Ceramide compounds containing chain carboxylic acid and preparation method and application thereof

Abstract

The present invention belongs to the field of biomedicine technology. It is a ceramide compound as shown in the chemical formula I: #imgabs0# wherein R 1 A residue selected from α-linolenic acid, γ-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, arachidonic acid, ximenynic acid, azelaic acid, jasmonic acid, threonic acid, 4-aminobutyric acid, erucic acid, glycolic acid, lactic acid, malic acid, tartaric acid, citric acid, lactobionic acid, rosmarinic acid, danshensu, sodium 3-(4-hydroxy-3-methoxyphenyl) lactate, p-hydroxyphenylacetic acid, palmitoleic acid, vitamin B5, vitamin H, ricinoleic acid, behenic acid, cis-11-eicosenoic acid, hexacosenoic acid-cis-17-enoic acid, cis-11,14-eicosadienoic acid, mandelic acid, or a condensation residue of a fruit acid, R 2 Select one of the following structures: -C 15 H 29 ,‑C 15 H 31 ,‑C 15 H 27 , ‑CHOHC 14 H 27 , ‑CHOHC 14 H 29 The present invention reacts a functional carboxylic acid with a sphingoid base to obtain a class of ceramide compounds with novel structures, thereby enhancing the original efficacy of such molecules and improving the physical and chemical properties of the ceramide compounds.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to ceramide compounds containing chain carboxylic acids, and preparation methods and applications thereof. Background Art

[0002] Ceramides are compounds composed of sphingosines and fatty acids bonded via an amide bond. The carbon chain length, degree of unsaturation, and number of hydroxyl groups in both the sphingosine and fatty acid moieties can vary, making ceramides a class of compounds rather than a single compound. Currently, 15 ceramides have been detected in the skin, which can be broadly divided into three categories: short-chain ceramides, long-chain ceramides, and keratinocyte-bonded ceramides. The fatty acid moiety primarily consists of saturated chain fatty acids and low-unsaturation chain fatty acids, and these compounds often face solubility issues.

[0003] It is reported that water evaporation from the epidermis is achieved through the moisture-retention function of ceramides present in intercellular lipids. When the concentration of ceramides in the skin decreases, the protective barrier function of the stratum corneum weakens, leading to various dermatological symptoms such as atopic dermatitis and psoriasis. In addition, due to the decrease in the amount of ceramides, dry skin occurs, and the skin's surface defense function is lost, making it easier for foreign substances to invade and cause secondary skin infections, thereby causing skin rejection reactions. Specifically, invading substances trigger the release of cytokines from surface cells such as keratinocytes, Langerhans cells, and melanocytes, causing inflammatory phenomena.

[0004] The importance of ceramides is widely recognized, and many cosmetic and pharmaceutical companies are conducting research to develop products utilizing them. However, natural ceramides are difficult to extract and purify, posing economic challenges. Consequently, some companies are working to develop ceramide analogs that are structurally similar to ceramides found in the skin and that can provide the same functional benefits. However, commercially available natural or ceramide analogs have limitations, such as low hydrophilicity and solubility, making it difficult to produce high-content products.

[0005] Due to the widespread demand for functional ceramides in the market, it is very necessary to quickly construct ceramide derivatives through chemical synthesis to further improve the properties of ceramides and enhance their efficacy. Summary of the Invention

[0006] The purpose of the present invention is to provide a novel ceramide compound containing a chain carboxylic acid, wherein the carbon atom directly connected to the carboxyl group is CH2- or CHR 3 -carboxylic acid, R 3 It is a substituent. The chain carboxylic acid may have a heterocyclic ring or an aromatic ring, but the ring carbon atoms of the heterocyclic ring or aromatic ring are not directly connected to the carboxyl group.

[0007] Another object of the present invention is to provide a method for synthesizing ceramide compounds.

[0008] Another object of the present invention is to provide uses of ceramide compounds.

