Pyridoxamine derivative as well as preparation method and application thereof

Abstract

The invention discloses a pyridoxamine derivative as well as a preparation method and application thereof, the pyridoxamine derivative has a structural general formula shown in the specification, and a substituent R is selected from ferulic acid, retinoic acid or nicotinic acid; the preparation method comprises the following steps: S1, dissolving a carboxylic acid compound in a solvent, adding a condensing agent and an activating agent, and stirring at 0 DEG C until the mixture is completely clarified and dissolved to obtain a reaction solution A; s2, dropwise adding alkali into the reaction liquid at the temperature of 0 DEG C, and reacting at the temperature of 0 DEG C to obtain reaction liquid B; s3, adding pyridoxamine hydrochloride into the reaction liquid B, and stirring and reacting for 12-48 hours at room temperature; and S4, after the pyridoxamine hydrochloride completely reacts, quenching the reaction with a saturated sodium bicarbonate aqueous solution, stirring and filtering a reaction solution, washing an organic phase with a saturated saline solution, extracting, collecting the organic phase, drying and concentrating, and purifying by column chromatography to finally obtain the target pyridoxamine derivative. The preparation method has the advantages of convenience in preparation and relatively low cost, and can be applied to preparation of cosmetics with anti-sugar, anti-oxidation and anti-aging effects.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pyridoxamine derivative preparation, and particularly relates to a pyridoxamine derivative, a preparation method and application thereof. BACKGROUND

[0002] With the continuous improvement of the level of social and economic development, the functional needs of consumers for cosmetics show a trend of refinement and complexity. Single-acting cosmetics have been difficult to meet the needs, and cosmetic raw materials with multiple effects have become a research hotspot in the field of skin science and cosmetics.

[0003] Due to high-sugar diet, irregular work and rest, and environmental stress, skin glycation damage and pigmentation problems have become increasingly prominent in the contemporary population, giving rise to an urgent need for active ingredients that can precisely target the glycation pathway and synergistically improve skin color.

[0004] From the perspective of skin physiology and molecular biology mechanisms, glycation damage and abnormal melanin deposition are closely related, and both contribute to the acceleration of skin aging phenotypes.

[0005] 1. The core mechanism of anti-sugar is to regulate the metabolic balance of advanced glycation end products (AGEs); 2. The antioxidant effect is to remove excess reactive oxygen species (ROS) in the skin and reduce their oxidative damage to cell structures; 3. The key to anti-aging focuses on the synthesis and loss balance of structural proteins such as collagen; Therefore, there is a possibility of cross-regulation between anti-sugar and multiple effects at the signal pathway level.

[0006] Under non-enzymatic reaction conditions, reduced sugars in the human body covalently bind to proteins and lipids, and through a series of complex reactions, brown-yellow AGEs are generated.

[0007] In the microenvironment of skin tissue, the progressive accumulation of AGEs can lead to structural damage and functional impairment of collagen and elastic fibers, thereby triggering skin laxity, deepening of wrinkles, and other signs of aging, and causing characteristic changes in skin color, such as dark yellow and dull.

[0008] The anti-glycation effect of pyridoxamine has been widely proven, and its mechanism mainly manifests in the following aspects: 1) Reducing AGEs: not only by inhibiting the initial combination of reducing sugars and proteins to reduce the production of AGEs precursors, but also by efficiently removing various toxic carbonyl compounds such as glyoxal (GO), methylglyoxal (MGO), and glycolaldehyde (GLA) generated during sugar metabolism and lipid peroxidation / degradation, thereby cutting off the key intermediate link in AGEs synthesis.

[0009] 2) Inhibit the glycosylation modification of collagen, while binding to early glycosylation products to prevent them from forming complexes with collagen and elastin, avoiding the destruction of protein structure, thus maintaining the integrity of collagen molecular structure and the order of spatial conformation, ensuring the normal function of skin support proteins such as elastin.

[0010] 3) By down-regulating the expression level of AGEs receptor (RAGE), it blocks the series of damage reactions mediated by AGEs-RAGE signal pathway, achieves the effect of anti-oxidation and soothing, and further delays the glycosylation aging process of skin at the molecular level.

[0011] Nicotinamide can effectively help maintain skin firmness, reduce fine lines and wrinkles caused by glycation, improve skin dullness and roughness, and make the skin appear full and healthy, transparent state, and delay the signs of skin aging caused by glycation from the deep level.

[0012] Retinoic acid is a classic anti-aging ingredient, and its anti-aging mechanism shows multi-dimensional targeting characteristics: 1) By activating the RAR / RXR signaling pathway, it precisely regulates the proliferation and differentiation cycle of basal keratinocytes, accelerates the metabolism of aged keratin, and promotes the orderly migration of new cells upward, effectively improving skin roughness and texture disorder problems; 2) Targeting dermal fibroblasts, on the one hand, up-regulating the expression of multiple collagen and elastin synthesis genes, increasing the collagen density of the dermis, on the other hand, strongly inhibiting the activity of matrix metalloproteinase (MMP-1 / -3 / -9), reducing the degradation and destruction of collagen fibers, improving skin laxity and wrinkle problems from the "synthesis-protection" dual dimensions.

[0013] However, the strong activity of retinoic acid is accompanied by significant irritation, which is the core limitation of its widespread application: it may activate the local stress response of the skin, especially when the skin barrier is weak or when it is used for the first time, it is easy to cause dryness, redness, itching, and other discomfort symptoms, and in severe cases, it can induce contact dermatitis; such irritation not only limits the use of sensitive skin, dry skin and other people, but also requires ordinary users to go through a long "tolerance" process, reducing the convenience of product use and user compliance.

