Eicosapentaenoic acid derivatives, their preparation methods and applications, and drugs for preventing and treating nervous system diseases

Abstract

The present invention provides eicosapentaenoic acid derivatives, their preparation methods and applications, and drugs for preventing and treating nervous system diseases, belonging to the field of pharmaceutical technology. The eicosapentaenoic acid derivatives provided by the present invention have the structure shown in Formula I. They are ester derivatives of eicosapentaenoic acid (EPA), have strong liposolubility, and can enhance the efficacy of EPA. Pharmacological experiments of the present invention show that the EPA derivatives have better neuroprotective effects than EPA, and are expected to expand the medicinal value of EPA. Formula I.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly to eicosapentaenoic acid derivatives, their preparation methods and applications, 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 organisms such as regulating metabolism, anti-inflammation, and anti-oxidation. EPA has been proven to be involved in the composition of cell membranes, affect cell signal transduction, and can also play anti-inflammatory, anti-oxidant, anti-apoptotic and other roles by regulating gene expression and affecting metabolic pathways. These biological characteristics make EPA show great potential in the research of multiple fields such as cardiovascular diseases and inflammatory diseases.

[0003] However, EPA is difficult to directly cross the tissue barrier and enter cells, resulting in the failure to fully exert its biological activity. Summary of the Invention

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

[0005] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0006] The present invention provides an eicosapentaenoic acid derivative having the structure shown in Formula I:

[0007] Formula I.

[0008] The present invention provides a preparation method of the eicosapentaenoic acid derivative described in the above technical solution, including the following steps:

[0009] Mix eicosapentaenoic acid, an activator and an organic solvent, and perform activation treatment to obtain an activated feed liquid;

[0010] Mix the activated feed liquid with tetrahydro-2-furanmethanol, and perform an esterification reaction to obtain an eicosapentaenoic acid derivative having the structure shown in Formula I.

[0011] Preferably, 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 min.

[0012] Preferably, the molar ratio of eicosapentaenoic acid to tetrahydro-2-furanmethanol is 1:1-1.5.

[0013] Preferably, the temperature of the esterification reaction is 10~40 °C, and the time is 1~3 h.

[0014] The present invention provides the use of the eicosapentaenoic acid derivative described in the above technical solution in the preparation of a drug for preventing and treating nervous system diseases.

[0015] Preferably, the nervous system diseases include neurodegenerative diseases.

[0016] Preferably, the neurodegenerative diseases include Alzheimer's disease or Parkinson's disease.

[0017] The present invention provides a drug for preventing and treating nervous system diseases, and the active ingredient includes the eicosapentaenoic acid derivative described in the above technical solution.

[0018] Preferably, the dosage form of the drug for preventing and treating nervous system diseases includes injection, pill or capsule.

[0019] The present invention provides an eicosapentaenoic acid derivative having the structure shown in Formula I. The eicosapentaenoic acid derivative provided by the present invention is an ester derivative of eicosapentaenoic acid, which has strong liposolubility and can improve the efficacy of EPA. The pharmacological experiments of the present invention show that the EPA derivative has better neuroprotective effects than EPA, and is expected to expand the medicinal value of EPA. Description of the Drawings

[0020] Figure 1 is the synthetic route diagram of the EPA derivative;

[0021] Figure 2 is the 1H NMR spectrum of the EPA derivative;

[0022] Figure 3 is the result diagram of the effect on the cell viability of HT22 detected after culturing cells for 24 h, treating with different concentrations of H2O2 for 4 h, and then continuing to culture for 24 h;

[0023] Figure 4 is the result diagram of the protective effect of different concentrations of EPA on the oxidative damage model of HT22 cells;

[0024] Figure 5 is the result diagram of the protective effect of different concentrations of EPA derivatives on the oxidative damage model of HT22 cells. Detailed Embodiments

[0025] The present invention provides an eicosapentaenoic acid derivative having the structure shown in Formula I:

[0026] Formula I.