[0009] In order to achieve one of the above purposes, the present invention adopts the following technical solutions:

[0010] In the first aspect of the present invention, a ceramide compound has a structure of the general formula I or an enantiomer or diastereomer of the general formula I:

[0011]

[0012] Among them, R 1 selected from the group consisting of α-linolenic acid, γ-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, arachidonic acid, ximenynic acid, azelaic acid, jasmonic acid, threonic acid, 4-aminobutyric acid, erucic acid, glycolic acid, lactic acid, malic acid, tartaric acid, citric acid, lactobionic acid, rosmarinic acid, danshensu, sodium 3-(4-hydroxy-3-methoxyphenyl) lactate, p-hydroxyphenylacetic acid, palmitoleic acid, vitamin B5, vitamin H, ricinoleic acid, behenic acid, cis-11-eicosenoic acid, hexacosenoic acid-cis-17-enoic acid, cis-11,14-eicosadienoic acid, mandelic acid, or a residue after condensation of a fruit acid,

[0013] R 2 Select one of the following structures:

[0014] -C 15 H 29 、-C 15 H 31 、-C 15 H 27 ,-CHOHC 14 H 27 ,-CHOHC 14 H 29 .

[0015] The residue after condensation refers to the remaining carboxylic acid fragment R after the carboxyl group of the corresponding carboxylic acid RCOOH condenses with the amino group of the sphingoid base to form a peptide bond. For example, the residue after condensation of α-linolenic acid is The residue after condensation of arachidonic acid is

[0016] Azelaic acid has antibacterial properties and can be used as a food preservative. It can be used in mouthwashes to prevent tooth decay and in soaps to prevent cracking. It also has good skin permeability and can enhance absorption in creams and cosmetics. It also has skin-lightening and whitening properties. Azelaic acid or its zinc salt, when combined with vitamin B6, is used in hair care products to treat male hormonal alopecia and stimulate hair growth.

[0017] 4-Aminobutyric acid (GABA) is an important inhibitory neurotransmitter in the central nervous system. It possesses good water solubility and thermal stability, making it safe for consumption and suitable for use in the production of beverages and other foods. Consuming a certain amount of GABA can improve sleep quality and lower blood pressure, among other physiological benefits.

[0018] Erucic acid can be used in the food industry or cosmetics.

[0019] Eicosapentaenoic acid (EPA) is the main component of fish oil and belongs to the ω-3 series of polyunsaturated fatty acids. It is an important nutrient that is indispensable to the human body. It helps lower the levels of cholesterol and triglycerides and promotes the metabolism of saturated fatty acids in the body, thereby reducing blood viscosity, improving blood circulation, and increasing tissue oxygen supply to eliminate fatigue.

[0020] Docosahexaenoic acid (DHA) is another important polyunsaturated fatty acid. It not only plays a very important role in the growth and development of brain nerves, infant visual development and intellectual development, but also has anti-allergic and immune-enhancing effects.

[0021] Furthermore, the R 2 Select one of the following structures:

[0022]

[0023] Furthermore, the R 2 Select one of the following structures:

[0024]

[0025] They correspond to sphingosine, dihydrosphingosine, and phytosphingosine respectively.

[0026] Furthermore, the R 1 The residue is selected from the condensation residues of α-linolenic acid, γ-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, arachidonic acid, erucic acid, p-hydroxyphenylacetic acid or palmitoleic acid.

[0027] Furthermore, the R 1 Selected from the residues after condensation of α-linolenic acid.

[0028] Furthermore, the R 1Selected from the residues after condensation of gamma-linolenic acid.

[0029] Furthermore, the R 1 Selected from the residues after condensation of eicosapentaenoic acid.

[0030] Furthermore, the R 1 Selected from the residues after condensation of docosahexaenoic acid.

[0031] Furthermore, the R 1 Selected from the residues after condensation of arachidonic acid.

[0032] Furthermore, the R 1 Selected from the residues after condensation of erucic acid.

[0033] Furthermore, the R 1 Selected from the residues after condensation of p-hydroxyphenylacetic acid.