[0014] Oxidative stress is the number one trigger for accelerating skin aging and inducing various skin problems; antioxidant mechanisms help to activate the young state of the skin, even out the skin tone, resist inflammation and promote repair by removing free radicals and breaking the oxidation reaction chain; in recent years, antioxidant cosmetic raw materials have caused a craze in the global beauty market, and their application status presents a multi-dimensional development trend.

[0015] Currently, the mainstream way to achieve multiple compound effects in the cosmetic industry is to compound multiple ingredients. However, this traditional compounding mode has many technical bottlenecks: First, the physicochemical properties of the components differ greatly, and antagonism is easy to occur, leading to inactivation of some active ingredients and reducing the overall efficacy. Second, the multi-component system has poor stability and is easy to separate and precipitate during storage, affecting product quality and shelf life. Third, the complex components destroy the skin microecology and easily increase the risk of skin sensitization, making it less suitable for sensitive skin. Fourth, the complex preparation process and high cost of raw materials result in high product pricing, limiting market popularization. Fifth, the components compete for penetration, resulting in low transdermal absorption efficiency and poor effect. SUMMARY

[0016] The purpose of the present application is to provide a pyridoxamine derivative, a preparation method and applications thereof. The present application has the advantages of convenient preparation, relatively low cost and application in the preparation of cosmetics with anti-sugar, anti-oxidation and anti-aging effects.

[0017] The technical solution of the present application is as follows: A pyridoxamine derivative, the general formula is as follows: Figure 29 Wherein the substituent group R is selected from ferulic acid, retinoic acid or nicotinic acid.

[0018] A preparation method of a pyridoxamine derivative, comprising the following steps: S1, dissolve 1.0-2.0 eq of carboxylic acid compound in a solvent, add 1.0-3.0 eq of condensing agent and 1.0-3.0 eq of activating agent, stir at 0℃ until completely clear and dissolved to obtain reaction liquid A; S2, add 1.0-3.0 eq of base dropwise to the reaction liquid at 0℃, and react at 0℃ for 30 min to obtain reaction liquid B; S3, add pyridoxamine hydrochloride to the reaction liquid B, and stir at room temperature for 12-48 hours, and monitor the reaction progress by TLC or HPLC; S4, after the pyridoxamine hydrochloride is completely reacted, quench the reaction with saturated sodium bicarbonate aqueous solution, extract after treatment, collect the organic phase, dry and concentrate, and then purify by column chromatography to obtain the target pyridoxamine derivative.

[0019] In the aforementioned pyridoxamine derivative, the reaction chemical formula is .

[0020] In the aforementioned pyridoxamine derivative, the carboxylic acid compound is one of ferulic acid, retinoic acid or nicotinic acid.

[0021] ​In the aforementioned pyridoxamine derivative, the condensing agent includes one or more of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, dicyclohexylcarbodiimide, diisopropylcarbodiimide, N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl) uronium hexafluorophosphate, 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate.

[0022] In the aforementioned pyridoxamine derivative, the activating agent includes one or more of N-hydroxy-7-azabenzotriazole, 1-hydroxybenzotriazole, 3-hydroxy-1,2,3-benzotriazin-4(3H)-one, N-hydroxysuccinimide, 4-dimethylaminopyridine, and benzotriazol-1-yloxytris-pyrrolidinophosphonium hexafluorophosphate.

[0023] In the aforementioned pyridoxamine derivative, the base includes one or more of N,N-diisopropylethylamine, triethylamine, N-methylmorpholine, pyridine, 2,6-lutidine, 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5-diazabicyclo[4.3.0]non-5-ene, sodium carbonate, sodium bicarbonate, sodium hydroxide, potassium hydroxide, and potassium carbonate.

[0024] In the aforementioned pyridoxamine derivative, the solvent includes one or more of petroleum ether, ethyl acetate, dichloromethane, methanol, ethanol, tetrahydrofuran, pyridine, diethyl ether, acetone, cyclohexane, toluene, N,N-dimethylformamide, and dimethyl sulfoxide.

[0025] Use of a pyridoxamine derivative in the manufacture of a cosmetic having anti-glycation efficacy, the substituent R of the pyridoxamine derivative being derived from ferulic acid, retinoic acid, or nicotinic acid.

[0026] Use of a pyridoxamine derivative in the manufacture of a cosmetic having anti-oxidation efficacy, the substituent R of the pyridoxamine derivative being derived from ferulic acid, retinoic acid, or nicotinic acid.

[0027] Use of a pyridoxamine derivative in the manufacture of a cosmetic having anti-aging efficacy, the substituent R of the pyridoxamine derivative being derived from ferulic acid, retinoic acid, or nicotinic acid.

[0028] Use of a pyridoxamine derivative in the manufacture of a cosmetic having soothing efficacy, the substituent R of the pyridoxamine derivative being derived from retinoic acid.

[0029] The application of a pyridoxamine derivative in cosmetics also includes tautomers of the pyridoxamine derivative, pharmaceutically acceptable salts, prodrugs, hydrates or solvates of the pyridoxamine derivative or the tautomers.

