to eicosapentaenoic acid derivatives, preparation methods and applications thereof, and drugs for preventing and treating nervous system diseases. Background Art

[0002] Eicosapentaenoic acid (EPA), as a special ω-3 fatty acid, plays multiple functions in the body, such as regulating metabolism, anti-inflammation, and anti-oxidation. EPA has been proven to participate in the formation of cell membranes, affect cell signal transduction, and play anti-inflammatory, anti-oxidation, and anti-apoptosis roles by regulating gene expression and affecting metabolic pathways. These biological characteristics make EPA show great potential in the research of cardiovascular diseases and inflammatory diseases.

[0003] However, EPA has difficulty directly crossing tissue barriers and entering cells, resulting in its biological activity not being fully exerted. Summary of the invention

[0004] The purpose of the present invention is to provide eicosapentaenoic acid derivatives and preparation methods and applications thereof, as well as drugs for preventing and treating nervous system diseases. The eicosapentaenoic acid derivatives provided by the present invention have good fat solubility and better neuroprotective effects than EPA.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides an eicosapentaenoic acid derivative having a structure shown in Formula I: Formula I.

[0006] The present invention provides a method for preparing the eicosapentaenoic acid derivatives described in the above technical solution, comprising the following steps: The eicosapentaenoic acid, the activating agent and the organic solvent are mixed and activated to obtain an activated liquid; The activated liquid is mixed with tetrahydro-2-furanmethanol to carry out an esterification reaction to obtain an eicosapentaenoic acid derivative having a structure shown in Formula I.

[0007] Preferably, the activator comprises N,N-diisopropylethylamine, 1-hydroxybenzotriazole and (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; the temperature of the activation treatment is 10-40° C., and the time is 20-40 min.

[0008] Preferably, the molar ratio of eicosapentaenoic acid to tetrahydro-2-furanmethanol is 1:1 to 1.5.

[0009] Preferably, the esterification reaction temperature is 10-40° C. and the time is 1-3 h.

[0010] The present invention provides the use of the eicosapentaenoic acid derivatives described in the above technical solution in the preparation of drugs for preventing and treating nervous system diseases.

[0011] Preferably, the neurological disease comprises a neurodegenerative disease.

[0012] Preferably, the neurodegenerative disease comprises Alzheimer's disease or Parkinson's disease.

[0013] The present invention provides a drug for preventing and treating nervous system diseases, wherein the active component comprises the eicosapentaenoic acid derivative described in the above technical solution.

[0014] Preferably, the dosage form of the drug for preventing and treating nervous system diseases includes injection, pill or capsule.

[0015] The present invention provides an eicosapentaenoic acid derivative having a structure shown in Formula I. The eicosapentaenoic acid derivative provided by the present invention is an ester derivative of eicosapentaenoic acid, which has strong fat solubility and can improve the efficacy of EPA. The pharmacological experiment of the present invention shows that the EPA derivative has a better neuron protective effect than EPA, and is expected to expand the medicinal value of EPA. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The synthetic route of EPA derivatives is shown below; Figure 2 is the H NMR spectrum of EPA derivatives; Figure 3 The results of the test on the cell viability of HT22 cells after culturing them for 24 hours and then treating them with different concentrations of H2O2 for 4 hours and then continuing to culture them for 24 hours; Figure 4 The figure shows the protective effect of different concentrations of EPA on the HT22 cell oxidative damage model; Figure 5 This is a graph showing the protective effects of different concentrations of EPA derivatives on the HT22 cell oxidative damage model. DETAILED DESCRIPTION

[0017] The present invention provides an eicosapentaenoic acid derivative having a structure shown in Formula I: Formula I.

[0018] EPA has a wide range of physiological activities, but its polarity is strong, and it is difficult to penetrate the hydrophobic phospholipid layer into the cell, resulting in its biological activity not being fully exerted. The present invention has developed and designed a new type of EPA derivative (specifically, an ester derivative of EPA) by structural modification of EPA. It has strong fat solubility and good absorption, can enter the cell in a targeted manner to exert its effects, has high bioavailability, and can achieve the improvement of EPA's efficacy, such as improving the metabolism and function of neurons, promoting nerve regeneration and repair, and having a certain preventive and therapeutic effect on nervous system diseases. The pharmacological experiments of the present invention show that EPA derivatives have better nerve cell protection than EPA, and are expected to expand the medicinal value of EPA.

[0019] The present invention provides a method for preparing the eicosapentaenoic acid derivatives described in the above technical solution, comprising the following steps: The eicosapentaenoic acid, the activating agent and the organic solvent are mixed and activated to obtain an activated liquid; The activated liquid is mixed with tetrahydro-2-furanmethanol to carry out an esterification reaction to obtain an eicosapentaenoic acid derivative having a structure shown in Formula I.