[0034] Furthermore, the R 1 Selected from the residues after condensation of palmitoleic acid.

[0035] Furthermore, the ceramide compound is selected from one of the following compounds:

[0036]

[0037]

[0038] A second aspect of the present invention provides a method for preparing a ceramide compound, comprising the following steps:

[0039]

[0040] Compound S1 reacts with p-nitrobenzenesulfonyl chloride and an organic base to obtain compound S2;

[0041] Compound S2 reacts with a sphingoid base to give compound I.

[0042] Furthermore, the molar ratio of the compound S1, p-nitrobenzenesulfonyl chloride, organic base, and sphingosine base is (1-2):(1-2):(2-6):1.

[0043] Furthermore, the condensing agent is triethylamine.

[0044] Furthermore, the solvent of the reaction is ethyl acetate.

[0045] A method for preparing a ceramide compound comprises the following steps:

[0046]

[0047] Compound S1 reacts with a sphingoid base and a condensing agent to obtain compound I.

[0048] Furthermore, the condensing agent includes EDCI and HOBT.

[0049] Furthermore, the molar ratio of the compound S1, EDCI, HOBT, and sphingoid base is 1:(1-2):(1-2):(0.8-1).

[0050] Furthermore, the solvent of the reaction is DCM.

[0051] The third aspect of the present invention is the use of ceramide compounds as antioxidants, especially in cosmetics, health products, and medicines.

[0052] The present invention has the following beneficial effects:

[0053] The present invention reacts functional carboxylic acids with sphingoid bases to obtain a class of novel structural ceramide compounds. The introduction of physiologically active molecular fragments of functional carboxylic acids into ceramides will enhance the original efficacy of such molecules and improve the physical and chemical properties of ceramide compounds. For example, increasing the unsaturation degree of long-chain fatty acids can improve their solubility, and the presence of multiple unsaturated bonds can enhance the antioxidant properties of the compounds.

[0054] Figures in the specification

[0055] Figure 1 This is the test result of the antioxidant activity of the ceramide compound of Example 14;

[0056] Figure 2 The test results of the cell proliferation method for the ceramide compound in Example 16 are as follows;

[0057] Figure 3 The test results of the LPS-induced cell method for the ceramide compound in Example 16 are as follows;

[0058] Figure 4 This is the test result of the cell scratch method for the ceramide compound in Example 16;

[0059] Figure 5 This is a bar graph of cell migration rate of the ceramide compound in Example 16. DETAILED DESCRIPTION

[0060] The present invention will be further described below with reference to specific embodiments.

[0061] All reactions were carried out under a nitrogen atmosphere. Unless otherwise stated, chemicals were purchased from commercial products and were not further purified. Dichloromethane and tetrahydrofuran used in the experiments were anhydrous solvents. Thin layer chromatography (TLC) used 60F254 silica gel plates. Silica gel column chromatography used Qingdao Marine Silica Gel (particle size 0.040-0.063 mm). TLC color development used UV light (254 nm) or iodine. NMR spectra were characterized using a Bruker DPX 400 nuclear magnetic resonance instrument. 1 H NMR is performed at 400 MHz, with deuterated methanol, deuterated DMSO or deuterated tetrahydrofuran as the solvent, and tetramethylsilane (TMS) as the internal standard. The unit of chemical shift is ppm, and the unit of coupling constant is Hz. 1 In H NMR, δ represents chemical shift, s represents a singlet, d represents a doublet, t represents a triplet, q represents a quartet, and m represents a multiplet.

[0062] EA refers to ethyl acetate, DCM refers to dichloromethane, EDCI refers to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and HOBT refers to 1-hydroxybenzotriazole.