[0027] Eicosapentaenoic acid (EPA) has a wide range of physiological activities. However, due to its strong polarity, it is difficult to penetrate the hydrophobic phospholipid layer and enter cells, resulting in the failure to fully exert its biological activity. In the present invention, through structural modification of EPA, a novel EPA derivative (specifically, an ester derivative of EPA) was developed and designed. It has strong liposolubility, good absorption, can target and enter cells to play a role, has a relatively high bioavailability, and can improve the efficacy of EPA, 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. Pharmacological experiments of the present invention show that the EPA derivative has better neuroprotective effects than EPA and is expected to expand the medicinal value of EPA.

[0028] The present invention provides a preparation method of the eicosapentaenoic acid derivative described in the above technical solution, comprising the following steps:

[0029] Mix eicosapentaenoic acid, an activator and an organic solvent, and perform activation treatment to obtain an activated liquid material;

[0030] Mix the activated liquid material with tetrahydro-2-furanmethanol and perform an esterification reaction to obtain an eicosapentaenoic acid derivative having the structure shown in Formula I.

[0031] 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.

[0032] In the present invention, eicosapentaenoic acid, an activator and an organic solvent are mixed and activation treatment is performed to obtain an activated liquid material. 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 dosage ratio of eicosapentaenoic acid to the organic solvent may be 9-10 mmol:10-20 mL, specifically 9.13 mmol:15 mL.

[0033] As an embodiment of the present invention, specifically, 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 may be 10-40 °C, specifically room temperature (25 °C); the time may be 20-40 min, specifically 30 min; the activation treatment may be carried out under stirring conditions.

[0034] After activation treatment, the present invention does not require any post-treatment. The obtained activated feed liquid is directly mixed with tetrahydro-2-furanmethanol for an esterification reaction to obtain an eicosapentaenoic acid derivative having the structure shown in Formula I. As an embodiment of the present invention, the molar ratio of the eicosapentaenoic acid to tetrahydro-2-furanmethanol can be 1:1 to 1.5, specifically 1:1.2. As an embodiment of the present invention, the temperature of the esterification reaction can be 10 to 40 °C, specifically room temperature; the time can be 1 to 3 h, specifically 2 h; and the esterification reaction can be carried out under stirring conditions. In the examples of the present invention, the reaction is specifically monitored by TLC.

[0035] As an embodiment of the present invention, after the esterification reaction, water can be added to the obtained product system, liquid separation is carried out, the aqueous phase is extracted with dichloromethane, the organic phase is collected, the organic phase is washed with saturated sodium chloride solution, then dried over anhydrous sodium sulfate, filtered, the solvent in the filtrate is rotary evaporated, and separation is carried out through a silica gel column (by volume ratio, the eluent used can be petroleum ether:ethyl acetate = 1:10) to obtain an eicosapentaenoic acid derivative having the structure shown in Formula I.

[0036] The present invention provides the application of the eicosapentaenoic acid derivative described in the above technical solution in the preparation of drugs for preventing and treating nervous system diseases. The EPA derivative provided by the present invention has strong liposolubility and relatively high bioavailability, and can realize the improvement of the efficacy of EPA. In the test examples of the present invention, specifically, an oxidative damage model of HT22 cells is constructed by inducing with H2O2, and then the protective effects of the EPA derivative and EPA on the cells are studied. The results show that the EPA derivative has stronger therapeutic potential than EPA in alleviating the oxidative damage of HT22 cells induced by H2O2.

[0037] As an embodiment of the present invention, the nervous system diseases can include neurodegenerative diseases; the neurodegenerative diseases can include Alzheimer's disease or Parkinson's disease.

[0038] The drug for preventing and treating nervous system diseases according to the present invention includes the eicosapentaenoic acid derivative (i.e., as an active ingredient) and pharmaceutically acceptable excipients. The present invention has no special limitation on the specific types of the pharmaceutically acceptable excipients, and pharmaceutically acceptable excipients well-known to those skilled in the art can be used. As an embodiment of the present invention, the dosage forms of the drug for preventing and treating nervous system diseases can include injections, pills or capsules.

[0039] The present invention provides a drug for preventing and treating nervous system diseases, and the active ingredient includes 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 can be the same as those in the above technical solution, and will not be elaborated here.

[0040] 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.

