An EPA derivative, its preparation method and uses

Abstract

The present invention provides an EPA derivative, a preparation method thereof and uses thereof. The EPA derivative is eicosapentaenoic acid-4-benzyloxybenzyl ester. The present invention belongs to the technical field of medicine and provides an EPA derivative with a novel structure. The EPA derivative is eicosapentaenoic acid-4-benzyloxybenzyl ester (abbreviated as EPA-BE), which is prepared by a one-step synthesis from 4-benzyloxybenzyl alcohol and eicosapentaenoic acid under conditions such as immobilized lipase. This derivative improves the bioavailability of EPA and enhances the therapeutic effect on atherosclerotic cardiovascular diseases.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to an EPA derivative and its preparation method and use. Background Art

[0002] Atherosclerotic cardiovascular disease (ASCVD) is one of the main causes of death and disability globally. ASCVD includes coronary artery disease (such as myocardial infarction), cerebrovascular disease (such as stroke), and peripheral artery disease, etc. Its main features include lipid deposition under the arterial intima, fibrous tissue hyperplasia, infiltration of inflammatory cells, and finally the formation of atherosclerotic plaques. The rupture and thrombosis of plaques can lead to acute occlusion of the blood vessel lumen, triggering serious events such as myocardial infarction or stroke. For the treatment of ASCVD patients, doctors may recommend the use of lipid-lowering drugs, antihypertensive drugs, antiplatelet drugs and other drug treatments to prevent or delay the occurrence of cardiovascular events. Although drugs such as statins have been widely used to reduce low-density lipoprotein cholesterol (LDL-C) and achieved significant clinical effects, the continuous risk of ASCVD still exists, indicating that simply reducing LDL-C is not sufficient to completely prevent the occurrence of cardiovascular events. In fact, according to the public data of REDUCE-IT and JELIS, compared with the treatment with statins alone, adding eicosapentaenoic acid (EPA) to statin treatment can significantly reduce major adverse cardiovascular events (MACE), which shows that EPA has a positive effect in the prevention and treatment of ASCVD. Moreover, it has long been a consensus in the industry that EPA has the effects of reducing blood lipids and maintaining cardiovascular health. EPA can protect cardiovascular health through various actions, such as reducing blood lipids, anti-inflammatory, antioxidant, improving endothelial function, inhibiting the progression of atherosclerosis, and regulating immune function.

[0003] As an important ω-3 polyunsaturated fatty acid, EPA is mainly present in deep-sea fish oils, such as salmon, sardines, and herring, and is one of the main components of fish oil. Currently, EPA products on the market mainly exist in the form of dietary supplements, such as fish oil capsules and concentrated fish oil liquids. These products usually contain EPA and other ω-3 fatty acids (such as docosahexaenoic acid, DHA). Although these dietary supplements have shown certain effects in preventing cardiovascular diseases, they cannot be used as drugs to treat cardiovascular diseases. Data shows that only high-purity EPA can provide cardiovascular protection for medical use. However, the latest research results indicate that the protective effect of high-dose EPA on the cardiovascular system seems to exceed its lipid-lowering effect, suggesting that EPA may reduce atherosclerosis and lower the risk of ASCVD through multiple mechanisms. However, the development and application of EPA as a drug ingredient are still relatively difficult, mainly because: ① The bioavailability of EPA in the human body is low, and the absorption effect is poor; ② EPA is easily oxidized and degraded, and its stability is poor.

[0004] In recent years, the derivatization research of EPA has become a hot topic. Currently, the derivatives of EPA are mainly ethyl ester-type EPA However, it also has problems such as poor bioavailability. There is only one product, eicosapentaenoic acid ethyl ester (EPA-EE, purity 96%) from Amarin Corporation in Ireland, used as a prescription drug for the treatment of hypertriglyceridemia. Chinese patent application CN 116407543 A discloses an EPA-EE nano-lipid composition, which makes EPA-EE into a nano-lipid composition to improve the bioavailability of EPA-EE.