[0063] General Synthesis Method of Ceramide Compounds

[0064] Method A

[0065]

[0066] Dissolve 1.2 eq (60 mmol) of p-nitrobenzenesulfonyl chloride in 70 ml of ethyl acetate. Dissolve 1.32 eq (66 mmol) of compound S1 and 3 eq (150 mmol) of triethylamine in 30 ml of ethyl acetate. Add the resulting mixture dropwise to the ethyl acetate solution of p-nitrobenzenesulfonyl chloride. Allow to react at 40°C for 12 h. TLC confirms the complete reaction of the p-nitrobenzenesulfonyl chloride. Add 2 eq (100 mmol) of triethylamine and 1 eq (50 mmol) of sphingoid base. Allow to react overnight at 40°C. TLC confirms the complete reaction of the sphingoid base.

[0067] Post-treatment: Add 100 mL of water, adjust the pH to 5-6 with 2N dilute hydrochloric acid, extract twice with 100 mL of saturated brine, dry over anhydrous sodium sulfate, filter, and concentrate in vacuo. The resulting residue is purified by silica gel column to obtain the product (50-70% yield).

[0068] Method B

[0069]

[0070] Compound S1 (50 mmol), EDCI (60-75 mmol, preferably 60 mmol), and HOBT (60-75 mmol, preferably 60 mmol) were placed in a 250 mL round-bottom flask, 100 mL of dichloromethane was added, and then stirred at room temperature for 1 hour. Subsequently, sphingoid base (40-50 mmol, preferably 45 mmol) was added to the reaction system and stirred at room temperature for 24-72 hours until the sphingoid base completely disappeared.

[0071] Workup: quenched by adding water. The organic layer was separated, dried, filtered and concentrated in vacuo. The residue thus obtained was purified by silica gel column to give the product (50-65% yield).

[0072] Example 1

[0073] The condensation product of α-linolenic acid and phytosphingosine by method A.

[0074]

[0075] 1 H NMR(400MHz,Methanol-d4)δ5.42–5.22(m,6H),4.07(td,J=5.8,4.3Hz,1H),3.79– 3.65(m,3H),3.58(t,J=5.8Hz,1H),3.52(ddd,J=9.6,5.6,2.4Hz,1H),2.80(t,J=6. 0Hz,4H),2.22(t,J=7.5Hz,2H),2.12–2.04(m,4H),1.91–1.80(m,1H),1.67–1.52( m, 4H), 1.35–1.28 (d, J = 22.7Hz, 31H), 0.97 (t, J = 7.6Hz, 3H), 0.89 (t, J = 7.6Hz, 3H).

[0076] Example 2

[0077] The condensation product of eicosapentaenoic acid and phytosphingosine by method B.

[0078]

[0079] 1H NMR(400MHz, Methanol-d4)δ5.49–5.25(m,1oH),4.11(q,J=5.5Hz,1H),3.75(qd,J=11.2,5.0Hz,2H),3.64–3.50(m,2H),2.94–2.77(m,8H), 2.32–2.22(m,2H),2.21–2.05(m,4H),1.70(dq,J=18.2,10.5,9.1Hz,3H),1.42–1.30(s,25H),1.00(t,J=7.5Hz,3H),0.92(t,J=6.7Hz,3H).

[0080] Example 3

[0081] The condensation product of docosahexaenoic acid and phytosphingosine by method B.

[0082]

[0083] 1 H NMR (400MHz, Methanol-d4) δ5.50–5.24(m,12H),4.11(q,J=5.3Hz,1H),3.82–3.69(m,2H),3.64–3.51(m,2H),2.87(dt,J=13.7,6.8Hz, 10H),2.49–2.36(m,2H),2.31(t,J=6.9Hz,2H),2.16–2.04(m,2H),1.32(d,J=8.7Hz,26H),0.99(t,J=7.5Hz,3H),0.92(t,J=6.7Hz,3H).

[0084] Example 4

[0085] The product of the condensation of a mixed acid of eicosapentaenoic acid and docosahexaenoic acid (mass ratio 22:51) with phytosphingosine by method B.

[0086] 1H NMR(400MHz, Methanol-d4)δ5.50–5.25(m,11.3H),4.17–4.06(m,1H),3.75(h,J=6.1,5.4Hz,2H),3.65–3.51(m,2H),2.95–2.75(m,9.2H),2.49 –2.36(m,1H),2.35–2.21(m,2H),2.11(h,J=7.2Hz,3H),1.78–1.62(m,2 H), 1.32 (d, J = 8.9Hz, 26H), 1.00 (t, J = 7.5Hz, 3H), 0.92 (t, J = 6.7Hz, 4H).