[0020] In the present invention, unless otherwise specified, the raw materials used are commercially available products well known to those skilled in the art or are prepared by methods well known to those skilled in the art.

[0021] The present invention mixes eicosapentaenoic acid, an activator and an organic solvent, performs activation treatment, and obtains an activated liquid. As an embodiment of the present invention, the activator may include N,N-diisopropylethylamine (DIPEA), 1-hydroxybenzotriazole (HOBT) and (1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl); the molar ratio of eicosapentaenoic acid, DIPEA, HOBT and EDC·HCl may be 1:2.5~3.5:1~2:1~2, specifically 1:3:1.5:1.5. As an embodiment of the present invention, the organic solvent may be N,N-dimethylformamide (DMF), and the amount ratio of eicosapentaenoic acid to the organic solvent may be 9~10mmol:10~20mL, specifically 9.13mmol:15mL.

[0022] As an embodiment of the present invention, the eicosapentaenoic acid is dissolved in an organic solvent, and then DIPEA, HOBT and EDC·HCl are added for activation treatment; the temperature of the activation treatment can be 10-40°C, specifically room temperature (25°C); the time can be 20-40min, specifically 30min; the activation treatment can be carried out under stirring conditions.

[0023] After the activation treatment, the present invention does not need to perform any post-treatment, and directly mixes the obtained activated liquid with tetrahydro-2-furanmethanol to perform an esterification reaction to obtain an eicosapentaenoic acid derivative having a structure shown in Formula I. As an embodiment of the present invention, the molar ratio of eicosapentaenoic acid to tetrahydro-2-furanmethanol can be 1:1~1.5, specifically 1:1.2. As an embodiment of the present invention, the temperature of the esterification reaction can be 10~40°C, specifically room temperature; the time can be 1~3h, specifically 2h; the esterification reaction can be carried out under stirring conditions. In the embodiment of the present invention, the reaction is specifically monitored by TLC.

[0024] As an embodiment of the present invention, after the esterification reaction, water can be added to the obtained product system, the liquids can be separated, the aqueous phase can be extracted with dichloromethane, the organic phase can be collected, the organic phase can be washed with a saturated sodium chloride solution, and then dried over anhydrous sodium sulfate, filtered, the solvent in the filtrate can be dried, and separated by a silica gel column (the eluent used can be petroleum ether: ethyl acetate = 1:10 by volume) to obtain an eicosapentaenoic acid derivative having a structure shown in Formula I.

[0025] The present invention provides the use of the eicosapentaenoic acid derivatives described in the above technical scheme in the preparation of drugs for preventing and treating nervous system diseases. The EPA derivatives provided by the present invention have strong fat solubility and high bioavailability, and can achieve the improvement of EPA efficacy. In the test example of the present invention, specifically, an HT22 cell oxidative damage model was constructed by H2O2 induction, and then the EPA derivatives and EPA's protective effects on cells were studied. The results showed that the EPA derivatives had stronger therapeutic potential than EPA in alleviating H2O2-induced HT22 cell oxidative damage.

[0026] As an embodiment of the present invention, the nervous system disease may include a neurodegenerative disease; the neurodegenerative disease may include Alzheimer's disease or Parkinson's disease.

[0027] The drug for preventing and treating nervous system diseases of the present invention comprises the eicosapentaenoic acid derivative (i.e., as an active ingredient) and a pharmaceutically acceptable excipient. The present invention does not specifically limit the specific types of the pharmaceutically acceptable excipients, and pharmaceutically acceptable excipients of the types familiar to those skilled in the art can be used. As an embodiment of the present invention, the dosage form of the drug for preventing and treating nervous system diseases may include an injection, a pill, or a capsule.

[0028] The present invention provides a drug for preventing and treating nervous system diseases, wherein the active component comprises the eicosapentaenoic acid derivative described in the above technical solution. As an embodiment of the present invention, the composition and dosage form of the drug for preventing and treating nervous system diseases may be consistent with the above technical solution, which will not be described in detail here.

[0029] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] The raw materials used in the following examples are commercially available products well known to those skilled in the art or are prepared by methods well known to those skilled in the art.

[0031] The reagents used in the following test examples include: hippocampal neuron HT22 cell line (Cybee Biotechnology Co., Ltd.), EPA derivatives (prepared in Example 1), EPA (MedChemExpress), fetal bovine serum (Thermo Fisher Scientific), DMEM high glucose medium (gibco), penicillin-streptomycin mixture (Solarbio), 0.25% trypsin (gibco), anhydrous ethanol (Guangdong Guanghua Technology Co., Ltd.), PBS powder (Biosharp), CCK-8 kit (Japan Tongren).