[0030] Compared with the prior art, the application has the following beneficial effects: 1) The application synthesizes a novel pyridoxamine derivative by introducing an acid structure with a corresponding cosmetic effect on the pyridoxamine functional group; the synthesis method of the pyridoxamine derivative of the application does not require a complex catalytic system, and the reaction can be carried out at room temperature and normal pressure, and the overall yield is high, effectively reducing the energy consumption and cost investment in the synthesis process; 2) The raw materials (pyridoxamine monomer, carboxylic acid reagent, etc.) used in the application are conventional bulk commodities in the chemical industry, which are simple to obtain, widely available and easy to control in purity, and can be directly used as basic raw materials for industrial production; by accurately introducing an acid structure with a corresponding cosmetic effect, not only the anti-glycation activity of the active skeleton of pyridoxamine itself is retained, but also the antioxidant and anti-aging activities are significantly improved; 3) In the pyridoxamine derivative prepared by the application, The feruloyl pyridoxamine shows excellent active oxygen scavenging ability in the in vitro antioxidant experiment, with a scavenging rate of 134%, and can also effectively inhibit the generation of advanced glycation end products (AGEs) with a scavenging rate of 101%; The nicotinoyl pyridoxamine shows excellent AGEs scavenging ability in the in vitro anti-glycation experiment, with a scavenging rate of 114.30% under high glucose conditions, and a scavenging rate of 246.21% under high galactose conditions, and can also effectively scavenge the ROS content generated by oxidation with a scavenging rate of 21.38%; The retinoyl pyridoxamine shows excellent AGEs scavenging ability in the in vitro anti-glycation experiment, with a scavenging rate of 196.66% under high glucose conditions, and a scavenging rate of 121.24% under high galactose conditions, and can also effectively scavenge the ROS content generated by oxidation with a scavenging rate of 85.86%; Therefore, the pyridoxamine derivative prepared by the application has a wide application prospect in cosmetics (such as anti-glycation and antioxidant skin care products); 4) The pyridoxamine derivative prepared by the application shows excellent anti-aging ability: in the in vitro experiment, it can improve a plurality of aging indicators under high glucose conditions; 5) Among the pyridoxamine derivatives prepared by the application, the retinoyl pyridoxamine also shows excellent soothing ability; In the in vitro cell experiment and epidermal skin model experiment, the IL-6 content is significantly reduced (by 21.86%), and the IL-8 content is significantly reduced (by 23.60%); Therefore, the present application has the advantages of convenient preparation, relatively low cost and application in preparation of cosmetics with anti-sugar, anti-oxidation and anti-aging effects. The retinyl pyridoxamine also has a soothing effect and can be applied in the preparation of cosmetics with a soothing effect. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 is a nuclear magnetic hydrogen spectrum result graph in Example 1 of the present application; Figure 2 is a nuclear magnetic carbon spectrum result graph in Example 1 of the present application; Figure 3 is a mass spectrum result graph in Example 1 of the present application; Figure 4 is a nuclear magnetic hydrogen spectrum result graph in Example 2 of the present application; Figure 5 is a nuclear magnetic carbon spectrum result graph in Example 2 of the present application; Figure 6 is a mass spectrum result graph in Example 2 of the present application; Figure 7 is a nuclear magnetic hydrogen spectrum result graph in Example 3 of the present application; Figure 8 is a nuclear magnetic carbon spectrum result graph in Example 3 of the present application; Figure 9 is a mass spectrum result graph in Example 3 of the present application; Figure 10 is a cytotoxicity test graph of ferula pyridoxamine in Experiment 1 of the present application; Figure 11 is a cytotoxicity test graph of nicotine pyridoxamine in Experiment 1 of the present application; Figure 12 is a cytotoxicity test graph of retinyl pyridoxamine in Experiment 1 of the present application; Figure 13 is a cell anti-sugar (glucose) test graph of ferula pyridoxamine in Experiment 2 of the present application; Figure 14 is a cell anti-sugar (glucose) test graph of nicotine pyridoxamine in Experiment 2 of the present application; Figure 15 is a cell anti-sugar (glucose) test graph of retinyl pyridoxamine in Experiment 2 of the present application; Figure 16 is a cell anti-sugar (galactose) test graph of ferula pyridoxamine in Experiment 3 of the present application; Figure 17 is a cell anti-sugar (galactose) test graph of nicotine pyridoxamine in Experiment 3 of the present application; Figure 18 is a cell anti-sugar (galactose) test graph of retinyl pyridoxamine in Experiment 3 of the present application; Figure 19 is a cell antioxidant test chart of feruloyl pyridoxal in experiment 4 of the present application; Figure 20 is a cell antioxidant test chart of nicotinoyl pyridoxal in experiment 4 of the present application; Figure 21 is a cell antioxidant test chart of retinoyl pyridoxal in experiment 4 of the present application; Figure 22 is a cell anti-glycosis-induced senescence test chart (glucose) of feruloyl pyridoxal in experiment 5 of the present application; Figure 23 is a cell anti-glycosis-induced senescence test chart (glucose) of nicotinoyl pyridoxal in experiment 5 of the present application; Figure 24 is a cell anti-glycosis-induced senescence test chart (galactose) of feruloyl pyridoxal in experiment 6 of the present application; Figure 25 is a cell anti-glycosis-induced senescence test chart (galactose) of nicotinoyl pyridoxal in experiment 6 of the present application; Figure 26 is a cell anti-glycosis-induced senescence test chart (galactose) of retinoyl pyridoxal in experiment 6 of the present application; Figure 27 is a cell soothing test chart of retinoyl pyridoxal in experiment 7 of the present application; Figure 28 is an epidermis soothing test chart of retinoyl pyridoxal in experiment 8 of the present application; Figure 29 is a structural general formula of pyridoxamine derivatives of the present application. DETAILED DESCRIPTION

[0032] The present application will be further described below in conjunction with the accompanying drawings and examples, but not as the basis for limiting the present application.

[0033] Example 1. A preparation method of pyridoxamine derivatives, specifically feruloyl pyridoxal, the steps are as follows: S1, 10 mmol of ferulic acid was dissolved in 50 mL of N, N-dimethylformamide, 12 mmol of 1- (3-dimethylamino propyl) -3-ethyl carbodiimide (EDCI) was added, 12 mmol of N-hydroxy-7-azabenzotriazole (HOAt) was added, and the reaction liquid A was obtained by stirring at 0℃ until complete clear dissolution; S2, 20 mmol of N, N-diisopropyl ethylamine (DIPEA) was added dropwise to the reaction liquid A at 0℃, and reacted for 30 min at 0℃ to obtain the reaction liquid B; S3, 12 mmol of pyridoxamine hydrochloride was added to the reaction liquid B, and the reaction was stirred at room temperature for 24 hours, and the reaction progress was monitored by TLC; S4, after the raw material pyridoxamine hydrochloride completely reacted, 50 mL of saturated sodium bicarbonate aqueous solution was added dropwise to quench the reaction, the reaction liquid was extracted with ethyl acetate, the organic phase was collected, dried with anhydrous sodium sulfate, concentrated, and then purified by column chromatography, and finally pyridoxylated ferula was obtained; the developing agent of column chromatography was dichloromethane:methanol=10:1(v / v).