[0087] Example 5

[0088] The product of the condensation of a mixed acid of eicosapentaenoic acid and docosahexaenoic acid (mass ratio 50:20) with phytosphingosine by method B.

[0089] 1 H NMR(400MHz, Methanol-d4)δ5.46–5.21(m,10.2H),4.08(dtt,J=6.3,4.4,2.2Hz, 1H),3.80–3.65(m,2H),3.57(t,J=5.8Hz,1H),3.55–3.46(m,1H),2.93–2.73(m,8. 3H),2.39(d,J=2.6Hz,0.7H),2.31–2.18(m,2H),2.18–1.99(m,3.4H),1.75–1.46( m, 4H), 1.29 (d, J = 7.2Hz, 26H), 0.97 (t, J = 7.5Hz, 3H), 0.93–0.86 (t, J = 7.5Hz, 3H).

[0090] Example 6

[0091] The condensation product of a mixture of eicosapentaenoic acid and docosahexaenoic acid (mass ratio 22:51) with sphingosine by method B.

[0092] 1H NMR (400MHz, Methanol-d4) δ5.69 (dt, J=14.2, 6.7Hz, 1H), 5.46 (ddd, J=15.4, 7.5, 2.6Hz, 1H), 5.41–5.21(m,11H),4.11–3.99(m,1H),3.90–3.76(m,1H),3.69(dd,J=5.5,3.7Hz,2H),2.84(dq ,J=14.8,6.1,5.4Hz,9H),2.37(dtd,J=10.5,5.8,5.2,2.5Hz,1H),2.31–2.15(m,3H),2.15–1. 94(m,6H),1.74–1.49(m,2H),1.41–1.19(m,31H),0.97(t,J=7.5Hz,3H),0.89(t,J=6.7Hz,4H).

[0093] Example 7

[0094] The condensation product of arachidonic acid and phytosphingosine by method B.

[0095]

[0096] 1 H NMR(400MHz, Methanol-d4)δ5.46–5.27(m,8H),4.08(td,J=5.8,4.4Hz,1H), 3.72(qd,J=11.2,5.0Hz,2H),3.57(t,J=5.8Hz,1H),3.52(ddd,J=9.5,5.7,2. 4Hz,1H),2.83(p,J=6.3,5.7Hz,6H),2.30–2.18(m,2H),2.09(dq,J=21.0,6. 5Hz, 4H), 1.74–1.59 (m, 3H), 1.40–1.20 (m, 31H), 0.90 (td, J = 6.9, 3.4Hz, 6H).

[0097] Example 8

[0098] The condensation product of α-linolenic acid and sphingosine by method A.

[0099]

[0100] 1H NMR(400MHz, Methanol-d4)δ5.74–5.63(m,1H),5.49–5.42(m,1H),5.41–5.24( m,6H),4.04(t,J=7.4Hz,1H),3.85(dt,J=7.5,5.0Hz,1H),3.68(d,J=5.0Hz,2H ),2.80(t,J=6.0Hz,4H),2.24–2.14(m,2H),2.14–1.97(m,6H),1.59(dt,J=8.0 ,3.9Hz,2H),1.41–1.22(m,30H),0.97(t,J=7.6Hz,3H),0.89(t,J=7.6Hz,3H).

[0101] Example 9

[0102] The condensation product of α-linolenic acid and dihydrosphingosine by method A.

[0103]

[0104] 1 H NMR (400MHz, Methanol-d4) δ5.41–5.24(m,6H),4.04(t,J=7.4Hz,1H),3.85(dt,J=7.5,5.0Hz,1H),3.68(d,J=5.0Hz,2H),2.80(t,J=6.0Hz ,4H),2.24–2.14(m,2H),2.14–1.97(m,4H),1.59(dt,J=8.0,3.9Hz,2H),1.41–1.22(m,36H),0.97(t,J=7.6Hz,3H),0.89(t,J=7.6Hz,3H).