[0032] The experimental instruments used in the following test examples include: a carbon dioxide incubator (Thermo Fisher Scientific), a multifunctional microplate reader (BioTek, USA), a constant temperature water bath (Beijing Medical Equipment Factory), and a microscope (OLYMPUS).

[0033] Other reagents and instruments not specifically mentioned can be purchased directly.

[0034] Example 1 Figure 1 The synthetic route of EPA derivatives is shown below. Figure 1 The preparation method of EPA derivatives is described in detail.

[0035] Eicosapentaenoic acid (EPA, 2.76 g, 9.13 mmol) was dissolved in 15 mL of N,N-dimethylformamide (DMF), and N,N-diisopropylethylamine (DIPEA, 3.54 g, 3 eq.), 1-hydroxybenzotriazole (HOBT, 1.85 g, 1.5 eq.) and (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 2.63 g, 1.5 eq.) were added at room temperature, stirred at room temperature for 30 min, and then tetrahydro-2-furanmethanol (1.12 g, 11 mmol, 1.2eq.), stirred at room temperature for 2h, and the reaction was completed after TLC monitoring; 30mL of water was added to the obtained product system, the liquid was separated, the aqueous phase was extracted with dichloromethane three times, the organic phases were combined, the organic phases were washed with a saturated sodium chloride solution, and then dried over anhydrous sodium sulfate, filtered, the solvent in the filtrate was dried, and separated by a silica gel column (the eluent used was petroleum ether: ethyl acetate = 1:10 by volume), to obtain an EPA derivative (EPA-2 tetrahydrofuran methyl ester), and its nuclear magnetic resonance hydrogen spectrum is as follows Figure 2 shown.

[0036] Test Example 1 1. Experimental methods (1) HT22 cell culture process 1.1. The HT22 cells were resuscitated by injecting 5 mL of DMEM high-glucose complete medium into a culture dish with a diameter of 60 mm. The culture dish was pre-prepared with 10% fetal bovine serum as a nutrient source and 1% penicillin-streptomycin mixture was added to ensure a sterile culture environment. The HT22 cells were then evenly inoculated into the culture dish. The inoculated culture dish was placed in a constant temperature incubator at a temperature of 37°C and a CO2 concentration of 5% for a preliminary culture of 24 h to promote cell adhesion and initial proliferation.

[0037] 1.2. When the cells are observed to grow to 80-90% coverage at the bottom of the culture dish, perform cell passaging to maintain an appropriate cell density. Prior to this, preheat the required culture medium, PBS buffer, and trypsin solution to 37°C to ensure temperature consistency during the operation. After removing the old culture medium, gently wash the cell surface with preheated PBS buffer to remove residual culture medium and non-adherent impurities. Subsequently, add 1 mL of trypsin solution to the culture dish, gently shake the culture dish to evenly cover the cell layer with trypsin, and then return the culture dish to the 37°C constant temperature incubator for digestion for 1 minute to effectively separate the adherent cells. After digestion is completed, immediately add 2 mL of fresh DMEM high-glucose complete medium to neutralize the activity of trypsin, and detach the cells from the bottom of the culture dish by gently blowing to form a cell suspension.

[0038] 1.3. Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge at 24°C and 1000×g for 3 min to allow the cells to settle at the bottom of the tube. Then, carefully remove the supernatant and add 2 mL of fresh DMEM high-glucose complete medium to the centrifuge tube, and gently pipette again to form a uniform cell suspension. Finally, take 1 mL of the cell suspension from the suspension and re-inoculate it into a new culture dish with a diameter of 60 mm, supplement it with 5 mL of DMEM high-glucose complete medium, and continue to place it in a 37°C constant temperature incubator for subsequent culture.

[0039] (2) Construction and evaluation of HT22 cell oxidative damage model 2.1. H2O2 concentration gradient treatment of HT22 cells In order to explore the effect of different concentrations of H2O2 on oxidative damage of HT22 cells, this experiment selected HT22 cells in the logarithmic growth phase and in good condition, and 3.5×10 3 The cells were precisely inoculated in a 96-well plate at a density of 100 cells / mL. After 24 hours of cell attachment and stabilization, the medium was changed, and each group of cells was exposed to DMEM high-glucose complete medium containing different final concentrations of H2O2 (0, 200μM, 400μM, 600μM). Four replicate wells were set up in each group to enhance the reliability of the experimental results. Subsequently, the well plate was placed in a constant temperature incubator at 37°C and a CO2 concentration of 5% for 4 hours in the dark to simulate the in vitro oxidative stress environment. After the culture was completed, all old culture media were removed and replaced with fresh DMEM high-glucose complete medium. After continuing to culture for 24 hours, the degree of cell oxidative damage was detected.