[0034] The structural formula of pyridoxylated ferula is .

[0035] The yield of pyridoxylated ferula prepared in Example 1 was 87%, and the HRMS (ESI) calculated for [C 18 H 20 N2O5+ H + ]: 345.1445, found: 345.1447.

[0036] The detection results of the nuclear magnetic resonance of the prepared pyridoxylated ferula are shown in the following table: Figures 1-3 Nuclear magnetic hydrogen spectrum analysis: ; Nuclear magnetic carbon spectrum analysis: .

[0037] The application of a pyridoxamine derivative in the preparation of cosmetics, the addition amount is 50-125 μg / mL.

[0038] The application of a pyridoxamine derivative in the preparation of cosmetics with anti-sugar, anti-oxidation and anti-aging effects.

[0039] Example 2. A preparation method of a pyridoxamine derivative, specifically pyridoxylated nicotine, the steps are: S1, 10 mmol of nicotinic acid was dissolved in 50 mL of N, N-dimethylformamide, 12 mmol of 1-(3-dimethylamino propyl)-3-ethyl carbodiimide (EDCI) was added, 12 mmol of N-hydroxy-7-azabenzotriazole (HOAt) was added, and the mixture was stirred at 0℃ until it was completely dissolved to obtain reaction liquid A; S2, 15 mmol of N, N-diisopropyl ethylamine (DIPEA) was added dropwise to reaction liquid A at 0℃, and the mixture was reacted at 0℃ for 30 min to obtain reaction liquid B; S3, 13 mmol of pyridoxamine hydrochloride was added to reaction liquid B, and the mixture was stirred at room temperature for 24 hours, and the reaction progress was monitored by TLC; ​S4, after the raw material pyridoxamine hydrochloride is completely reacted, 50 mL of saturated sodium bicarbonate aqueous solution is added dropwise to quench the reaction, and after stirring for 30 min, the reaction solution is filtered, and the reaction solution is extracted with ethyl acetate, the organic phase is washed with saturated brine, extracted, and the organic phase is collected, dried with anhydrous sodium sulfate, concentrated, and then purified by column chromatography to obtain pyridoxamine; the developing agent of column chromatography is dichloromethane:methanol = 10:1 (v / v).

[0040] The structural formula of pyridoxamine is .

[0041] The yield of pyridoxamine prepared in Example 2 is 73%, and the HRMS (ESI) calculated for [C 14 H 15 N3O3+H + ]: 274.1186, found: 274.1187.

[0042] The detection results of the nuclear magnetic resonance of the prepared pyridoxamine are as shown in Figures 4-6 . Nuclear magnetic hydrogen spectrum analysis: ; Nuclear magnetic carbon spectrum analysis: .

[0043] The application of a pyridoxamine derivative in the preparation of a cosmetic, the addition amount is 25-1000 μg / mL.

[0044] The application of a pyridoxamine derivative in the preparation of a cosmetic with anti-sugar, anti-oxidation and anti-aging effects.

[0045] Example 3. A preparation method of a pyridoxamine derivative, specifically retinyl pyridoxamide, the steps are: S1, 10 mmol of retinoic acid is dissolved in 50 mL of N, N-dimethylformamide, 12 mmol of 1-(3-dimethylamino propyl)-3-ethyl carbodiimide (EDCI) is added, 12 mmol of N-hydroxy-7-azabenzotriazole (HOAt) is added, and the mixture is stirred at 0°C until it is completely dissolved to obtain reaction solution A; S2, 20 mmol of N, N-diisopropyl ethylamine (DIPEA) is added dropwise to reaction solution A at 0°C, and the mixture is reacted at 0°C for 30 min to obtain reaction solution B; S3, 12 mmol of pyridoxamine hydrochloride is added to reaction solution B, and the mixture is stirred at room temperature for 24 hours, and the reaction progress is monitored by TLC; S4, after the raw material pyridoxamine hydrochloride is completely reacted, 50 mL of saturated sodium bicarbonate aqueous solution is added dropwise to quench the reaction, and after stirring for 30 min, the reaction solution is filtered, and the reaction solution is extracted with ethyl acetate, the organic phase is washed with saturated brine, extracted, and the organic phase is collected, dried with anhydrous sodium sulfate, concentrated, and then purified by column chromatography to obtain retinyl pyridoxamide; the developing agent for column chromatography is dichloromethane:methanol = 20:1 (v / v).

[0046] The structural formula of retinyl pyridoxamide is .

[0047] The yield of retinyl pyridoxamide prepared in Example 3 is 92%, and the HRMS (ESI) calculated for [C 28 H 38 N2O3+ H + ]: 451.2955, found: 451.2956.

[0048] The detection results of the nuclear magnetic resonance of the prepared retinyl pyridoxamide are shown in the following table: Figures 7-9 Nuclear magnetic hydrogen spectrum analysis: ; Nuclear magnetic carbon spectrum analysis: .

[0049] The application of a pyridoxamine derivative in the preparation of a cosmetic, with an addition amount of 10-30 μg / mL.

[0050] The application of a pyridoxamine derivative in the preparation of a cosmetic with anti-sugar, anti-oxidation, soothing and anti-aging effects.