[0105] Example 10

[0106] The condensation product of erucic acid and sphingosine by method A.

[0107]

[0108] 1H NMR (400MHz, Methanol-d4) δ5.68(dt,J=14.1,6.8Hz,1H),5.44(dt,J=14.1,6.8Hz,1H),5.33(t,J=4.9Hz,2H),4.03(t,J=7.6Hz,1H),3.89– 3.80(m,1H),3.68(d,J=5.0Hz,2H),2.18(t,J=7.5Hz,2H),2.09–1.91(m,6H),1.58–1.52(m,2H),1.33–1.26(m,50H),0.89(t,J=6.7Hz,6H).

[0109] Example 11

[0110] The condensation product of 4-hydroxyphenylacetic acid and phytosphingosine by method B.

[0111]

[0112] 1 H NMR(400MHz, Methanol-d4)δ7.11(d,J=8.5Hz,2H),6.72(d,J=8.5Hz,2H),4.12–4.01(m,1H),3.79–3.65(m ,3H),3.53(dd,J=7.2,4.5Hz,1H),3.48–3.40(m,3H),1.63–1.41(m,2H),1.28(s,24H),0.95–0.82(m,3H).

[0113] Example 12

[0114] The condensation product of palmitoleic acid and sphingosine by method A.

[0115]

[0116] 1 H NMR (400MHz, Methanol-d4) δ5.68(dt,J=15.5,6.7Hz,1H),5.45(dt,J=15.5,6.7Hz,1H),5.33(t,J=4.9Hz,2H),4.03(t,J=7.5Hz,1H),3.85(dt,J=7 .6,5.0Hz,1H),3.68(d,J=5.0Hz,2H),2.18(t,J=7.5Hz,2H),2.03(q,J=6 .2Hz, 6H), 1.65–1.49 (m, 2H), 1.30 (d, J = 14.6Hz, 38H), 0.94–0.83 (m, 6H).

[0117] Example 13

[0118] The condensation product of gamma-linolenic acid and phytosphingosine by method A.

[0119]

[0120] 1 H NMR(400MHz,Methanol-d4)δ5.52–5.33(m,6H),4.09–4.02(m,1H),3.72–3.6 8(m,3H),3.53(t,J=5.8Hz,1H),3.50–3.44(m,1H),2.76(t,J=6.2Hz,4H),2. 18(t,J=7.6Hz,2H),2.10–2.02(m,4H),1.94–1.82(m,1H),1.69–1.54(m,4H) ,1.32–1.25(d,J=22.4Hz,31H),0.92(t,J=7.6Hz,3H),0.83(t,J=7.6Hz,3H).

[0121] Example 14

[0122] The antioxidant activity of the ceramide compounds prepared in Examples 1, 2, 4, 6, and 8 was tested using the antioxidant-ABTS method.

[0123] Principle: The use of ABTS (2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonicacid)diammonium salt) to evaluate the antioxidant capacity of samples was originally proposed by Miller et al. (1993). The method currently used is generally the improved method by Re et al. (1999). This method utilizes the fact that ABTS can be oxidized by a series of compounds, such as potassium persulfate, hydrogen peroxide, and manganese dioxide, to generate a blue-green ABTS+ cation radical with a maximum absorption peak at 734 nm. In the presence of antioxidants, ABTS+ is reduced to colorless ABTS. The antioxidant capacity of the reactant can be determined by measuring the absorbance at 734 nm.

[0124] ABTS free radical scavenging assay: Mix 3 mL of 12 mmol / L ABTS aqueous solution with 3 mL of 2.45 mmol / L potassium persulfate solution. Stir well and incubate at room temperature in the dark for 12–16 hours. Use DMSO as the dilution medium and adjust the potassium persulfate solution to an absorbance of 0.700 ± 0.025 at 734 nm. Add samples of varying concentrations, shake well, incubate in the dark for 10 minutes, and measure absorbance at 734 nm.

