CN115304509A - Ceramide compound containing chain carboxylic acid and preparation method and application thereof - Google Patents

Ceramide compound containing chain carboxylic acid and preparation method and application thereof Download PDF

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CN115304509A
CN115304509A CN202210939884.1A CN202210939884A CN115304509A CN 115304509 A CN115304509 A CN 115304509A CN 202210939884 A CN202210939884 A CN 202210939884A CN 115304509 A CN115304509 A CN 115304509A
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ceramide
sphingosine
linolenic
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杨超文
叶柳
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Shenzhen Dikeman Biotechnology Co ltd
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Abstract

The invention belongs to the technical field of biological medicines, and discloses a ceramide compound shown as a chemical formula I:
Figure DDA0003785097780000011
wherein R is 1 Selected from alpha-linolenic acid, gamma-linolenic acid, eicosapentaenoic acid and docosahexenoic acidHexaenoic 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, hexacosan-17-enoic acid, cis-11, 14-eicosadienoic acid, residue obtained by condensation of mandelic acid or tartaric acid, R 2 Selected from 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 invention reacts functional carboxylic acid with sphingosine alkali to obtain a ceramide compound with a novel structure, thereby improving the original effect of the molecule and improving the physicochemical property of the ceramide compound.

Description

Ceramide compound containing chain carboxylic acid and preparation method and application thereof
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a ceramide compound containing chain carboxylic acid and a preparation method and application thereof.
Background
Ceramides are compounds in which sphingoid is bonded to fatty acids via amide bonds. The carbon chain length, unsaturation and hydroxyl number of the sphingosine moiety and the fatty acid moiety can all be varied, so that ceramide is a class of compound rather than a single compound. Currently, 15 ceramides are detected in the skin, and can be roughly classified into three types, short-chain ceramides, long-chain ceramides, and keratinocyte-bonded ceramides. Wherein the fatty acid moiety mainly comprises saturated chain fatty acids and chain fatty acids of low unsaturation, and these compounds tend to face solubility problems.
It is reported that the evaporation of water from the epidermis is achieved by the water-maintaining action of ceramide present in intercellular lipids, and that when the concentration of ceramide in the skin is decreased, the protective barrier function of the stratum corneum is weakened, resulting in the occurrence of various dermatological symptoms such as atopic dermatitis and psoriasis. In addition, xerosis cutis occurs due to the decrease of the amount of ceramide, the defense function of the skin surface is lost, foreign substances are more easily invaded and secondary infection of the skin is caused, thereby causing skin rejection. Specifically, the invader causes cytokine release from cells such as keratinocytes of the epidermal cells, langerhans cells, and melanocytes, thereby causing an inflammatory phenomenon and the like.
The importance of ceramides is well known and many cosmetic and pharmaceutical companies are conducting research to develop products using it. However, natural ceramides are difficult to extract and purify, and have economic problems, and some companies are making efforts to develop ceramides having a structure similar to that of ceramides present in the skin and functionally providing the same effects. It is known that commercially available natural or ceramide-like compounds have a limitation in that it is difficult to prepare high-content products due to low hydrophilicity, solubility, and the like.
Due to the wide market demand for functional ceramides, it is necessary to rapidly construct ceramide derivatives by chemical synthesis, so as to further improve the properties and enhance the efficacy of ceramide.
Disclosure of Invention
The invention aims to provide a ceramide compound with a novel structure, which contains chain carboxylic acid, wherein the chain carboxylic acid refers to that the carbon atom directly connected with carboxyl is CH 2 -or CHR 3 Carboxylic acid of (A), R 3 Is a substituent, the chain carboxylic acid may have a heterocyclic or aromatic ring, but the ring carbon atom of the heterocyclic or aromatic ring is not directly bonded to the carboxyl group.
Another objective of the invention is to provide a synthesis method of the ceramide-like compound.
Another object of the present invention is to provide the use of ceramide-like compounds.