[0051] Verification experiment Experimental reagents: phosphate buffer solution (PBS, Biosharp), high-sugar DMEM medium (Gibco), low-sugar DMEM medium (Gibco), 0.25% trypsin solution (Gibco), antibiotic penicillin-streptomycin (Gibco), fetal bovine serum (FBS, Biyun Tian), cck8 reagent (Bi Yun Tian), dimethyl sulfoxide DMSO (China Pharmaceutical), NaOH (China Pharmaceutical), reactive oxygen detection kit (Bi Yun Tian), AGEs content detection Elisa kit (enzyme-linked biological), resuscitation medium (Boxi Biological, PY3031), IL-6 Elisa kit (Bi Yun Tian), IL-8 ELISA detection kit (Solaybao); ​Experimental cells: human immortalized keratinocytes (HaCaT), provided by Sibionics (Shanghai) Biotech Co., Ltd.; human primary fibroblasts HDF, provided by Boxi Biotech Co., Ltd.; epidermal skin model culture medium, provided by Boxi Biotech (PY3021).

[0052] The corresponding experimental verification was carried out for feruloylpyridoxamine, nicotinoylpyridoxamine and retinoylpyridoxamine.

[0053] Experimental 1 cytotoxicity test Logarithmic growth period human primary fibroblasts HDF were taken and added to a 96-well plate (8*10 3 cells were attached overnight in an incubator (37℃, 5% CO2), different concentrations of pyridoxamine derivatives were added, specifically: The drug concentration gradient of feruloylpyridoxamine was 0, 3.9, 7.8, 15.6, 31.2, 62.5, 125, 250, 500, 1000 μg / mL; The drug concentration gradient of nicotinoylpyridoxamine was 0, 3.9, 7.8, 15.6, 31.2, 62.5, 125, 250, 500, 1000 μg / mL; The drug concentration gradient of retinoylpyridoxamine was 0, 2.5, 5, 10, 20, 30, 40 μg / mL; After 24h of incubation in an incubator, CCK8 reagent was prepared into working solution according to the instructions, 100 μL was added to each well, incubated for 1h, and then the absorbance value was detected by an enzyme marker, and the relative cell viability was calculated, Cell relative viability (%) = (sample absorbance - reagent control absorbance) / (blank group absorbance - reagent control absorbance) * 100%.

[0054] The results are shown in Tables 1, 2 and 3,

[0055]

[0056]

[0057] From Table 1 and the attached Figure 10 It can be seen that the cell viability of feruloylpyridoxamine obtained in Example 1 decreased significantly when the concentration was above 250 μg / mL; From Table 2 and the attached Figure 11 It can be seen that nicotinoylpyridoxamine obtained in Example 2 had no obvious cytotoxicity when the concentration was below 1000 μg / mL; From Table 3 and the attached Figure 12It can be seen that the cell viability of the retinyl pyridoxamine obtained in Example 3 significantly decreased when the concentration was above 30 μg / mL.

[0058] Experiment 2 Cell Anti-glycation Test (Glucose) Experimental grouping: blank control group (complete culture medium), negative group (125 mM glucose), sample group; The sample group is specifically: The ferulic pyridoxamine experiment: a mixture of 50 μg / mL concentration of Example 1 and 125 mM glucose, and a mixture of 100 μg / mL concentration of Example 1 and 125 mM glucose; The nicotinic pyridoxamine experiment: a mixture of 50 μg / mL concentration of Example 2 and 125 mM glucose, and a mixture of 100 μg / mL concentration of Example 2 and 125 mM glucose; The retinyl pyridoxamine experiment: a mixture of 20 μg / mL concentration of Example 3 and 125 mM glucose; Experimental method: take human primary fibroblast HDF in logarithmic growth phase, add to a 6-well plate, 2.0*10 5 cells per well, and incubate in an incubator (37 °C, 5% CO2) overnight; According to the above grouping, the drug is administered, incubated for 24 h in culture, and the supernatant is collected. The Elisa kit for AGEs is used, and the supernatant is reacted in the coated plate according to the kit instruction steps. After complete reaction, the absorbance is detected using an enzyme label instrument at 450 nm, and the AGEs concentration C in the sample is calculated through the curve calibrated by the standard sample in the kit. The AGEs clearance rate calculation formula is, AGEs clearance rate (%) = (negative group C-sample group C) / (negative group C-blank control C) * 100%.

[0059] The results are shown in Tables 4, 5 and 6,

[0060]

[0061]

[0062] From the above Table 4 and the attached Figure 13 It can be seen that compared with the glucose modeling NC group, the ferulic pyridoxamine obtained in Example 1 can remove the content of AGEs, and when 100 μg / mL of ferulic pyridoxamine sample is used, the inhibition rate of AGEs content is 129.89%; From the above Table 5 and the attached Figure 14It can be seen that the retinyl pyridoxamine obtained in Example 3 can remove the content of AGEs compared with the glucose modeling NC group, wherein the removal rate of AGEs content is 196.66% when using 20 μg / mL of retinyl pyridoxamine sample; From Table 6 above and the attached Figure 15 It can be seen that the retinyl pyridoxamine obtained in Example 3 can remove the content of AGEs compared with the glucose modeling NC group, wherein the removal rate of AGEs content is 196.66% when using 20 μg / mL of retinyl pyridoxamine sample; It can be seen that the retinyl pyridoxamine obtained in Example 3 can remove the content of AGEs compared with the glucose modeling NC group, wherein the removal rate of AGEs content is 196.66% when using 20 μg / mL of retinyl pyridoxamine sample;

[0063] Experiment 3 Cell Anti-glycation Test (Galactose) Experimental grouping: blank control group (complete culture medium), negative group (40 mg / mL galactose), sample group; The sample group is specifically: Ferula pyridoxamine experiment: mixture of 100 μg / mL concentration of Example 1 and 40 mg / mL galactose; Nicotinamide experiment: mixture of 100 μg / mL concentration of Example 2 and 40 mg / mL galactose; Retinyl pyridoxamine experiment: mixture of 20 μg / mL concentration of Example 3 and 40 mg / mL galactose; Experimental method: take logarithmic growth period human primary fibroblast HDF, add 6 well plate, 2.0*10 5 Individuals / well, culture box (37 ℃, 5% CO2), cell adhesion overnight; According to the above grouping, the drug is given, incubated in culture for 24 h, the supernatant is collected, the Elisa kit of AGEs is used, the supernatant is reacted in the coated plate according to the kit instruction steps, after complete reaction, the enzyme label instrument 450 nm is used to detect the absorbance, through the curve calibrated by the standard sample in the kit, the AGEs concentration C in the sample is calculated; The formula for calculating the removal rate of AGEs is, AGEs removal rate (%) = (negative group C-sample group C) / (negative group C-blank control C) * 100%.