[0125] The results are as follows Figure 1 As shown, control 1 is ceramide 3 and control 2 is ceramide 3B.

[0126] It can be seen that the compounds of the present invention have better antioxidant effects than existing ceramide compounds, and the scavenging rate of free radicals is generally 15-40% higher.

[0127] Example 15

[0128] Solubility test

[0129] 10 mg of the ceramide compounds prepared in Examples 1, 2, 3, 7, and 8 were dissolved in 0.5 mL of ethanol, respectively. All of them were completely dissolved. For comparison, Control 1 was slightly soluble with many particles, while Control 2 was completely dissolved.

[0130] Example 16

[0131] Tissue repair testing

[0132] 1. Testing the Tissue Repair Performance of the Ceramide Compounds and Ceramide 3 Prepared in Examples 2 and 4 Using a Cell Proliferation Assay

[0133] MTT assay to detect cell proliferation activity: HaCaT cells were plated at 1×10 4 Cells were seeded at a density of 100 μL / well in a 96-well plate and incubated overnight in an incubator. After 24 hours, the supernatant was discarded and 100 μL of culture medium containing samples (or blank) of different concentrations was added. After incubation for another 24 hours, the culture medium was removed and 100 μL of MTT was added to each well. The absorbance at 450 nm was measured and the cell survival rate was calculated as A. 给药孔 / A 空白孔 ×100%.

[0134] The results are as follows Figure 2 As shown, compared with solvent group M, low concentrations (0.156-0.625 mM) of the compounds of Example 2 and Example 4 significantly increased HaCaT cell viability, indicating that within this concentration range, the compounds of Example 2 and Example 4 are safe and non-toxic to cells, effectively promote keratinocyte proliferation, and have the potential to repair damaged skin barriers. However, within the same concentration range, ceramide 3 reduced HaCaT cell viability and inhibited keratinocyte proliferation.

[0135] 2. LPS-induced cell assay to detect the anti-inflammatory and repair effects of the ceramide compounds and ceramide 3 prepared in Examples 2 and 4

[0136] Interleukin 6 (IL-6) is the most typical cytokine associated with inflammation. It plays an important role in host defense by regulating the immune and inflammatory responses. Inflammation affects the skin barrier, increasing epidermal water loss and affecting the growth of keratinocytes. Once the barrier is damaged, it is difficult to recover. It also breaks down the extracellular matrix, causing skin collapse, and inhibits collagen synthesis, making the skin loose and wrinkled. Therefore, effectively reducing the production of interleukin IL-6 in keratinocytes and fibroblasts caused by external damage and ultraviolet rays, and reducing the inflammatory response, is crucial to restoring the skin barrier and protecting skin elasticity and stability.

[0137] B16 mouse melanoma cells were cultured at a density of 1×10 4 Cells were seeded in 96-well plates at 100 μg / well and allowed to adhere overnight in an incubator. After 24 hours, the supernatant was discarded and 100 μL of samples diluted in DMEM medium at different concentrations were added. The negative control group was treated with drug-free DMEM medium. Three replicates were plated in each group and incubated in an atmosphere of 5% CO2 and 37°C. Two hours after drug administration, 10 μg / mL LPS was added to the lipopolysaccharide model group and the experimental group and incubated for 24 hours. After the reaction, 50 μL of the cell supernatant was collected and intracellular IL-6 gene expression was detected using an IL-6 ELISA kit.

[0138] The results are as follows Figure 3 As shown, compared to the solvent group, all three ceramides within a concentration range (0.0625-0.5 mM) effectively reduced the expression of the inflammatory cytokine IL-6, demonstrating anti-inflammatory activity. In particular, within the same concentration range, the compound in Example 2 exhibited superior inhibitory efficiency, suggesting its potential for treating other skin diseases caused by inflammatory factors.