In order to achieve one of the purposes, the invention adopts the following technical scheme;
in a first aspect of the invention, a ceramide compound having the structure of formula I or an enantiomer, diastereomer of formula I:
Figure BDA0003785097760000021
wherein R is 1 Selected from the group consisting of alpha-linolenic acid, gamma-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, 3- (4-hydroxy-4-carboxylic acid3-methoxyphenyl) sodium lactate, p-hydroxyphenylacetic acid, palmitoleic acid, vitamin B5, vitamin H, ricinoleic acid, behenic acid, cis-11-eicosenoic acid, hexacosan-17-enoic acid, cis-11, 14-eicosadienoic acid, mandelic acid or tartaric acid,
R 2 selected from 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 residue after condensation is the residue of the carboxylic acid fragment R, e.g.after condensation of the carboxyl group of the corresponding carboxylic acid RCOOH with the amino group of the sphingoid base to form a peptide bond, e.g.the residue after condensation of alpha-linolenic acid
Figure BDA0003785097760000022
The residue after condensation of arachidonic acid is
Figure BDA0003785097760000023
Wherein, the azelaic acid has antibacterial property, can be used as food preservative, is beneficial to preventing and treating decayed teeth when being used in gargle, and can avoid cracking of the surface of soap when being used in perfumed soap; the cosmetic has good permeability to skin, and can increase absorption function of skin when used in cream cosmetics; has skin brightening and whitening effects. Azelaic acid or its zinc salt and vitamin B6 are combined for hair care product, and are suitable for treating male hormone alopecia with vigorous male endocrine, and simultaneously stimulating hair growth.
4-aminobutyric acid is an important central nervous system inhibitory neurotransmitter, has good water solubility and thermal stability, is safe to eat, and can be used for producing foods such as beverages. The intake of a certain amount of GABA has the physiological effects of improving the sleep quality of the organism, reducing the blood pressure and the like.
Erucic acid can be applied to food industry or cosmetics.
Eicosapentaenoic acid EPA is the main component of fish oil, belongs to omega-3 series polyunsaturated fatty acid, is an indispensable important nutrition for human body, and has the functions of helping to reduce the content of cholesterol and triglyceride and promoting the metabolism of saturated fatty acid in the body, thereby reducing the blood viscosity, promoting the blood circulation, improving the oxygen supply of tissues and eliminating fatigue.
Docosahexaenoic acid (DHA) is another important polyunsaturated fatty acid, plays an important role in the growth and development of cranial nerves, the visual development and intelligent development of infants, and also has the effects of resisting allergy, enhancing immunity and the like.
Further, said R 2 Selected from one of the following structures:
Figure BDA0003785097760000031
further, said R 2 Selected from one of the following structures:
Figure BDA0003785097760000032
corresponding to sphingosine, dihydrosphingosine and phytosphingosine respectively.
Further, said R 1 Selected from the group consisting of residues resulting from the condensation of alpha-linolenic acid, gamma-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, arachidonic acid, erucic acid, p-hydroxyphenylacetic acid, or palmitoleic acid.
Further, said R 1 Selected from residues resulting from condensation of alpha-linolenic acid.
Further, said R 1 Selected from residues resulting from condensation of gamma-linolenic acid.
Further, said R 1 Selected from residues resulting from condensation of eicosapentaenoic acid.
Further, said R 1 Selected from residues resulting from condensation of docosahexaenoic acid.
Further, said R 1 Selected from residues after condensation of arachidonic acid.
Further, said R 1 Selected from residues after erucic acid condensation.
Further, said R 1 Selected from residues of condensed p-hydroxyphenylacetic acid.
Further, said R 1 Selected from residues after condensation of palmitoleic acid.
Further, the ceramide compound is selected from one of the following compounds:
Figure BDA0003785097760000041
Figure BDA0003785097760000051
in a second aspect of the present invention, a method for preparing a ceramide-like compound, comprising the steps of:
Figure BDA0003785097760000052
reacting the compound S1 with p-nitrobenzenesulfonyl chloride and organic base to obtain a compound S2;
reacting the compound S2 with sphingosine alkali to obtain a compound I.
Further, the molar ratio of the compound S1 to the p-nitrobenzenesulfonyl chloride to the organic base to the sphingosine base is (1-2): (1-2): (2-6): 1.
further, the condensing agent is triethylamine.