[0064] The results are shown in Tables 7, 8 and 9,

[0065]

[0066]

[0067] From Table 7 above and the attached Figure 16It can be seen that the retinoic pyridoxamine obtained in Example 3 can remove the content of AGEs compared with the galactose modeling NC group, and when 20 μg / mL of the retinoic pyridoxamine sample is used, the inhibition rate of AGEs content is 121.24%; From Table 8 above and the attached Figure 17 It can be seen that the retinoic pyridoxamine obtained in Example 3 can remove the content of AGEs compared with the galactose modeling NC group, and when 20 μg / mL of the retinoic pyridoxamine sample is used, the inhibition rate of AGEs content is 121.24%; From Table 9 above and the attached Figure 18 It can be seen that the retinoic pyridoxamine obtained in Example 3 can remove the content of AGEs compared with the galactose modeling NC group, and when 20 μg / mL of the retinoic pyridoxamine sample is used, the inhibition rate of AGEs content is 121.24%; Thus it is proved that pyridoxamine derivatives have anti-glycation effect.

[0068] Experiment 4 Cell Antioxidant Test Experimental grouping: set up blank control group, negative group, sample group, and set at least 3 replicate holes for each group; Experimental method: take HaCaT cells in logarithmic growth phase, inoculate cells into 12-well plates at a seeding amount of 3.0×10 5 Holes, and place in an incubator (37 ℃, 5% CO2) for incubation overnight; after overnight incubation, the sample group, blank group, and negative control group are still replaced with complete culture medium; The sample group is specifically: Retinoic pyridoxamine experiment: 100 μg / mL concentration of Example 1; Pyridoxamine experiment: 100 μg / mL concentration of Example 2; Retinoic pyridoxamine experiment: 10 μg / mL concentration of Example 3, 20 μg / mL concentration of Example 3; After 24 h of incubation in the incubator, use a UVB irradiation instrument with 1000 mJ / cm 2 After incubation of the probe according to the specific operation steps of the reactive oxygen species detection kit, perform flow cytometry detection to obtain the fluorescence intensity value DM (DCF (ROS) MFI) of each group, and the ROS removal rate calculation formula is, ROS removal rate (%) = (negative group DM-sample group DM) / (negative group DM-blank control DM) x 100%.

[0069] The results are shown in Tables 10, 11, and 12,

[0070]

[0071]

[0072] From Table 10 and the accompanying Figure 19 It can be seen that, compared with the NC group irradiated by UVB, the feruloyl pyridoxal phosphate obtained in Example 1 can significantly scavenge the active oxygen generated, and the ROS scavenging rate is 134.66% using 100 μg / mL of feruloyl pyridoxal phosphate; it shows that feruloyl pyridoxal phosphate has antioxidant effect; From Table 11 and the accompanying Figure 20 It can be seen that, compared with the NC group irradiated by UVB, the nicotinoyl pyridoxal phosphate obtained in Example 2 can significantly scavenge the active oxygen generated, and the ROS scavenging rate is 21.38% using 100 μg / mL of nicotinoyl pyridoxal phosphate; it shows that nicotinoyl pyridoxal phosphate has antioxidant effect; From Table 12 and the accompanying Figure 21 It can be seen that, compared with the NC group irradiated by UVB, the retinoyl pyridoxal phosphate obtained in Example 3 can significantly scavenge the active oxygen generated, and the ROS scavenging rate is 85.86% using 20 μg / mL of retinoyl pyridoxal phosphate; it shows that retinoyl pyridoxal phosphate has antioxidant effect; Therefore, it shows that pyridoxamine derivatives have antioxidant efficacy.

[0073] Experiment 5 Cell Anti-sugar (glucose) aging test Experimental grouping: blank control group (complete culture medium), negative group (125 mM glucose), sample group; The sample group is specifically: Feruloyl pyridoxal phosphate experiment: mixture of 100 μg / mL concentration of Example 1 and 125 mM glucose; Nicotinoyl pyridoxal phosphate experiment: mixture of 100 μg / mL concentration of Example 2 and 125 mM glucose; Experimental method: take human primary fibroblasts HDF in logarithmic growth phase, add to a 6-well plate, 2.0*10 5 6 / well, in a cell culture incubator (37 °C, 5% CO2), the cells are attached overnight; According to the above grouping, the drug is administered, incubated in the cell culture incubator for 24 h, and the RNA is extracted and reverse transcribed according to the Vazyme kit instructions; according to the qPCR kit instructions, the PCR reaction system of GAPDH, COL-I, MMP-1, MMP-3, p21, p16, etc. of the blank group, the negative group, and the sample group is configured in a 96-well plate (the specific primer sequences are shown in Table 13 below), after the PCR system is configured, the system is added to the PCR 96-well plate according to the well plate design, the sealing film is attached, and the mixture is gently mixed; Put the 96-well plate into the fluorescence qRT-PCR instrument for Real Time PCR reaction; After amplification, GAPDH was used as an internal reference gene, and the CT value of the target gene expression was obtained by comparing with the control group. The relative quantitative analysis was performed by 2-△△CT method, and the data analysis was performed by Excel software.