[0005] Therefore, it is of great significance to provide a novel-structured EPA derivative with the effect of treating atherosclerosis. Summary of the Invention

[0006] To solve the problems existing in the prior art, the present invention provides a novel-structured EPA derivative, which is eicosapentaenoic acid-4-benzyloxybenzyl ester (abbreviated as EPA-BE), and is synthesized by a one-step method from 4-benzyloxybenzyl alcohol and eicosapentaenoic acid under conditions such as immobilized lipase. This derivative improves the bioavailability of EPA and improves the treatment effect of atherosclerotic cardiovascular diseases. The object of the present invention will be further described in the following detailed description.

[0007] The present invention provides an EPA derivative, which is eicosapentaenoic acid-4-benzyloxybenzyl ester, and its structural formula is:

[0008] With the above technical solution, the EPA derivative provided by the present invention has a novel structure, and experiments have proved that this compound has obvious effects of improving atherosclerosis, anti-inflammation and maintaining vascular health.

[0009] Preferably, for the said EPA derivative, its synthesis route is as Figure 1 shown, and the preparation method comprises the following steps:

[0010] A) Weigh 4-benzyloxybenzyl alcohol and eicosapentaenoic acid, dissolve them in anhydrous tetrahydrofuran, stir, and then add immobilized lipase and molecular sieve, and continuously stir and react at 35 - 45 °C;

[0011] B) Monitor the reaction using thin-layer chromatography. After reacting for 35 - 45 h, filter while it is hot, collect the filtrate, rinse the filter residue with an organic solvent, combine the filtrates and evaporate and concentrate to obtain a crude product of a pale yellow liquid; then use a Pure flash chromatography purification system for separation and purification, and elute to obtain the EPA derivative.

[0012] Correspondingly, the present invention also provides a preparation method for the said EPA derivative, comprising the following steps:

[0013] A) Weigh 4-benzyloxybenzyl alcohol and eicosapentaenoic acid, dissolve them in anhydrous tetrahydrofuran, stir, and then add immobilized lipase and molecular sieve, and continuously stir and react at 35 - 45 °C;

[0014] B) Monitor the reaction using thin-layer chromatography. After reacting for 35 - 45 h, filter while it is hot, collect the filtrate, rinse the filter residue with an organic solvent, combine the filtrates and evaporate and concentrate to obtain a crude product of a pale yellow liquid; then use a Pure flash chromatography purification system for separation and purification, and elute to obtain the EPA derivative.

[0015] Preferably, the said immobilized lipase is selected from Novozym 435 or Lipozyme TL IM or Lipozyme RMIM.

[0016] Preferably, the said molecular sieve is activated molecular sieve.

[0017] Preferably, the temperature of the said reaction is 38 - 42 °C, and the time is 35 - 42 h.

[0018] Preferably, the Pure flash chromatography purification system uses a silica gel-packed chromatography column.

[0019] Preferably, the said organic solvent uses petroleum ether and / or ethyl acetate.

[0020] Preferably, the elution is carried out using petroleum ether / ethyl acetate as the eluent, and the volume percentages of petroleum ether and ethyl acetate are 94% and 6% respectively.

[0021] In addition, the present invention also provides the use of the EPA derivative in the preparation of a drug for preventing and treating atherosclerotic cardiovascular diseases.

[0022] Compared with the prior art, the beneficial effects of the present invention include:

[0023] (1) The present invention provides a novel-structured EPA derivative, which is eicosapentaenoic acid-4-benzyloxybenzyl ester (EPA-BE), and is prepared by one-step synthesis from 4-benzyloxybenzyl alcohol and eicosapentaenoic acid under conditions such as immobilized lipase.

[0024] (2) The present invention provides a preparation method of the EPA derivative. The reaction conditions are mild, the preparation steps are relatively simple, one-step synthesis preparation is achieved, and the yield is relatively high.

