A retinoid derivative, its preparation and use

Abstract

The application belongs to the technical field of cosmetics and skin external preparation, and discloses a retinoid, a preparation method and application thereof. The compound is formed by covalently coupling a Schiff base or a kinin releasing enzyme cleavable peptide bond (L) and a free L-amino acid, a di- to hexapeptide or a C6-C22 fatty acid ester (P) of the retinol parent (R), and the structural general formula is R-L-P. The compound has a 12-week stability rate of ≥80% in a 25℃ aqueous preparation, is recognized by a keratin layer carboxylic acid esterase and a kinin releasing enzyme KLK-5 / KLK-7 after being applied to the skin, is subjected to enzymatic cutting in a skin acidic cuticle (pH 4.5-5.5) microenvironment, and releases free retinol at a fixed point. Compared with retinol palmitate, the in-situ release efficiency of the compound of the application is increased by 2.5-4.5 times, while the risk of systemic absorption is significantly reduced, and the incidence of sensitive skin irritation is ≤6.7%.

Description

Technical Field

[0001] This invention relates to the field of cosmetics and topical skin preparations, specifically to an enzyme-sensitive retinaldehyde derivative, its preparation method, and its application. Background Technology

[0002] Vitamin A and its metabolic family (retinol, retinal, and retinoic acid) are recognized as active molecules for skin anti-aging and anti-acne. Retinic acid is explicitly prohibited by China's "Cosmetic Safety Technical Specifications." Retinol and retinal are permitted components in cosmetics, but they suffer from technical problems such as poor stability, high irritation, and low release efficiency. In existing technologies, the enzymatic release rate of retinyl palmitate is only 10%-20%, and the direct esterification product of retinol has defects such as poor skin targeting and easy to cause irritation to sensitive skin; at the same time, the synthesis of retinal derivatives often uses acidic deprotection processes, which easily lead to the degradation of retinal, resulting in low product purity and yield; amino acid / peptide raw materials use carbamate protecting groups, which require acidic deprotection conditions, easily causing the degradation of the vitamin A parent compound, and incomplete deprotection can easily introduce impurities. Summary of the Invention

[0003] This invention provides a one-step unprotected synthesis process for retinaldehyde Schiff base. The resulting product is stable in aqueous formulations in vitro. After being applied to human skin, it is enzymatically cleaved by keratinase and kallikrein to release free retinaldehyde in situ.

[0004] A retinaldehyde derivative has the following general structural formula: RLP; Wherein, R is selected from all-trans retinal; L is selected from Schiff base; and P is selected from free L-amino acid, dipeptide to hexapeptide, or one of their C6-C22 fatty acid esters.

[0005] The fatty acid ester is selected from one of linoleic acid ester, isopalmitic acid ester, or stearic acid ester; the free L-amino acid is one of L-cysteine ​​or L-lysine.

[0006] Retinaldehyde derivatives are prepared through the following steps: (1) Dissolve all-trans-retinaldehyde in an anhydrous solvent and stir in an ice bath in the dark until completely dissolved; (2) Add free L-amino acids, dipeptides to hexapeptides, or one of their C6-C22 fatty acid esters, stir and disperse, adjust the pH of the system to 7.2 with triethylamine, stir at room temperature in the dark for 3 h, and monitor the complete reaction of retinaldehyde by thin-layer chromatography (TLC). (3) The reaction solution was purified by reversed-phase HPLC, eluted with acetonitrile-water gradient, concentrated under reduced pressure at 35°C in the dark, and dried under vacuum to obtain the target compound.

[0007] In the preparation method, the molar ratio of all-trans retinal to free L-amino acids, dipeptides to hexapeptides, or their C6-C22 fatty acid esters is 1:1 to 1.5; the solvent is ethanol, and the volume molar ratio of the solvent to all-trans retinal is 10-12 mL / mmol; the reversed-phase HPLC purification uses a C18 column; the acetonitrile-water volume ratio is 60:40 to 90:10, using a gradient elution method, and the total volume of eluent is 15 to 25 times the volume of the sample to be purified; the reduced pressure concentration temperature is 30 to 35℃, and the vacuum degree is -0.08 to -0.1 MPa; the vacuum drying is performed at a vacuum degree of -0.09 to -0.1 MPa, a temperature of 25 to 30℃, and a time of 12 to 24 h.