[0041] 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.

[0042] 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).

[0043] 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).

[0044] Other reagents and instruments not specifically mentioned can be purchased directly.

[0045] Example 1

[0046] Figure 1 The synthetic route of EPA derivatives is shown below. Figure 1 The preparation method of EPA derivatives is described in detail.

[0047] Eicosapentaenoic acid (EPA, 2.76 g, 9.13 mmol) was dissolved in 15 mL of N,N-dimethylformamide (DMF). At room temperature, 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. The mixture was stirred at room temperature for 30 min, then tetrahydro-2-furanmethanol (1.12 g, 11 mmol, 1.2 eq.) was added, and the mixture was stirred at room temperature for 2 h. The reaction was monitored by TLC until completion. 30 mL of water was added to the resulting product system, and the layers were separated. The aqueous phase was extracted three times with dichloromethane. The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the solvent in the filtrate was evaporated. The residue was separated by silica gel column chromatography (the eluent used was petroleum ether:ethyl acetate = 1:10 by volume) to obtain the EPA derivative (EPA-2 tetrahydrofuran methyl ester), and its 1H NMR spectrum is specifically as follows Figure 2 as shown.

[0048] Test Example 1

[0049] 1. Experimental Method

[0050] (1) Cultivation process of HT22 cells

[0051] 1.1. The HT22 cells were resuscitated. 5 mL of DMEM high-glucose complete medium, which was pre-prepared with 10% fetal bovine serum as a nutrient source and supplemented with 1% penicillin-streptomycin mixture to ensure a sterile culture environment, was injected into a culture dish with a diameter of 60 mm. Subsequently, the HT22 cells were evenly inoculated into the culture dish, and the inoculated culture dish was placed in a constant temperature incubator at 37 °C with a CO2 concentration of 5% for 24 h of preliminary culture to promote cell adhesion and preliminary proliferation.

[0052] 1.2. When it was observed that the cells grew adherently on the bottom of the culture dish to a coverage rate of 80 - 90%, cell passage operation was performed to maintain an appropriate cell density. Before that, the required medium, PBS buffer solution, and trypsin solution were preheated to 37 °C to ensure temperature consistency during the operation. After removing the old medium, the cell surface was gently washed with preheated PBS buffer solution to remove residual medium and non-adherent impurities. Subsequently, 1 mL of trypsin solution was added to the culture dish, and the culture dish was gently shaken to evenly cover the cell layer with trypsin. Then the culture dish was placed back in the 37 °C constant temperature incubator for digestion for 1 min to effectively separate the adherent cells. After digestion was completed, 2 mL of fresh DMEM high-glucose complete medium was immediately added to neutralize the trypsin activity, and the cells were separated from the bottom of the culture dish by gentle pipetting to form a cell suspension.

[0053] 1.3. Transfer the cell suspension to a 15 mL centrifuge tube and centrifuge it at 24 °C and 1000×g for 3 min to precipitate the cells at the bottom of the tube. Subsequently, carefully remove the supernatant, add 2 mL of fresh high-glucose DMEM complete medium to the centrifuge tube, and gently pipette again to form a uniform cell suspension. Finally, aspirate 1 mL of the cell suspension from this suspension and re-inoculate it into a new 60 mm diameter culture dish, and supplement it with 5 mL of high-glucose DMEM complete medium, and continue to place it in a 37 °C constant temperature incubator for subsequent culture.

[0054] (2)Construction and evaluation of HT22 cell oxidative damage model

[0055] 2.1. Treatment of HT22 cells with H2O2 concentration gradient

[0056] To explore the effect of different concentrations of H2O2 on the oxidative damage of HT22 cells, in this experiment, HT22 cells in the logarithmic growth phase and in good condition were selected and precisely inoculated into a 96-well plate at a density of 3.5×10 3 cells per well. After 24 h of cell attachment and stabilization, the medium was changed to expose the cells in each group to high-glucose DMEM complete medium containing different final concentrations of H2O2 (0, 200 μM, 400 μM, 600 μM), and 4 replicate wells were set in each group to enhance the reliability of the experimental results. Subsequently, the plate was placed in a constant temperature incubator at 37 °C and 5% CO2 concentration for light-shielded culture for 4 h to simulate the in vitro oxidative stress environment. After the culture was completed, all the old medium was removed and replaced with fresh high-glucose DMEM complete medium, and the degree of cell oxidative damage was detected after continuing to culture for 24 h.