[0025] (3) The present invention provides the use of the EPA derivative in the preparation of a drug for preventing and treating atherosclerotic cardiovascular diseases. Experiments have demonstrated the therapeutic effect of EPA-BE on atherosclerotic cardiovascular diseases, and it is significantly superior to EPA-EE in terms of the therapeutic effect on existing plaques and anti-inflammatory effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Synthesis route diagram of the EPA derivative provided by the present invention.

[0027] Figure 2 1H NMR spectrum of EPA-BE 1 1H NMR spectrum.

[0028] Figure 3 13C NMR spectrum of EPA-BE 13 13C NMR spectrum.

[0029] Figure 4 Results diagram of the content changes of pro-inflammatory cytokines (NF-α, IL-1β and IL-6) and anti-inflammatory cytokine (IL-10) in rats before and after drug treatment intervention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0030] The present invention will be further described in detail below with reference to the drawings and embodiments.

[0031] In the present invention, the reagents and instruments involved are all conventional commercially available products, or can be obtained by conventional technical means in the art. The Pure flash chromatographic purification system is purchased from BUCHI, Switzerland.

[0032] Synthesis of Eicosapentaenoic Acid-4-benzyloxybenzyl Ester (EPA-BE) in Example 1

[0033] Weigh 5.00 g of 4-benzyloxybenzyl alcohol (23.33 mmol) and 10.59 g of eicosapentaenoic acid (35.00 mmol), and dissolve them in 60 mL of anhydrous tetrahydrofuran. The mixed solution is stirred at 40 °C for 5 min, and then 1.5 g of Novozym 435 enzyme and 1 g of activated molecular sieve are added. Stir continuously at 40 °C and react for 36 h. Monitor the reaction using thin-layer chromatography (developer: petroleum ether and ethyl acetate in a volume ratio of 15:1, R f = 0.5). After the reaction is completed, filter while it is hot, collect the filtrate, wash the filter residue with petroleum ether and ethyl acetate three times each, combine the filtrates and evaporate and concentrate to obtain a crude light yellow liquid; then use the BUCHIPureflash chromatography purification system for separation and purification. Use petroleum ether / ethyl acetate as the eluent (the volume percentages of petroleum ether and ethyl acetate are 94% and 6% respectively) to elute to obtain the corresponding components, collect the corresponding fractions and perform vacuum distillation to obtain a light yellow liquid product (EPA-BE). Then perform NMR analysis on the obtained product. Using CDCl3 as the solvent, the 1 1H NMR spectrum of EPA-BE is as Figure 2 shown, and the 13 13C NMR spectrum of EPA-BE is as Figure 3 shown. Based on 4-benzyloxybenzyl alcohol, the molar yield is 68.3%.

[0034] EPA-BE: 1 1H NMR (300 MHz, Chloroform-d) δ 7.47 - 7.34 (m, 5H, Ph-), 7.32 - 7.27 (m, 2H, H-2, H-6), 7.02 - 6.93 (m, 2H, H-3, H-5), 5.46 - 5.25 (m, 10H, -CH2C H =C H CH2-), 5.06 (d, J = 5.5 Hz, 4H, PhC H 2-), 2.82 (dtd, J = 15.5, 5.7, 2.1 Hz, 8H, -CH=CH-C H 2-CH=CH-), 2.35 (t, J = 7.5 Hz, 2H, H-2), 2.15 - 2.01 (m, 4H, H-4', -C H 2CH3), 1.72 (p, J = 7.5 Hz, 2H, H-3'), 0.98 (t, J = 7.5 Hz, 3H, -CH2C H 3). 1313C NMR (75 MHz, Chloroform-d) δ 173.53 (C1”), 158.82 (C4), 136.85 (C1'), 132.07, 130.11, 128.98, 128.85, 128.64, 128.60, 128.28, 128.26, 128.14, 128.04, 127.91, 127.47, 127.04, 114.88 (C3), 70.03 (Ph- C -H2-), 65.96 (-Ph- C -H2-), 33.75 (C2”), 31.47 (C4”), 26.57, 25.65, 25.63, 25.57, 24.79 (C3”), 20.59 (C19”), 14.32 (C20”). MS (ESI, +ve): m / z 521.3 [M+Na + .