[0008] The core synthesis strategy of this invention is as follows: The Schiff base reaction involving retinaldehyde uses free amino acids as raw materials. It requires no protection or deprotection steps and is synthesized in a one-step unprotected method, avoiding side reactions and retinaldehyde degradation. The process is simple and the product has high purity.

[0009] The retinaldehyde derivatives prepared by this method have the following characteristics: (1) The hydrolysis half-life under pH 4.5-5.5 conditions does not exceed 1 / 10 of that under pH 7.4 conditions; (2) After incubation at pH 4.5 and 37℃ for 30 minutes, the release rate of free retinaldehyde was ≥70%; (3) The residual mass after 12 weeks of storage in an aqueous preparation at 25°C is ≥80%.

[0010] A cosmetic composition containing a retinaldehyde derivative has a pH value of 5.5-6.5; the retinaldehyde derivative has a mass content of 0.01%-2.0%. Preferably, the mass content of the retinaldehyde derivative is 0.1%-0.5%; the cosmetic composition does not contain parabens or ethanol.

[0011] The cosmetic composition dosage form is selected from aqueous gels, serums, lotions, creams, lyophilized powders, or masks. The lyophilized powder dosage form is prepared by freeze-drying a solution containing retinaldehyde derivatives and reconstituted with an aqueous matrix at pH 5.5-6.5 before use.

[0012] Cosmetic compositions containing retinaldehyde derivatives are applied to human skin. The retinaldehyde derivatives undergo enzymatic cleavage in the acidic epidermal microenvironment of the stratum corneum (pH 4.5-5.5) or upon contact with endogenous carboxylesterases, kallikrein KLK-5, KLK-7, sebaceous triglyceride lipases, or Propionibacterium acnes lipases in the stratum corneum, releasing free retinaldehyde. The retinaldehyde derivatives are used in the preparation of cosmetics for photoaging repair, post-acne inflammation repair, or sensitive skin barrier repair.

[0013] Cosmetic compositions for repairing post-acne inflammatory pigmentation can be used in combination with niacinamide, 4-n-butylresorcinol, or α-arbutin; cosmetic compositions for repairing photoaging wrinkles can be used in combination with peptides and hyaluronic acid oligosaccharides.

[0014] The cosmetics are used for the repair of sensitive skin, and the total incidence of irritation reaction was verified by a patch test on 30 volunteers with sensitive skin to be ≤6.7%.

[0015] The beneficial effects of this invention are: (1) The stability of the aqueous phase is significantly improved, and the mass residue rate of retinaldehyde derivative in the aqueous formulation at 25℃ is ≥80% after 12 weeks; (2) pH-responsive release: Under the conditions of pH 4.5 / 37℃ / 30 min, the release rate of free retinaldehyde is ≥70%; (3) Highly efficient skin enzymatic digestion, with the target product released at a rate of up to 82.3% within 30 minutes; (4) High in situ active concentration in the skin, with the cumulative amount of free active substances being 2.5-4.5 times that of the retinyl palmitate control group; (5) Significantly reduces the risk of dermal penetration and systemic absorption; (6) The incidence of sensitive skin irritation reaction is ≤6.7%, which is suitable for the anti-aging needs of sensitive skin. Detailed Implementation

[0016] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0017] Example 1: Synthesis of all-trans-retinaldehyde-L-cysteine ​​Schiff base Feed quantities: 284.4 mg (1.0 mmol) all-trans retinaldehyde, 121.2 mg (1.0 mmol) free L-cysteine, 12 mL anhydrous ethanol, appropriate amount of triethylamine, 5 mL purified water. Operating procedures: (1) Dissolve all-trans-retinaldehyde in anhydrous ethanol and stir in an ice bath in the dark until completely dissolved; (2) Add free L-cysteine, stir and disperse, then adjust the pH of the system to 7.2 with triethylamine, stir at room temperature in the dark for 3 h, and monitor the reaction of retinaldehyde by TLC until it is complete. The whole process uses free amino acids as raw materials, without any protection or deprotection steps. (3) The reaction solution was purified by reversed-phase HPLC (C18 column, acetonitrile-water gradient elution), the target peak was collected, concentrated under reduced pressure at 35℃ in the dark, and dried under vacuum. Yield: 86%, HPLC purity: 99.0%, retinaldehyde degradation rate: <1%.