Further, the solvent of the reaction is ethyl acetate.
A process for preparing a ceramide-like compound, comprising the steps of:
Figure BDA0003785097760000061
reacting the compound S1 with sphingosine alkali and a condensing agent to obtain a compound I.
Further, the condensing agent includes EDCI and HOBT.
Further, the molar ratio of the compound S1, EDCI, HOBT, sphingosine base is 1: (1-2): (1-2): (0.8-1).
Further, the solvent of the reaction is DCM.
In a third aspect of the present invention, ceramide compounds are used as antioxidants, especially in cosmetics, health products, and pharmaceuticals.
The invention has the following beneficial effects:
the invention obtains a class of ceramide compounds with novel structures by reacting functional carboxylic acid with sphingosine base, the original effect of the class of molecules can be improved by introducing a molecular fragment with physiological activity of the functional carboxylic acid into ceramide, the physicochemical property of the ceramide compounds can also be improved, for example, the solubility of long-chain fatty acid can be improved by increasing the unsaturation degree of the long-chain fatty acid, and the oxidation resistance of the compounds can be improved by the existence of a plurality of unsaturated bonds.
Drawings
FIG. 1 is the results of the oxidation resistance test of the ceramide compound of example 14;
FIG. 2 is the results of the cell proliferation assay of ceramide compound of example 16;
FIG. 3 shows the results of the LPS-induced cell assay of the ceramide compound of example 16;
FIG. 4 shows the results of the cell scratching method for the ceramide compound of example 16;
FIG. 5 is a histogram of the cell mobility of the ceramide compound of example 16.
Detailed Description
The present invention will be further described with reference to the following specific examples.
All reactions were carried out under a nitrogen atmosphere. Unless otherwise stated, chemicals were purchased from commercial products and were not further purified. The dichloromethane and tetrahydrofuran used in the experiment are anhydrous solvents. Thin Layer Chromatography (TLC) used 60F254 silica gel plates. The silica gel column chromatography uses Qingdao marine silica gel (particle size 0.040-0.063 mm). TLC color development was performed with UV light (254 nm) or iodine. NMR spectra Using Bruker DPX 400 Nuclear magnetismThe resonance apparatus is characterized in that the resonance apparatus is characterized, 1 h NMR is 400MHz, solvent is deuterated methanol, deuterated DMSO or deuterated tetrahydrofuran, and Tetramethylsilane (TMS) is used as internal standard. Chemical shifts are in ppm and coupling constants are in Hz. In that 1 In H NMR, δ represents chemical shift, s represents singlet, d represents doublet, t represents triplet, q represents quartet, and m represents multiplet.
EA means ethyl acetate, DCM means dichloromethane, EDCI means 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, and HOBT means 1-hydroxybenzotriazole.
General synthesis method of ceramide compound
Method A
Figure BDA0003785097760000071
Dissolving 1.2eq (60 mmol) of p-nitrobenzenesulfonyl chloride in 70ml of ethyl acetate, dissolving 1.32eq (66 mmol) of compound S1 and 3eq (150 mmol) of triethylamine in 30ml of ethyl acetate, dropwise adding the mixture into an ethyl acetate solution of p-nitrobenzenesulfonyl chloride, reacting at 40 ℃ for 12h, and detecting the completion of the reaction of p-nitrobenzenesulfonyl chloride by TCL. 2eq (100 mmol) of triethylamine and 1eq (50 mmol) of sphingosine base were added and the reaction was carried out overnight at 40 ℃ and the completion of the sphingosine base reaction was checked by TLC.
And (3) post-treatment: adding 100mL of water, adjusting the pH value to 5-6 by 2N diluted hydrochloric acid, extracting twice by 100mL of saturated saline solution, drying by anhydrous sodium sulfate, filtering and concentrating in vacuum. The residue thus obtained was purified by silica gel column to give the product (50-70% yield).