[0035] Synthesis of Eicosapentaenoic Acid-4-benzyloxybenzyl Ester (EPA-BE) in Example 2

[0036] Weigh 5.00 g of 4-benzyloxybenzyl alcohol (23.33 mmol) and 10.59 g of eicosapentaenoic acid (35.00 mmol), and dissolve them in 60 mL of anhydrous tetrahydrofuran. The mixed solution is stirred at 40 °C for 5 min, and then 2 g of Lipozyme TL IM enzyme and 1 g of activated molecular sieve are added. Stir continuously at 40 °C and react for 40 h. Monitor the reaction using thin-layer chromatography (developer: petroleum ether and ethyl acetate in a volume ratio of 15:1, R f = 0.5). After the reaction is completed, filter while it is hot, collect the filtrate, wash the filter residue with petroleum ether and ethyl acetate three times each, combine the filtrates and evaporate and concentrate to obtain a crude product of a pale yellow liquid; then use the BUCHI Pureflash chromatography purification system for separation and purification, use petroleum ether / ethyl acetate as the eluent (the volume percentages of petroleum ether and ethyl acetate are 94% and 6% respectively) to elute to obtain the corresponding components, collect the corresponding fractions and perform vacuum distillation to obtain a pale yellow liquid product (EPA-BE). Based on 4-benzyloxybenzyl alcohol, the molar yield is 60.6%.

[0037] Efficacy Test of Eicosapentaenoic Acid-4-benzyloxybenzyl Ester (EPA-BE) in Test Example 1

[0038] Animal experiment: Sixty SPF-grade male SD rats with a body weight of 220 g ± 19.5 g were randomly divided into six groups: a control group, an atherosclerosis model group, an EPA-EE group, a low-dose EPA-BE group, a medium-dose EPA-BE group, and a high-dose EPA-BE group. Among them, rats were induced to develop atherosclerosis by a high-fat diet to establish an atherosclerosis model. On this basis, drug intervention was carried out on the atherosclerosis model group. The EPA-EE treatment group was given intragastric administration of 200 mg / kg EPA-EE for treatment intervention on the basis of a high-fat diet. The EPA-BE group was given intragastric administration of EPA-BE drugs at low dose (10 mg / kg), medium dose (100 mg / kg), and high dose (200 mg / kg) respectively. At the same time, the control group was fed with a normal diet and given intragastric administration of the same amount of normal saline as the above drugs. All treatment groups were given the same amount of feed every day, and the experiment lasted for 12 weeks. After the experiment, blood samples and tissue samples were collected for analysis. The blood lipid levels in the serum or tissues of rats were detected by an automatic biochemical analyzer. The contents of inflammatory factors (NF-α, IL-1β, IL-6, and IL-10) were detected by enzyme-linked immunosorbent assay (ELISA). Indexes such as the area and thickness of atherosclerotic plaques were measured by an image analyzer to evaluate the degree of atherosclerosis and the changes in related physiological indexes of rats in each group.

[0039] 1. Detection of atherosclerotic plaque changes

[0040] Table 1 Changes in the contents of atherosclerotic plaques in rats before and after drug treatment intervention

[0041]

[0042]

[0043] Note: EPA-EE group (200 mg / kg), low-dose EPA-BE group (10 mg / kg), medium-dose EPA-BE group (100 mg / kg), high-dose EPA-BE group (200 mg / kg).