[0018] Example 2: Synthesis of all-trans-retinaldehyde-L-lysine Schiff base Feed amounts: 284.4 mg (1.0 mmol) all-trans retinaldehyde, 146.2 mg (1.0 mmol) free L-lysine, 10 mL anhydrous ethanol, and appropriate amount of triethylamine. Procedure: Same as Example 1, except the amino acid was replaced with free L-lysine. The Schiff base reaction was completed by stirring at room temperature in the dark for 3.5 h. Free amino acids were used as the raw material throughout the process, without any protection or deprotection steps. Yield: 83%, HPLC purity: 98.7%.

[0019] Example 3: Synthesis of all-trans-retinaldehyde-Cys-Lys dipeptide Schiff base Feeding amounts: 284.4 mg (1.0 mmol) of all-trans retinal, 249.3 mg (1.0 mmol) of free Cys-Lys dipeptide, 12 mL of anhydrous ethanol, 3 mL of purified water, and appropriate amount of triethylamine. Operating steps: (1) Dissolve all-trans retinal in anhydrous ethanol and stir in an ice bath in the dark until completely dissolved; (2) Add free Cys-Lys dipeptide, add purified water dropwise and stir to disperse, adjust the pH of the system to 7.2 with triethylamine, stir at room temperature in the dark for 3.5 h, monitor the retinal reaction by TLC until complete, use free dipeptide as raw material throughout the process, without any protection or deprotection steps; (3) Purify the reaction solution by reverse-phase HPLC (C18 column, acetonitrile-water gradient elution), collect the target peak, concentrate under reduced pressure in the dark at 35℃, and dry under vacuum. Yield: 80%, HPLC purity 98.6%, retinal degradation rate <1.5%.

[0020] Example 4: Synthesis of all-trans-retinaldehyde-Ala-Ala-Pro-Phe tetrapeptide Schiff base Feeding amounts: 284.4 mg (1.0 mmol) of all-trans retinaldehyde, 445.5 mg (1.0 mmol) of free Ala-Ala-Pro-Phe tetrapeptide, 15 mL of anhydrous ethanol, 2 mL of N,N-dimethylformamide, and appropriate amount of triethylamine. Operation steps: (1) Dissolve all-trans retinal in a mixed solvent of anhydrous ethanol and DMF, and stir in an ice bath in the dark until completely dissolved; (2) Add free Ala-Ala-Pro-Phe tetrapeptide, stir to disperse, and adjust the pH of the system to 7.2 with triethylamine. Stir at room temperature in the dark for 4 h, and monitor the retinal reaction by TLC until complete; This peptide is a specific recognition sequence of kallikrein KLK-5 / KLK-7, which can realize the dual release of Schiff base pH response and peptide bond cleavage, without any protection or deprotection steps; (3) The reaction solution is purified by reverse-phase HPLC (C18 column, acetonitrile-water gradient elution), the target peak is collected, concentrated under reduced pressure in the dark at 35℃, and dried under vacuum. Yield: 74%, HPLC purity 98.2%, retinal degradation rate <2%.

[0021] Example 5: Synthesis of all-trans-retinaldehyde-Lys-Gly-His-Lys-Gly-Pro hexapeptide Schiff base Feed amounts: 284.4 mg (1.0 mmol) of all-trans retinal, 681.8 mg (1.0 mmol) of free Lys-Gly-His-Lys-Gly-Pro hexapeptide, 15 mL of anhydrous ethanol, 5 mL of purified water, and appropriate amount of triethylamine. Operation steps: (1) Dissolve all-trans retinal in anhydrous ethanol and stir in an ice bath in the dark until completely dissolved; (2) Add free hexapeptide, add purified water dropwise and stir to disperse, adjust the pH of the system to 7.2 with triethylamine, stir at room temperature in the dark for 4.5 h, monitor the retinal reaction by TLC until complete, use free hexapeptide as raw material throughout the process, without any protection or deprotection steps; (3) Purify the reaction solution by reverse-phase HPLC (C18 column, acetonitrile-water gradient elution), collect the target peak, concentrate under reduced pressure in the dark at 35℃, and dry under vacuum. Yield: 70%, HPLC purity 98.0%, retinal degradation rate <2.2%.