[0057] 2.2. Detection of cell viability based on the CCK-8 method

[0058] Based on the experiments in 2.1, after the end of the predetermined culture time, the CCK-8 method was used to quantitatively evaluate the survival rate of cells in each group. First, the old culture medium in the well plate was removed, and a mixture of 100 μL of high-glucose DMEM basal medium and 10 μL of CCK-8 solution was added to each well. At the same time, a blank control group (i.e., containing only the culture medium and CCK-8 solution, without cells) was set up to correct the background absorbance value. Subsequently, the well plate was incubated in the dark for 2 h under the same conditions to allow CCK-8 to fully react with the dehydrogenase in living cells, resulting in a detectable color change. After the incubation, the absorbance values (OD) of each well were measured at wavelengths of 450 nm and 610 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and the interference of non-specific absorption was eliminated by calculating the difference (OD450nm - OD610nm). The formula for calculating the cell survival rate is: [(OD value of the experimental well - OD value of the blank well) / (OD value of the control well - OD value of the blank well)] × 100%. Among them, the experimental well represents the mixture of HT22 cells in each group after drug administration and CCK-8; the control well is the mixture of normal HT22 cells without any treatment and CCK-8; the blank well contains only the culture medium and CCK-8 solution to eliminate background interference. By comparing the cell survival rates under different H2O2 concentration treatments, suitable oxidative damage conditions can be screened out, providing a reliable basis for subsequent in-depth research.

[0059] (3)Screening of the protective concentration of EPA and its derivatives against the HT22 model damaged by H2O2

[0060] 3.1 Determination of H2O2 concentration and experimental grouping

[0061] Based on the experimental results in 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:

[0062] Normal control group (Control): Cultured with high-glucose DMEM complete medium;

[0063] Model group (H2O2 group): Co-cultured with 400 μM H2O2 and high-glucose DMEM complete medium;

[0064] Experimental group: Multiple concentration gradients of EPA and its derivatives were set (0 μM, 10 μM, 20 μM, 40 μM, 80 μM), and each concentration group was used in combination with 400 μM H2O2 and high-glucose DMEM complete medium.

[0065] 3.2 Determination of the effective administration concentration of EPA and its derivatives

[0066] HT22 cells in the logarithmic growth phase with good growth state were selected, and according to 3.5×10 per well 3Cells were precisely inoculated into 96-well plates according to the density. After culturing the cells adherently for 24 h, the culture medium was changed, and the cells in each group were respectively exposed to complete culture medium containing different final concentrations (0 μM, 10 μM, 20 μM, 40 μM, 80 μM) of EPA and EPA derivatives. EPA and EPA derivatives were pre-dissolved in absolute ethanol for preparation, and the final concentration of absolute ethanol was <0.1%. After continuous culture for 24 h, the old culture medium was aspirated, and DMEM high-glucose complete culture medium containing 400 μM H2O2 was added to each group. At the same time, a normal control group (containing only DMEM high-glucose complete culture medium) was established. Four replicate wells were set in each group, and they were cultured in the dark for 4 h in a constant temperature incubator at 37 °C and a CO2 concentration of 5%. Subsequently, all the culture medium was removed and replaced with fresh DMEM high-glucose complete culture medium, and CCK-8 cell viability detection was performed after continuous culture for 24 h. The detection method followed the standard procedure described in 2.2.