[0044] As can be seen from Table 1, atherosclerotic plaques successfully appeared in the rats fed with a high-fat diet, while no abnormalities were found in the rats fed with a normal diet. After drug intervention, varying degrees of changes occurred in the plaque size and thickness in the plaque model group, as well as the content of related indicators. Among them, both the plaque volume and lipid length in the rats in the EPA-EE group decreased, indicating that the plaque symptoms were improved during the treatment process. Meanwhile, the lipid components in the plaque affect the stability of the plaque. Excessive lipid content is likely to cause plaque rupture, which is the main cause of cardiovascular events. At the same time, the fibrous cap thickness of the plaque is the fibrous cap layer on the surface of the atherosclerotic plaque. The thickness of the fibrous cap can reflect the stability of the plaque. The greater the thickness, the more stable the plaque and the less likely it is to rupture. Judging from the data, the fibrous cap thickness of the plaque in the rats increased by 13.7% after EPA-EE intervention, enhancing the plaque stability. Therefore, EPA-EE intervention helps to improve atherosclerotic plaques and reduce the risk of cardiovascular events. At the same time, after intervention with different doses of EPA-BE, the plaque volume and lipid length in the rats decreased to varying degrees. The higher the EPA-BE dose, the lower the plaque volume and lipid length, and the higher the fibrous cap thickness of the plaque. At the same drug concentration, the effect of EPA-BE in reducing the plaque volume and lipid content is about twice that of EPA-EE, and the effect of increasing the fibrous cap thickness of the plaque is about three times that of EPA-EE. This shows that EPA-BE is significantly superior to EPA-EE in the treatment effect on existing plaques.

[0045] Atherosclerosis is an inflammatory process that is a response to lipid accumulation within the arterial wall. Its pathogenesis begins with elevated cholesterol levels, leading to altered permeability of arterial endothelial cells, enabling lipids, especially "cholesterol-containing low-density lipoprotein (LDL)", to enter the arterial wall. Within the arterial wall, these lipids bind to the extracellular proteoglycan-rich matrix and aggregates, thereby triggering a series of inflammatory responses and pathological changes. Therefore, the LDL particle and cholesterol crystal content can reflect the degree of atherosclerosis. As a competitive inhibitor of arachidonic acid cyclooxygenase and lipoxygenase, EPA plays a key role in regulating inflammation. It is generally believed that EPA has an anti-inflammatory effect, while metabolites derived from arachidonic acid have a pro-inflammatory effect, exacerbating atherosclerotic conditions. Therefore, by supplementing EPA to change the ratio of EPA to arachidonic acid, it can help shift metabolism from a pro-inflammatory state to an anti-inflammatory state. As can be seen from Table 1, after drug intervention with EPA-EE and EPA-BE, both the LDL and cholesterol contents in the plaques decreased significantly. Similarly, lower LDL particle and cholesterol contents were observed after intervention with the same dose of EPA-BE compared to EPA-EE. At the same time, there were also significant differences in the EPA content in the rats. Under the same dose condition, the EPA content in the rats of the EPA-BE group was approximately 10 times that of the EPA-EE group. This may be because EPA-BE has lower polarity and is more likely to enter the outer phospholipid bilayer of cells than EPA-EE, and then remove the prodrug group to exert its effect. Therefore, under the same amount of action, EPA-BE has a more significant therapeutic effect than EPA-EE.

[0046] 2. Detection of Inflammatory Factors and Nitric Oxide (NO) Content

[0047] Inflammation is a core factor in the occurrence of cardiovascular events associated with dyslipidemia. Inflammation can promote the occurrence of arterial calcification, increase the instability of atherosclerotic plaques, promote endothelial dysfunction, foam cell formation, chronic inflammation, etc., and ultimately exacerbate the occurrence of atherosclerosis. NF-α (tumor necrosis factor α) and IL-1β (interleukin 1β) are pro-inflammatory cytokines that can induce inflammatory responses and exacerbate the progression of atherosclerosis. IL-6 (interleukin 6) is also a pro-inflammatory cytokine, and an increase in its level is associated with the progression of atherosclerosis and an increased risk of cardiovascular events. In contrast, IL-10 (interleukin 10) is an anti-inflammatory cytokine, and an increase in its level helps to inhibit the inflammatory response related to atherosclerosis. At the same time, high-sensitivity C-reactive protein (hsCRP) is an important inflammatory marker that can reflect the systemic inflammatory state of the body. A high level of hsCRP indicates the presence of a persistent inflammatory response, which is closely related to the occurrence and progression of atherosclerosis. Nitric oxide, as a vasodilator, is a recognized mediator of vascular health. Nitric oxide regulates vascular diastolic tension by reducing endothelin, regulating vascular permeability, platelet aggregation and adhesion, and the recruitment and adhesion of immune cells in the blood circulation, maintaining normal endothelial function, and thus playing an anti-atherosclerotic role.