[0074]

[0075] The results of feruloylpyridoxamine are shown in Tables 14-18 below,

[0076]

[0077]

[0078]

[0079]

[0080] From the above Tables 14-18 and the attached Figure 22 As can be seen, compared with the glucose glycation model group, the feruloylpyridoxamine obtained in Example 1 can significantly improve the changes in aging indicators caused by excessive glycation (glucose). Using 100 μg / mL of nicotinoylpyridoxamine, the relative expression of COL-I mRNA was increased by 32.62%, the relative expression of MMP-1 mRNA was decreased by 46.29%, the relative expression of MMP-3 mRNA was decreased by 35.74%, the relative expression of p21 mRNA was decreased by 30.48%, and the relative expression of p16 mRNA was decreased by 41.18%.

[0081] The results of nicotinoylpyridoxamine are shown in Tables 19-22 below,

[0082]

[0083]

[0084]

[0085] From the above Tables 19-22 and the attached Figure 23It can be seen that, compared with the glucose sugarization modeling group, the nicotinamide pyridoxamide obtained in Example 2 can significantly improve the changes in aging indicators caused by excessive sugarization (glucose). Using 100 μg / mL of nicotinamide pyridoxamide, the relative expression of COL-I mRNA is increased by 48.71%, the relative expression of MMP-1 mRNA is reduced by 31.62%, the relative expression of MMP-3 mRNA is reduced by 8.59%, and the relative expression of p21 mRNA is reduced by 24.58%.

[0086] Experiment 6 Cell Anti-sugar (galactose) Aging Test Experimental grouping: blank control group (complete culture medium), negative group (40 mg / mL galactose), sample group; The sample group is specifically: Crude feruloyl pyridoxamide experiment: mixture of 100 μg / mL of Example 1 and 40 mg / mL of galactose; Nicotinamide pyridoxamide experiment: mixture of 100 μg / mL of Example 2 and 40 mg / mL of galactose; Retinyl pyridoxamide experiment: mixture of 20 μg / mL of Example 3 and 40 mg / mL of galactose; Experimental method: take logarithmic growth period human primary fibroblasts HDF, add to 6-well plates, 2.0*10 5 cells per well, incubate in an incubator (37 °C, 5% CO2) overnight; According to the above grouping, the drug is administered, incubated in an incubator for 24 h, and the RNA is extracted according to the Vazyme kit instructions, and the reverse transcription is performed; according to the qPCR kit instructions, the PCR reaction system of GAPDH, COL-I, COL-III, MMP-1, and MMP-3 of the blank group, negative group, and sample group is configured in a 96-well plate (the specific primer sequences are shown in Table 13), after the PCR system is configured, the system is added to the PCR 96-well plate according to the plate design, the sealing film is attached, and the mixture is gently mixed; the 96-well plate is placed in a fluorescent qRT-PCR instrument for Real Time PCR reaction; After amplification, GAPDH is used as an internal reference gene, compared with the control group, the CT value of the target gene expression is obtained by result output, 2-△△CT method is used for relative quantitative analysis, and Excel software is used for data analysis.

[0087] The results of the feruloyl pyridoxamide experiment are shown in Tables 23-26,

[0088]

[0089]

[0090]

[0091] From Tables 23-26 and Appendix Figure 24 As can be seen, compared with the galactose glycation model, the ferulopyridoxamide obtained in Example 1 can significantly improve the changes in aging indicators caused by excessive glycation (galactose). Using 100 μg / mL of ferulopyridoxamide, the relative expression level of COL-I mRNA was increased by 304.38%, the relative expression level of COL-III mRNA was increased by 270.62%, the relative expression level of MMP-1 mRNA was decreased by 94.71%, and the relative expression level of MMP-3 mRNA was decreased by 55.88%.

[0092] The experimental results of nicotinamide are shown in Tables 27-29.

[0093]

[0094]

[0095] From Tables 27-29 and Appendix Figure 25 As can be seen, compared with the galactose glycation model, the nicotinamide obtained in Example 2 can significantly improve the changes in aging indicators caused by excessive glycation (galactose). Using 100 μg / mL of nicotinamide, the relative expression level of COL-I mRNA was increased by 244.31%, the relative expression level of MMP-1 mRNA was reduced by 92.99%, and the relative expression level of MMP-3 mRNA was reduced by 56.21%.

[0096] The results of the retinylpyridinium assay are shown in Tables 30-32.

[0097]

[0098]

[0099] From Tables 30-32 and Appendix Figure 26It can be seen that, compared with the galactose glycosylation modeling group, the retinyl pyridoxamide obtained in Example 3 can significantly improve the changes in aging indicators caused by excessive glycosylation (galactose). Using 20 μg / mL of retinyl pyridoxamide, the relative expression of COL-I mRNA increased by 111.70%, the relative expression of COL-III mRNA increased by 102.06%, and the relative expression of MMP-1 mRNA decreased by 12.49%.

[0100] Experiment 7 Cell Soothing Test Experimental grouping: blank control group, negative group, sample group, at least 3 replicate wells in each group; Experimental method: take HaCaT cells in logarithmic growth phase, inoculate cells into 12-well plates at a seeding amount of 3.0 x 10 5 cells / well, and incubate in an incubator (37 °C, 5% CO2) overnight; after overnight incubation, the sample group is given 20 μg / mL of Example 3, the blank group and the negative control group are still replaced with complete culture medium; After 24 h of incubation in the incubator, use a UVB irradiator with an energy of 20 mJ / cm 2 According to the experimental grouping, perform the experiment, incubate at 37 °C, 5% CO2 for 24 h, collect the supernatant, use an IL-6 Elisa kit, follow the kit instructions, react the supernatant in the coated plate, after complete reaction, use a microplate reader to detect the absorbance at 450 nm and 630 nm wavelengths, and calculate the IL-6 concentration secreted by each sample according to the standard sample calibration curve in the kit; Reduction rate (%) = (negative group - sample group) / negative group * 100%.