[0022] Example 6: Synthesis of all-trans-retinaldehyde-L-lysine stearate Schiff base Feeding amounts: 284.4 mg (1.0 mmol) of all-trans retinal, 412.7 mg (1.0 mmol) of L-lysine stearate, 15 mL of anhydrous ethanol, and appropriate amount of triethylamine. Procedure: (1) Dissolve all-trans retinal in anhydrous ethanol and stir in an ice bath in the dark until completely dissolved; (2) Add L-lysine stearate (C18 fatty acid ester, belonging to the C6-C22 range), stir and disperse, then adjust the pH of the system to 7.2 with triethylamine, stir at room temperature in the dark for 3 h, monitor the retinal reaction by TLC until complete, and there are no protection and deprotection steps; (3) Purify the reaction solution by reverse-phase HPLC (C18 column, acetonitrile-water gradient elution), collect the target peak, concentrate under reduced pressure in the dark at 35℃, and dry under vacuum. Yield: 78%, HPLC purity 98.4%, retinal degradation rate <1.2%.

[0023] Example 7: pH-responsive hydrolysis kinetics test Test method: Prepare phosphate buffer solutions (0.1 M) with pH values ​​of 4.5, 5.5, 6.5, and 7.4; take 10 mg of the test compound and add 10 mL of buffer solution with different pH values, and shake at 37℃ for 30 min; detect the free retinaldehyde content by HPLC and calculate the release rate.

[0024] Table 1. pH response hydrolysis data (release rate %) at 30 min Example 8: Stability test of aqueous gel formulation Preparation method: 0.2% of the analyte, 0.2% of carbomer 940, 5% of butylene glycol, 3% of glycerol, and triethanolamine were added to adjust the pH to 5.5. Purified water was added to bring the solution to 100% to prepare an aqueous gel. Testing method: Samples were collected at 0, 4, 8, and 12 weeks after sealing and protecting from light at 25°C. The residual mass of the compound was determined by HPLC.

[0025] Table 2. Stability data for 12 weeks (25℃) Example 9: Franz diffusion cell porcine skin permeability test Test method: (1) Take fresh pig ear skin, remove subcutaneous fat, fix it in a Franz diffusion cell, and use pH 5.5 phosphate buffer as the receiving solution. Cycle at 37℃. (2) Add 1 g of 0.2% test compound gel to the supply cell and permeate at 37℃ for 24 h. (3) Separate the stratum corneum + epidermis and dermis of the skin and extract and detect the cumulative amount of free retinaldehyde.

[0026] Table 3. 24-hour cumulative permeability (μM) Example 10: Sensitive Skin Irritation Test Test method: Thirty volunteers with sensitive skin (Fitzpatrick skin type II-III, confirmed by positive lactic acid sting test) were selected. A closed patch test was used, in which 0.1% of the test compound sample was applied to the upper back of the subjects. The patch was removed after 48 hours. Skin reactions such as erythema, edema, burning, and itching were observed at 30 min and 24 h after removal. The total irritation response rate was calculated according to the CTFA standard.

[0027] Table 4 Comparison of irritation to sensitive skin (30 cases) Example 11: Preparation of freeze-dried powder Formula: 0.5% retinaldehyde-Ala-Ala-Pro-Phe tetrapeptide Schiff base, 5% mannitol, 2% trehalose, and purified water to 100 mL. Procedure: (1) Add mannitol and trehalose to purified water, stir until completely dissolved, and filter aseptically through a 0.22 μm filter membrane; (2) Add retinaldehyde-Ala-Ala-Pro-Phe tetrapeptide Schiff base, stir in the dark until completely dissolved, and filter aseptically again through a 0.22 μm filter membrane; (3) Dispense into sterile vials, 1 mL per vial; (4) Transfer to a freeze dryer, pre-freeze at -40℃ for 4 h, program temperature rise and vacuum freeze dry for 24 h, and seal with stopper.