[0040] 2.2 Cell viability detection based on CCK-8 method Based on the experiment in 2.1, after the predetermined culture time, the CCK-8 method was used to quantitatively evaluate the survival rate of each group of cells. First, the old culture medium in the well plate was removed, and a mixture of 100 μL DMEM high-glucose basal medium and 10 μL CCK-8 solution was added to each well. At the same time, a blank control group (i.e., only culture medium and CCK-8 solution, without cells) was set up to correct the background absorbance value. Subsequently, the well plate was placed in the same conditions and incubated in the dark for 2 hours to allow CCK-8 to fully react with the dehydrogenase in the living cells to produce a detectable color change. After the incubation, the absorbance value (OD) of each well was measured at 450nm and 610nm wavelengths using an enzyme reader, and the interference of nonspecific absorption was eliminated by calculating the difference (OD450nm-OD610nm). The calculation formula for cell survival rate is: [(OD value of experimental well-OD value of blank well) / (OD value of control well-OD value of blank well)]×100%. Among them, the experimental wells represent the mixed solution of HT22 cells and CCK-8 in each group after administration; the control wells are the mixed solution of normal HT22 cells and CCK-8 without any treatment; the blank wells contain only culture medium and CCK-8 solution to eliminate background interference. By comparing the cell survival rate under different H2O2 concentrations, the appropriate oxidative damage conditions can be screened out, providing a reliable basis for subsequent in-depth research.

[0041] (3) Screening of protective concentrations of EPA and EPA derivatives against H2O2-induced damage in the HT22 model 3.1. Determination of H2O2 concentration and experimental grouping Based on the experimental results of 2.2, the appropriate concentration required for the preparation of the H2O2-induced HT22 cell oxidative damage model was determined. The experimental grouping was set as follows: Normal control group (Control): cultured with DMEM high-glucose complete medium; Model group (H2O2 group): 400 μM H2O2 and DMEM high-glucose complete medium were used for co-culture; Experimental group: multiple EPA and EPA derivative concentration gradients (0μM, 10μM, 20μM, 40μM, 80μM) were set up, and each concentration group was used in combination with 400μM H2O2 and DMEM high-glucose complete medium.

[0042] 3.2 Determination of effective dosing concentration of EPA and EPA derivatives HT22 cells in the logarithmic growth phase and in good growth condition were selected and cultured at a rate of 3.5 × 10 3The cells were seeded in a 96-well plate at a density of 100 cells / well. After 24 hours of cell adherence culture, the culture medium was replaced, and the cells in each group were exposed to complete culture medium containing different final concentrations (0μM, 10μM, 20μM, 40μM, 80μM) of EPA and EPA derivatives. EPA and EPA derivatives were dissolved in anhydrous ethanol in advance, and the final concentration of anhydrous ethanol was <0.1%. After 24 hours of continuous culture, the old culture medium was removed, and DMEM high-glucose complete culture medium containing 400μM H2O2 was added to each group. At the same time, a normal control group (containing only DMEM high-glucose complete culture medium) was set up. Each group was set up with 4 replicates and cultured in a constant temperature incubator at 37°C and 5% CO2 for 4 hours in the dark. Subsequently, all culture medium was removed and replaced with fresh DMEM high-glucose complete culture medium. After 24 hours of continuous culture, CCK-8 cell viability detection was performed. The detection method followed the standard process described in 2.2.

[0043] 2. Experimental results By adopting the H2O2 induction strategy, this experiment successfully constructed an HT22 cell oxidative damage model, and after systematic evaluation, 400μM H2O2 treatment for 4h was established as the optimal condition for constructing the model. Subsequently, in the experimental group, HT22 cells were pre-treated with EPA and EPA derivatives of different concentration gradients for 24h, then exposed to H2O2 for 4h oxidative damage treatment, and then replaced with fresh culture medium for 24h. The experimental results showed that EPA showed an unexpected inhibitory effect on cell viability under low concentration range (10μM, 20μM), while it showed a protective trend under higher concentration (40μM, 80μM), but this effect did not reach statistical significance. On the contrary, EPA derivatives showed a stronger protective effect on cells with increasing concentration, especially at a concentration of 80μM, the protective effect reached statistical significance (P<0.01). The following is a detailed description.