[0067] 2. Experimental Results

[0068] By adopting the H2O2 induction strategy, an oxidative damage model of HT22 cells was successfully constructed in this experiment. After systematic evaluation, it was established that treatment with 400 μM H2O2 for 4 h was the optimal condition for constructing this model. Subsequently, in the experimental group, HT22 cells were pre-treated with different concentration gradients of EPA and EPA derivatives for 24 h, then exposed to H2O2 for 4 h for oxidative damage treatment, and then replaced with fresh culture medium and continued to be cultured for 24 h. The experimental results showed that EPA had an unexpected inhibitory effect on cell viability under low concentration ranges (10 μM, 20 μM), while it showed a protective trend under higher concentrations (40 μM, 80 μM), but this effect was not statistically significant. On the contrary, EPA derivatives showed a stronger protective effect on cells with increasing concentration. Especially when the concentration was 80 μM, its protective effect reached statistical significance (P < 0.01). The following is a detailed description.

[0069] (1) Effects of Different Concentrations of H2O2 on the Viability of HT22 Cells

[0070] The effects of different concentrations of H2O2 on the viability of HT22 cells were systematically evaluated using the CCK-8 method. Figure 3 Figure showing the results of the effect of treating HT22 cells with different concentrations of H2O2 for 4 h after culturing for 24 h and then continuing to culture for 24 h on cell survival rate (compared with the control group, **P < 0.01, ***P < 0.001, ****P < 0.0001), from Figure 3It can be seen that with the increase in the concentration of H2O2, the viability of HT22 cells decreased significantly. Specifically, treatment with 200 μM H2O2 has led to significant inhibition of cell viability (P<0.01), while the inhibitory effect of 400 μM H2O2 is more significant (P<0.001), and 600 μM H2O2 further exacerbated this trend (P<0.0001). Based on the above results, 400 μM H2O2 treatment for 4 h was selected as the standard model condition for inducing oxidative damage in HT22 nerve cells in this experiment.

[0071] (2) Effects of different concentrations of EPA on the viability of the HT22 oxidative damage model

[0072] The effects of different concentrations of EPA on the viability of cells in the HT22 oxidative damage model were detected using the CCK-8 method. Figure 4 Figure showing the protective effects of different concentrations of EPA on the HT22 cell oxidative damage model (compared with the model group, ****P<0.0001). It can be seen from Figure 4 that compared with the H2O2 model group, EPA did not show obvious protective effects at concentrations of 10 μM and 20 μM. At concentrations of 40 μM and 80 μM, the protective effects of EPA on cells gradually increased, but did not reach statistical significance.

[0073] (3) Effects of different concentrations of EPA derivatives on the viability of the HT22 oxidative damage model

[0074] The effects of different concentrations of EPA derivatives on the viability of cells in the HT22 oxidative damage model were further evaluated. Figure 5 Figure showing the protective effects of different concentrations of EPA derivatives on the HT22 cell oxidative damage model (compared with the model group, ***P<0.01, **P<0.001). It can be seen from Figure 5 that compared with the H2O2 model group, all concentration groups of EPA derivatives (10 μM, 20 μM, 40 μM, 80 μM) showed enhanced cell protective effects with increasing concentration. In particular, the 80 μM EPA derivative group showed significant statistical differences (P<0.01), indicating that it has a stronger protective effect on H2O2-induced oxidative damage in HT22 cells.

[0075] From the above results, it can be seen that the oxidative damage model of HT22 cells was successfully constructed by H2O2 induction, and a significant decrease in cell survival rate was observed. After determining that the treatment with 400 μM H2O2 for 4 h was the optimal model establishment condition, the cytoprotective effects of EPA and EPA derivatives under pretreatment conditions were further explored. The experimental results showed that EPA exhibited a certain protective trend at high concentrations, but it did not reach statistical significance. In contrast, the cytoprotective effect of EPA derivatives was significantly enhanced with increasing concentration, especially showing a significant statistical difference at a concentration of 80 μM, suggesting that EPA derivatives have stronger therapeutic potential than EPA in alleviating H2O2-induced oxidative damage of HT22 cells. This finding provides new ideas and directions for the treatment of related neurological diseases such as neurodegenerative diseases.

[0076] The above description is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope 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 activating agent is 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. A drug for preventing and treating nervous system diseases, characterized in that: The active component comprises the eicosapentaenoic acid derivative according to claim 1.

7. The drug for preventing and treating nervous system diseases according to claim 6, characterized in that: The dosage form of the drug for preventing and treating nervous system diseases is injection, pill or capsule.

Images