Method B
Figure BDA0003785097760000072
Compound S1 (50 mmol), EDCI (60-75 mmol, preferably 60 mmol), HOBT (60-75 mmol, preferably 60 mmol) were placed in a 250mL addition round bottom flask, 100mL of dichloromethane was added, followed by stirring at room temperature for 1 hour, followed by adding sphingosine base (40-50 mmol, preferably 45 mmol) to the reaction, and stirring at room temperature for 24-72 hours until the sphingosine base completely disappeared.
And (3) post-treatment: quenched by the addition of 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).
Example 1
The product of the condensation of alpha-linolenic acid with phytosphingosine by method A.
Figure BDA0003785097760000081
1 H NMR(400MHz,Methanol-d 4 )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)。
Example 2
The product of the condensation of eicosapentaenoic acid with phytosphingosine by method B.
Figure BDA0003785097760000082
1 H NMR(400MHz,Methanol-d 4 )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)。
Example 3
The product of the condensation of docosahexaenoic acid with phytosphingosine by method B.
Figure BDA0003785097760000083
1 H NMR(400MHz,Methanol-d 4 )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)。
Example 4
Condensation product of eicosapentaenoic acid and docosahexaenoic acid mixed acid (mass ratio 22.
1 H NMR(400MHz,Methanol-d 4 )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,2H),1.32(d,J＝8.9Hz,26H),1.00(t,J＝7.5Hz,3H),0.92(t,J＝6.7Hz,4H)。
Example 5
Condensation product of eicosapentaenoic acid and docosahexaenoic acid mixed acid (50 mass ratio.
1 H NMR(400MHz,Methanol-d 4 )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)。
Example 6
Condensation product of eicosapentaenoic acid and docosahexaenoic acid mixed acid (mass ratio 22.
1 H NMR(400MHz,Methanol-d 4 )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)。
Example 7
The product of the condensation of arachidonic acid with phytosphingosine by method B.
Figure BDA0003785097760000091
1 H NMR(400MHz,Methanol-d 4 )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)。
Example 8
The product of the condensation of alpha-linolenic acid with sphingosine by method A.
Figure BDA0003785097760000101
1 H NMR(400MHz,Methanol-d 4 )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)。
Example 9
The product of the condensation of alpha-linolenic acid with sphinganine by method A.
Figure BDA0003785097760000102
1 H NMR(400MHz,Methanol-d 4 )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)。
Example 10
The product of the condensation of erucic acid with sphingosine by method a.
Figure BDA0003785097760000103
1 H NMR(400MHz,Methanol-d 4 )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)。
Example 11
The product of the condensation of 4-hydroxyphenylacetic acid with phytosphingosine by method B.
Figure BDA0003785097760000111
1 H NMR(400MHz,Methanol-d 4 )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)。
Example 12
The product of the condensation of palmitoleic acid with sphingosine by method a.
Figure BDA0003785097760000112
1 H NMR(400MHz,Methanol-d 4 )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)。
Example 13
The product of the condensation of gamma-linolenic acid with phytosphingosine by method A.
Figure BDA0003785097760000113
1 H NMR(400MHz,Methanol-d 4 )δ5.52–5.33(m,6H),4.09–4.02(m,1H),3.72–3.68(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)。
Example 14
The ceramide compounds prepared in examples 1, 2, 4, 6 and 8 were tested for oxidation resistance by the antioxidant-ABTS method
The experimental principle is as follows: evaluation of the antioxidant capacity of samples using ABTS (2, 2' -Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) dimonium salt) was originally proposed by Miller et al (1993) and generally followed by a later Re et al (1999) improvement. The method utilizes ABTS to be oxidized by a series of compounds such as potassium persulfate, hydrogen peroxide, manganese dioxide and the like to generate blue-green ABTS + cation free radicals, and has a maximum absorption peak at 734 nm. ABTS + is reduced to colorless ABTS under the action of an antioxidant. The oxidation resistance of the reactant can be judged by measuring the light absorption value at 734 nm.
And (3) ABTS free radical scavenging and detecting oxidation resistance: 3mL of ABTS aqueous solution (12 mmol/L) and 3mL of potassium persulfate solution (2.45 mmol/L) are uniformly mixed and are stabilized for 12 to 16 hours at room temperature in a dark place. DMSO is used as a dilution medium, a proper dilution ratio is selected, and the absorbance of the potassium persulfate solution at the wavelength of 734nm is adjusted to be 0.700 +/-0.025. Adding samples with different concentrations, shaking, standing in dark for 10min, and measuring absorbance at 734 nm.