[0028] Example 12: pH-responsive hydrolysis kinetics test Test method: Prepare phosphate buffer solutions (0.1 M) with pH values ​​of 4.5, 5.5, 6.5, and 7.4. Take 10 mg of the compound to be tested, add 10 mL of buffer solution with different pH values, and shake at 37 °C for 30 min. HPLC was used to detect the free retinaldehyde content and calculate the release rate.

[0029] Table 1. pH response hydrolysis data (release rate %) at 30 min Example 13: Stability test of aqueous gel formulation Preparation method: 0.2% of the test compound, 0.2% of carbomer 940, 5% of butylene glycol, 3% of glycerol, and triethanolamine were added to adjust the pH to 5.5. Purified water was added to bring the solution to 100% to prepare an aqueous gel. Testing method: Samples were collected at 0, 4, 8, and 12 weeks after sealing and protecting from light at 25°C. The residual mass of the compound was determined by HPLC.

[0030] Table 2. Stability data for 12 weeks (25℃) Example 14: Franz diffusion cell porcine skin permeability test Test method: (1) Take fresh pig ear skin, remove subcutaneous fat, fix it in a Franz diffusion cell, and use pH 5.5 phosphate buffer solution as the receiving solution. Circulate at a constant temperature of 37℃. (2) Add 1 g of 0.2% of the test compound gel to the supply tank and allow it to permeate at a constant temperature of 37℃ for 24 h; (3) Separate the stratum corneum, epidermis and dermis of the skin, and extract and detect the cumulative amount of free retinaldehyde.

[0031] Table 3. 24-hour cumulative permeability (μM) Example 15: Sensitive Skin Irritation Test Test method: Thirty volunteers with sensitive skin (Fitzpatrick skin type II-III, confirmed by positive lactic acid sting test) were selected. A closed patch test was used, in which 0.1% of the test compound sample was applied to the upper back of the subjects. The patch was removed after 48 hours. Skin reactions such as erythema, edema, burning, and itching were observed at 30 minutes and 24 hours after removal. The total irritation response rate was calculated according to the CTFA standard.

[0032] Table 4 Comparison of irritation to sensitive skin (30 cases) Example 16: Emulsion Preparation Formula: Retinaldehyde-Cys Schiff base 0.1%, squalane 10%, jojoba oil 5%, glyceryl stearate 2%, Tween-80 2%, xanthan gum 0.1%, glycerin 4%, purified water to 100%. Operating steps: (1) Preparation of oil phase: Squalane, jojoba oil, glyceryl stearate and Tween-80 are mixed, heated to 80°C and stirred until completely melted and homogeneous; (2) Aqueous phase preparation: Xanthan gum and glycerol were added to purified water, stirred to swell, and heated to 80°C and kept warm; (3) Homogenization and emulsification: The aqueous phase is slowly added to the oil phase, homogenized at high speed at 80℃ for 5 min, and then stirred and cooled to 40℃; (4) Add retinaldehyde-Cys Schiff base, stir evenly in the dark, and cool to room temperature before discharging.

[0033] Example 17: Preparation of water-based matrix by reconstitution of lyophilized powder Formula (mass percentage): Butylene glycol 5%, glycerin 3%, sodium hyaluronate 0.1%, p-hydroxyacetophenone 0.3%, citrate-triethanolamine buffer system to adjust pH to 5.5-6.5, purified water to 100%. Operating steps: (1) Add sodium hyaluronate, butylene glycol and glycerin to purified water and stir until completely swollen and dissolved; (2) Add p-hydroxyacetophenone and stir until the system is homogeneous and transparent; (3) Finely adjust the pH value to 5.5-6.5 with citrate or triethanolamine, and filter aseptically through a 0.22μm filter membrane to obtain the reconstituted aqueous matrix.