[0048] From Figure 4 It can be seen that after the intervention of EPA-EE and EPA-BE in the atherosclerosis model, the contents of NF-α, IL-1β and IL-6 in the body all decreased to varying degrees. Among them, with the increase in the dose of EPA-BE, the contents of the three pro-inflammatory cytokines further decreased. Under the intervention of the same dose, the contents of the three pro-inflammatory cytokines after EPA-BE intervention were about 50% - 100% lower than those after EPA-EE.

[0049] Table 2 Changes in the contents of nitric oxide and high-sensitivity C-reactive protein in rats before and after drug treatment intervention

[0050]

[0051] As can be seen from Table 2, compared with the model group, the hsCRP content in the EPA-EE and EPA-BE groups decreased significantly. Under the intervention of high-dose EPA-BE, the hsCRP content decreased from 2 times higher than that of the normal group to about 80% of the normal group, indicating that EPA-BE has a good anti-inflammatory effect. At the same time, the IL-10 content increased significantly after the intervention of EPA-EE and EPA-BE. Compared with the control group, the high-dose EPA-BE intervention increased the IL-10 content by about 30%. In summary, EPA-BE has a significant anti-inflammatory effect, and it is more significant than EPA-EE.

[0052] As can be seen from Table 2, the NO content in the model group was about 30% lower than that in the normal group. After treatment with EPA-EE, the NO content in the rats recovered to a level slightly higher than that in the normal group. After intervention with the corresponding dose of EPA-BE, the NO content increased by a further 15%. Thus, it can be seen that EPA-BE has a good effect on improving vascular health.

[0053] The above content is a further detailed description of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention pertains, without departing from the concept of the present invention, several simple deductions or substitutions can still be made, and all should be regarded as belonging to the protection scope of the present invention.

Claims

1. An EPA derivative, characterized in that: The EPA derivative is eicosapentaenoic acid-4-benzyloxybenzyl ester, and its structural formula is:

2. The method for preparing the EPA derivative according to claim 1, characterized in that: The method comprises the following steps: A) weighing 4-benzyloxybenzyl alcohol and eicosapentaenoic acid, dissolving them in anhydrous tetrahydrofuran, stirring, then adding immobilized lipase and molecular sieves, and continuously stirring and reacting at 35-45° C.; B) Monitor the reaction using thin layer chromatography, filter while hot after reacting for 32-45 hours, collect the filtrate, rinse the filter residue with an organic solvent, combine the filtrate and evaporate and concentrate to obtain a pale yellow liquid crude product; then separate and purify using a Pure flash chromatography purification system to elute to obtain an EPA derivative.

3. The method for preparing the EPA derivative according to claim 2, characterized in that: The immobilized lipase is selected from Novozym 435, Lipozyme TLIM or Lipozyme RM IM.

4. The method for preparing the EPA derivative according to claim 2, characterized in that: The molecular sieve is an activated molecular sieve.

5. The method for preparing the EPA derivative according to claim 2, characterized in that: The reaction temperature is 38-42°C and the reaction time is 35-42h.

6. The method for preparing the EPA derivative according to claim 2, characterized in that: The Pure flash chromatography purification system uses a chromatography column filled with silica gel.

7. The method for preparing the EPA derivative according to claim 2, characterized in that: The organic solvent is petroleum ether and / or ethyl acetate.

8. The method for preparing the EPA derivative according to claim 2, characterized in that: The elution uses petroleum ether / ethyl acetate as the eluent, and the volume percentages of petroleum ether and ethyl acetate are 94% and 6% respectively.

9. Use of the EPA derivative according to claim 1 in the preparation of a drug for preventing and treating atherosclerotic cardiovascular diseases.

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