The results are shown in FIG. 1, in which control 1 was ceramide 3 and control 2 was ceramide 3B.
Therefore, compared with the existing ceramide compound, the compound of the invention has better antioxidant effect, and the clearance rate of free radicals is generally higher by 15-40%.
Example 15
Solubility test
10mg of the ceramide compounds prepared in examples 1, 2, 3, 7, and 8 were dissolved in 0.5mL of ethanol, respectively, and were completely dissolved, and as a comparison, control 1 was slightly soluble and had many particles; control 2 was completely dissolved.
Example 16
Tissue repair test
1. Cell proliferation method for testing tissue repair performance of ceramide compound and ceramide 3 prepared in examples 2 and 4
Detecting cell proliferation activity by using an MTT method: haCaT cells were cultured at 1X 10 4 The density of cells/well was plated in 96-well plates overnight in an incubator. After 24h, the supernatant was discarded, 100. Mu.L of medium containing samples (or blanks) of different concentrations was added, incubation was continued for 24h and the medium was removed, 100. Mu.L of thiazole blue (MTT) was added to each well, absorbance at 450nm was measured, and cell survival = A was calculated Medicine feeding hole /A Blank hole ×100％。
The results are shown in fig. 2, and compared with solvent group M, the low concentration (0.156-0.625 mM) of the compounds of example 2 and example 4 significantly improves the activity of HaCaT cells, indicating that in 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 of repairing damaged skin barrier. However, in the same concentration range, ceramide 3 decreased HaCaT cell viability and inhibited keratinocyte proliferation.
2. Anti-inflammatory repair efficacy of ceramide compound and ceramide 3 prepared in examples 2 and 4, respectively, by LPS-induced cell assay
Interleukin 6 (Interleukin 6, IL-6), is the most typical cytokine associated with inflammation. It plays an important role in host defense by modulating immune and inflammatory responses. Inflammation affects the skin barrier, which enhances epidermal water loss, and also affects the growth of keratinocytes, which are difficult to recover after the barrier is damaged. It also decomposes extracellular matrix, causes skin collapse, and inhibits collagen synthesis. The skin is relaxed and has wrinkles. Therefore, the preparation method effectively reduces the generation of interleukin IL-6 in keratinocytes and fibroblasts caused by external injury and ultraviolet, reduces inflammatory reaction, and is vital to recovering skin barrier and protecting skin elasticity and stability.
B16 mouse melanoma cells at a density of 1X 10 4 Planting each well in 96-well plate, placing in incubator, wall-sticking overnight, removing supernatant after 24 hr, adding 100 μ L samples with different concentrations diluted with DMEM medium, negative control group is DMEM medium without medicine, each group has 3 multiple wells, and the weight percent is 5% 2 And incubating at 37 ℃. After 2h of administration, the lipopolysaccharide model groups and the experimental groups added 10. Mu.g/mL LPS and incubated together for 24h. After the reaction, 50. Mu.L of cell supernatant was collected and the expression of IL-6 gene in the cells was detected by using IL-6ELISA kit.
As shown in FIG. 3, all three ceramides in the concentration range (0.0625-0.5 mM) were effective in reducing the expression of IL-6 inflammatory factor and had anti-inflammatory activity, as compared to the solvent group. In particular, the compound of example 2 showed better inhibitory efficiency in the same concentration range, indicating that it has a certain potential in other skin diseases caused by inflammatory factors.
3. Wound healing of ceramide compound and ceramide 3 prepared in examples 2 and 4 was examined by the cell scratching method
Cell scratch assay is an in vitro assay to study cell migration. When the keratinocytes grow to be fused into a single-layer state, a blank area (scratch) is artificially manufactured on the fused single-layer cells, the cells at the edge of the scratch gradually enter the blank area to heal the scratch, the migration process of the epidermal keratinocytes is simulated to a certain extent, and the migration capability of the cells is judged by observing the cell states of the scratch areas at different periods, so that the method is an important method in an in-vitro experiment for researching the healing and repairing of skin wounds.