[0044] (1) Effects of different concentrations of H2O2 on HT22 cell viability The CCK-8 method was used to systematically evaluate the effects of different concentrations of H2O2 on the viability of HT22 cells. Figure 3 The results of the cell viability test of HT22 cells after culturing for 24 hours and then treating them with different concentrations of H2O2 for 4 hours and then continuing to culture them for 24 hours (compared with the control group, **P<0.01, ***P<0.001, ****P<0.0001). Figure 3It can be seen that with the increase of H2O2 concentration, the viability of HT22 cells decreased significantly. Specifically, 200μM H2O2 treatment has led to significant inhibition of cell viability (P<0.01), while the inhibitory effect of 400μM H2O2 is more significant (P<0.001), and 600μM H2O2 further aggravates this trend (P<0.0001). Based on the above results, this experiment selected 400μM H2O2 treatment for 4h as the standard model condition for inducing oxidative damage in HT22 neurons.

[0045] (2) Effects of different concentrations of EPA on the activity of HT22 oxidative damage model The CCK-8 method was used to detect the effects of different concentrations of EPA on the cell viability of HT22 oxidative damage model cells. Figure 4 The protective effect of different concentrations of EPA on the HT22 cell oxidative damage model (compared with the model group, ****P<0.0001) is shown in the figure. Figure 4 It can be seen that compared with the H2O2 model group, EPA did not show obvious protective effects at concentrations of 10μM and 20μM. At concentrations of 40μM and 80μM, the protective effect of EPA on cells gradually increased, but it has not yet reached statistical significance.

[0046] (3) Effects of different concentrations of EPA derivatives on the activity of HT22 oxidative damage model The effects of different concentrations of EPA derivatives on the viability of HT22 oxidative damage model cells were further evaluated. Figure 5 The protective effect of different concentrations of EPA derivatives on the HT22 cell oxidative damage model (compared with the model group, ***P<0.01, **P<0.001). Figure 5 It can be seen that compared with the H2O2 model group, each concentration group of EPA derivatives (10μM, 20μM, 40μM, 80μM) showed enhanced cell protection as the concentration increased. In particular, the 80μM EPA derivative group showed a significant statistical difference (P<0.01), indicating that it has a stronger protective effect on H2O2-induced oxidative damage in HT22 cells.

[0047] From the above results, it can be seen that the present invention successfully constructed an HT22 cell oxidative damage model by H2O2 induction, and a significant decrease in cell survival rate was observed. After determining that 400μM H2O2 treatment for 4h was the optimal modeling condition, the cell protective effect of EPA and EPA derivatives under pretreatment conditions was further explored. The experimental results showed that EPA showed a certain protective trend at high concentrations, but it did not reach statistical significance. On the contrary, EPA derivatives significantly enhanced the cell protection effect with increasing concentration, especially at a concentration of 80μM, showing significant statistical differences, suggesting that EPA derivatives have stronger therapeutic potential than EPA in alleviating H2O2-induced HT22 cell oxidative damage. This discovery provides new ideas and directions for the treatment of related nervous system diseases such as neurodegenerative diseases.

[0048] 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. An eicosapentaenoic acid derivative, characterized in that: It has the structure shown in formula I: Formula I.

2. The method for preparing the eicosapentaenoic acid derivative according to claim 1, characterized in that: The following steps are involved: The eicosapentaenoic acid, the activator and the organic solvent are mixed and activated to obtain an activated liquid; The activated liquid is mixed with tetrahydro-2-furanmethanol to carry out an esterification reaction to obtain an eicosapentaenoic acid derivative having a structure shown in Formula I.

3. The preparation method according to claim 2, characterized in that: The activator includes N,N-diisopropylethylamine, 1-hydroxybenzotriazole and (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; the temperature of the activation treatment is 10-40°C and the time is 20-40 minutes.

4. The preparation method according to claim 2, characterized in that: The molar ratio of eicosapentaenoic acid to tetrahydro-2-furanmethanol is 1:1-1.

5.

5. The preparation method according to claim 2, characterized in that: The temperature of the esterification reaction is 10-40° C. and the time is 1-3 hours.

6. Use of the eicosapentaenoic acid derivative according to claim 1 in the preparation of drugs for preventing and treating nervous system diseases.

7. The use according to claim 6, characterized in that: The nervous system diseases include neurodegenerative diseases.

8. The use according to claim 7, characterized in that: The neurodegenerative disease includes Alzheimer's disease or Parkinson's disease.

9. A drug for preventing and treating nervous system diseases, characterized in that: The active component comprises the eicosapentaenoic acid derivative according to claim 1.

10. The drug for preventing and treating nervous system diseases according to claim 9, characterized in that: The dosage form of the drug for preventing and treating nervous system diseases includes injection, pill or capsule.

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