[0139] 3. Cell scratch assay to detect wound healing of the ceramide compounds and ceramide 3 prepared in Examples 2 and 4

[0140] The cell scratch test is an in vitro method for studying cell migration. When keratinocytes grow to a fused monolayer, a blank area (scratch) is artificially created on the fused monolayer. Cells at the edge of the scratch gradually move into the blank area, healing the "scratch." This, to a certain extent, simulates the migration process of epidermal keratinocytes. By observing the state of cells in the scratch area at different times, the migration ability of cells can be determined. This is an important in vitro method for studying skin wound healing and repair.

[0141] Here’s how to do it:

[0142] 1. Marking the culture plate: First, use a marker pen to draw horizontal lines evenly on the back of the 6-well plate, using a ruler to measure the lines. Draw horizontal lines approximately every 0.5 to 1 cm across the holes, with at least 5 lines crossing each hole. Be careful not to make the lines too thick when marking.

[0143] 2. Cell plating: Add about 5×10 5 cells (the number of different cells varies and is adjusted according to the growth rate of the cells). The inoculation principle is that the fusion rate reaches 100% after overnight.

[0144] 3. Cell scratching: On the second day, use the tip of the pipette, perpendicular to the cell plane, to scratch the cell layer along the line drawn on the back of the plate the day before (it is best to use the same pipette tip between different wells).

[0145] 4. Wash cells: After the scratch is completed, wash the cells three times with sterile PBS to wash away the cells that are not attached to the wall, that is, the cells that are crossed during the scratching process, so that the gaps left after the scratching are clearly visible, and then replace with fresh serum-free culture medium.

[0146] 5. Cell culture and observation: Place the cells in a 37°C, 5wt% CO2 incubator. Then take out the cells after 24 hours, observe and measure the width of the scratch under a microscope, and take pictures. The results are as follows: Figure 4 IMAGE J software was used to analyze and calculate the cell migration rate. The results are shown in Figure 5 shown.

[0147] After 24 hours of co-incubation with cells, all three ceramides within a concentration range (1.0 mM) effectively enhanced the migration ability of keratinocytes. At the same concentration, the compound of Example 4 showed better ability to promote cell healing, indicating that it has a better effect in promoting the healing and repair of damaged epidermis.

[0148] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A ceramide compound having the structure of general formula I, or a condensation product of α-linolenic acid and phytosphingosine, a condensation product of α-linolenic acid and dihydrosphingosine, a condensation product of erucic acid and sphingosine, a condensation product of arachidonic acid and phytosphingosine, or an enantiomer or diastereomer thereof: in, R 1 The residue selected from the group consisting of γ-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, erucic acid, ricinoleic acid, cis-11-eicosenoic acid, hexacosenoic acid, and cis-11,14-eicosadienoic acid condensation residues, R 2 Select one of the following structures:

2. The ceramide compound according to claim 1, characterized in that Select one of the following compounds:

3. A method for preparing the ceramide compound according to claim 1 or 2, characterized in that: The following steps are involved: Compound S1 reacts with p-nitrobenzenesulfonyl chloride and an organic base to obtain compound S2; Compound S2 reacts with sphingoid base to obtain compound I; R 1 、R 2 As defined in claim 1 or 2.

4. The method according to claim 3, characterized in that The molar ratio of the compound S1, p-nitrobenzenesulfonyl chloride, organic base and sphingosine base is (1-2): (1-2): (2-6): 1; the organic base is triethylamine; and the solvent of the reaction is ethyl acetate.

5. A method for preparing the ceramide compound according to claim 1 or 2, characterized in that: The following steps are involved: Compound S1 reacts with a sphingoid base and a condensing agent to obtain compound I; R 1 、R 2 As defined in claim 1 or 2.

6. The method according to claim 5, characterized in that The condensing agent includes EDCI and HOBT, and the molar ratio of the compound S1, EDCI, HOBT, and sphingoid base is 1:(1-2):(1-2):(0.8-1); the solvent of the reaction is DCM.

7. Use of the ceramide compound according to claim 1 or 2 in the preparation of an antioxidant.

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