The operation method comprises the following steps:
1. and (3) streaking the culture plate: firstly, a Marker pen is used at the back of a 6-hole plate, a straight ruler is used for uniformly drawing transverse lines, the transverse lines are drawn approximately every 0.5-1 cm, the transverse lines cross through holes, at least 5 lines pass through each hole, and the attention lines are not too thick when the lines are drawn.
2. Cell paving: about 5X 10 additions to the wells 5 Is smallCells (different cell numbers are different and adjusted according to the growth speed of the cells) are inoculated according to the principle that the fusion rate reaches 100 percent after the night.
3. Cell division: the next day, the cell layer is scored using a tip, perpendicular to the cell plane, along the line drawn on the back of the plate on the first day (preferably the same tip is used between different wells).
4. Washing cells: after the scoring was completed, cells were washed 3 times with sterile PBS, and non-adherent cells, i.e., cells streaked during streaking, where the gaps left after streaking were clearly visible, were washed away, and then replaced with fresh serum-free medium.
5. Cell culture and observation: placing the cells at 37 ℃ and 5wt% CO 2 Culturing in an incubator. Then, the cells were taken out at 24 hours, and the width of the scratch was observed and measured by a microscope line and photographed, and the result is shown in fig. 4. Cell mobility was calculated using IMAGE J software analysis and the results are shown in figure 5.
Three ceramides in the concentration range (1.0 mM) were all effective in improving the migration ability of keratinocytes after 24 hours of incubation with the cells. At the same concentration, the compound in example 4 shows better ability for promoting cell healing, which indicates that the compound has better effect for promoting healing and repairing of damaged epidermis.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are also within the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims (10)

1. A ceramide-like compound having the structure of formula I or an enantiomer, diastereomer of formula I:
Figure FDA0003785097750000011
wherein R is 1 Selected from alpha-flaxAcid, gamma-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, hexacosan-17-enoic acid, cis-11, 14-eicosadienoic acid, mandelic acid or tartaric acid,
R 2 selected from 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 。
2. The ceramide-like compound according to claim 1, wherein R is 2 Selected from one of the following structures:
Figure FDA0003785097750000012
3. the ceramide-like compound of claim 2, wherein R is 2 Selected from one of the following structures:
Figure FDA0003785097750000021
4. the ceramide-like compound of claim 1, wherein R is 1 Selected from the group consisting of residues resulting from the condensation of alpha-linolenic acid, gamma-linolenic acid, eicosapentaenoic acid, docosahexaenoic acid, arachidonic acid, erucic acid, p-hydroxyphenylacetic acid or palmitoleic acid.
5. The ceramide-like compound according to claim 1, selected from one of the following compounds:
Figure FDA0003785097750000022
Figure FDA0003785097750000031
6. a process for preparing a ceramide-like compound according to any one of claims 1 to 5, comprising the steps of:
Figure FDA0003785097750000032
reacting the compound S1 with p-nitrobenzenesulfonyl chloride and organic base to obtain a compound S2;
reacting the compound S2 with sphingosine alkali to obtain a compound I;
R 1 、R 2 as defined in claims 1-5.
7. The method according to claim 6, wherein the molar ratio of the compound S1 to the p-nitrobenzenesulfonyl chloride to the organic base to the sphingoid base is (1-2): (1-2): (2-6): 1; the condensing agent is triethylamine; the solvent for the reaction is ethyl acetate.
8. A process for producing the ceramide compound according to any one of claims 1 to 5, comprising the steps of:
Figure FDA0003785097750000041
reacting the compound S1 with sphingosine alkali and a condensing agent to obtain a compound I;
R 1 、R 2 as defined in claims 1-5.
9. The method of claim 8, wherein the condensing agent comprises EDCI and HOBT, and the molar ratio of the compound S1, EDCI, HOBT, sphingosine base is 1: (1-2): (1-2): (0.8-1); the solvent for the reaction was DCM.
10. Use of the ceramide-like compound as claimed in any one of claims 1 to 5 as an antioxidant.
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