[0034] The above description is merely a few preferred embodiments of the present invention and is not intended to limit the scope of protection of the present invention. It should be noted that those skilled in the art can make various modifications, equivalent substitutions, or improvements to the above embodiments without departing from the core ideas and technical principles of the present invention. Any such changes and modifications made within the spirit and principles of the present invention should be considered as included within the scope of protection defined by the claims of the present invention.

Claims

1. A retinaldehyde derivative, characterized in that, It has the following general structural formula: RLP; Wherein, R is selected from all-trans retinal; L is selected from Schiff base; and P is selected from free L-amino acid, dipeptide to hexapeptide, or one of their C6-C22 fatty acid esters.

2. The retinaldehyde derivative according to claim 1, characterized in that, The fatty acid ester is selected from linoleic acid ester, isopalmitic acid ester, or stearic acid ester; the free L-amino acid is selected from L-cysteine ​​or L-lysine.

3. The retinaldehyde derivative according to claim 1, characterized in that, The retinaldehyde derivative is prepared by the following steps: (1) Dissolve all-trans-retinaldehyde in an anhydrous solvent and stir in an ice bath in the dark until completely dissolved; (2) Add free L-amino acids, dipeptides to hexapeptides, or one of their C6-C22 fatty acid esters, stir and disperse, adjust the pH of the system to 7.2 with triethylamine, stir at room temperature in the dark for 3 h, and monitor the complete reaction of retinaldehyde by thin-layer chromatography. (3) The reaction solution was purified by reversed-phase HPLC, eluted with acetonitrile-water gradient, concentrated under reduced pressure at 35°C in the dark, and dried under vacuum to obtain the target compound.

4. The method for preparing retinaldehyde derivatives according to claim 3, characterized in that, The molar ratio of all-trans retinal to free L-amino acids, dipeptides to hexapeptides, or their C6-C22 fatty acid esters is 1:1 to 1.

5. The solvent is ethanol, and the volume molar ratio of the solvent to all-trans retinal is 10-12 mL / mmol. The reversed-phase HPLC purification uses a C18 column. The acetonitrile-water volume ratio is 60:40 to 90:10, using a gradient elution method, with the total eluent volume being 15 to 25 times the volume of the sample to be purified. The reduced-pressure concentration temperature is 30 to 35°C, and the vacuum degree is -0.08 to -0.1 MPa. The vacuum drying is performed at a vacuum degree of -0.09 to -0.1 MPa, a temperature of 25 to 30°C, and a time of 12 to 24 hours.

5. A cosmetic composition, characterized in that, The composition comprises the retinaldehyde derivative according to any one of claims 1-4, wherein the pH value of the composition is 5.5-6.5; the mass content of the retinaldehyde derivative is 0.01%-2.0%; and the composition is free of parabens and ethanol.

6. The cosmetic composition according to claim 5, characterized in that, The retinaldehyde derivative is preferably present in a mass content of 0.1%-0.5%.

7. The cosmetic composition according to claim 5, characterized in that, The cosmetic composition dosage form is selected from aqueous gel, serum, lotion, cream, lyophilized powder or mask.

8. The cosmetic composition according to claim 7, characterized in that, The lyophilized powder formulation is prepared by freeze-drying a solution containing the retinaldehyde derivative, and is reconstituted with an aqueous matrix at pH 5.5-6.5 before use; the aqueous matrix is ​​an aqueous solution containing a polyol humectant and does not contain ethanol or paraben preservatives.

9. The cosmetic composition according to claim 8, characterized in that, The polyol moisturizer in the aqueous matrix is ​​selected from one or more of butylene glycol, glycerin, 1,3-propanediol, and 1,2-hexanediol; the aqueous matrix may also contain one or more water-soluble skin care ingredients selected from sodium hyaluronate and panthenol.

10. The cosmetic composition according to claim 9, characterized in that, The cosmetic composition can be used in combination with niacinamide, 4-butylresorcinol or α-arbutin for the repair of post-acne inflammatory pigmentation; the cosmetic composition can also be used in combination with peptides and hyaluronic acid oligosaccharides for the repair of photoaging wrinkles.

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