[0101] The results are shown in Table 33,

[0102] From the above Table 33 and the attached Figure 27 It can be seen that, compared with the UVB modeling NC group, the retinyl pyridoxamide obtained in Example 3 can significantly reduce the IL-6 content. Using 20 μg / mL of retinyl pyridoxamide, the IL-6 secretion content of the cells decreased by 21.86%.

[0103] Experiment 8 Epidermal Soothing Test Experimental grouping: blank control group (model culture medium), negative control group (600 mJ / cm 2 UVB irradiation + model culture medium), sample group (600 mJ / cm 2 UVB irradiation + model culture medium + 30 μg / mL of Example 3); Experimental procedure: After receiving the 3D skin model, use the recovery medium to stabilize and incubate overnight at 37℃, 5% CO2; After overnight incubation, the experiment was carried out according to the experimental grouping, and the supernatant was collected after 24h culture at 37℃, 5% CO2. The IL-8 Elisa kit was used according to the kit instruction steps, and the supernatant was reacted in the coated plate. After complete reaction, the absorbance was detected by enzyme-labeled instrument at 450nm and 630nm wavelength. Through the standard sample calibration curve in the kit, the IL-8 concentration secreted by each sample was calculated; Reduction rate (%) = (negative group - sample group) / negative group * 100%.

[0104] The results are shown in Table 34,

[0105] From the above Table 34 and the attached Figure 28 It can be seen that compared with the UVB modeling NC group, the retinol pyridoxamine obtained in Example 3 can significantly reduce the content of IL-8. The use of 30μg / mL of retinol pyridoxamine can reduce the IL-8 secretion content of cells by 23.60%.

[0106] In summary, the pyridoxamine derivative provided by the present application shows excellent active oxygen scavenging ability in in vitro antioxidant experiments, and can effectively inhibit the generation of advanced glycation end products (AGEs), indicating that it has a wide application prospect in the preparation of cosmetics with anti-glycation, antioxidant and anti-aging effects.

Claims

1. A pyridoxine derivative, characterized in that: The general structural formula is The substituent R is derived from ferulic acid, retinic acid, or nicotinic acid.

2. The method for preparing a pyridoxine derivative according to claim 1, characterized in that, Includes the following steps: S1. Dissolve 1.0-2.0 eq of a carboxylic acid compound in a solvent, add 1.0-3.0 eq of a condensing agent and 1.0-3.0 eq of an activator, and stir at 0°C until completely clear and dissolved to obtain reaction solution A; S2. Add 1.0-3.0 eq of base dropwise to the reaction solution at 0℃ and react at 0℃ for 30 min to obtain reaction solution B; S3. Add pyridoxine hydrochloride to reaction solution B, stir the reaction at room temperature for 12-48 hours, and monitor the reaction progress by TLC or HPLC. After S4 and pyridoxine hydrochloride react completely, the reaction is quenched with saturated sodium bicarbonate aqueous solution. After treatment, the mixture is extracted, the organic phase is collected, dried and concentrated, and then purified by column chromatography to finally obtain the target pyridoxine derivative.

3. The method for preparing a pyridoxine derivative according to claim 2, characterized in that: The carboxylic acid compound is one of ferulic acid, retinoic acid, or niacin.

4. The method for preparing a pyridoxine derivative according to claim 2, characterized in that: The condensing agent includes one or more of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, dicyclohexylcarbodiimide, diisopropylcarbodiimide, N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate, 6-chlorobenzotriazol-1,1,3,3-tetramethylurea hexafluorophosphate, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate, and 2-(1H-benzotriazol-L-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate.

5. The method for preparing a pyridoxine derivative according to claim 2, characterized in that: The activator includes one or more of N-hydroxy-7-azabenzotriazole, 1-hydroxybenzotriazole, 3-hydroxy-1,2,3-benzotriazine-4(3H)-one, N-hydroxysuccinimide, 4-dimethylaminopyridine, and benzotriazine-1-yloxytripyrrolidinylphosphonium hexafluorophosphate.

6. The method for preparing a pyridoxine derivative according to claim 2, characterized in that: The base includes one or more of N,N-diisopropylethylamine, triethylamine, N-methylmorpholine, pyridine, 2,6-dimethylpyridine, 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5-diazabicyclo[4.3.0]non-5-ene, sodium carbonate, sodium bicarbonate, sodium hydroxide, potassium hydroxide, and potassium carbonate.

7. The method for preparing a pyridoxine derivative according to claim 2, characterized in that: The solvent includes one or more of petroleum ether, ethyl acetate, dichloromethane, methanol, ethanol, tetrahydrofuran, pyridine, diethyl ether, acetone, cyclohexane, toluene, N,N-dimethylformamide, and dimethyl sulfoxide.

8. The application of a pyridoxine derivative according to claim 1 in the preparation of cosmetics with anti-glycation effects, characterized in that: The substituent R of the pyridoxine derivative is derived from ferulic acid, retinoic acid, or nicotinic acid.

9. The application of a pyridoxine derivative according to claim 1 in the preparation of cosmetics with antioxidant effects, characterized in that: The substituent R of the pyridoxine derivative is derived from ferulic acid, retinoic acid, or nicotinic acid.

10. The application of a pyridoxine derivative according to claim 1 in the preparation of cosmetics with anti-aging effects, characterized in that: The substituent R of the pyridoxine derivative is derived from ferulic acid, retinoic acid, or nicotinic acid.

11. The application of a pyridoxine derivative according to claim 1 in the preparation of cosmetics with soothing effects, characterized in that: The substituent R in the pyridoxine derivative is derived from retinoic acid.

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