All-trans retinoic acid benzyl ester, its preparation method and application

Abstract

The present application relates to a kind of all-trans retinoic acid benzyl ester and its preparation method and application, its structural formula is as shown in following formula: the all-trans retinoic acid benzyl ester of the present application is RAR gamma and RXR-alpha agonist, side effect is small and also has the activity of retinoic acid, can promote epithelial cell differentiation and exfoliation, regulate and prevent keratinization and inhibit the secretion of sebum, with good application value in anti-inflammatory, improve keratinization abnormal disease etc..

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of vitamin A, and particularly relates to a phenylmethyl ester of all-trans retinoic acid and a preparation method and application thereof. BACKGROUND

[0002] All-trans-retinoic acid (ATRA), also known as tretinoin or vitamin A acid, is one of the main physiologically active metabolites of vitamin A. The compound has extremely wide uses in the fields of drugs and cosmetics. Specifically, in the field of drugs, all-trans-retinoic acid can be used not only for treating skin keratinization abnormality diseases such as acne, but also for treating skin tumors and early myeloblast leukemia. In the field of cosmetics, all-trans-retinoic acid has the effects of anti-aging and anti-wrinkle.

[0003] In the research on the target, mechanism of action and in vivo metabolism of all-trans-retinoic acid, it is found that all-trans-retinoic acid (Szymański Skopek R, Palusińska M, et al. Retinoic acid and its derivatives in skin [J]. Cells, 2020, 9(12): 2660.) has an in vivo half-life of about 1 hour and can be rapidly metabolized by cytochrome P450 enzymes (CYP26s). CYP is involved in the hydroxylation of ATRA, thereby inactivating its function. ATRA is not only a substrate for CYP26 enzymes, but also an effective inducer of CYP26, thus producing a negative feedback loop. ATRA deficiency mediated by CYP or other mechanisms is associated with cancer progression and various skin diseases.

[0004] Retinoic acid receptors (RARs) are key developmental regulators that act as molecular switches in many developmental processes, including skin development. Six RARs have been identified, namely retinoic acid receptors alpha, beta, and gamma (RAR-alpha, RAR-beta, and RAR-gamma) and retinoid X receptors alpha, beta, and gamma (RXR-alpha, RXR-beta, and RXR-gamma). RARalpha, RARgamma, and RXR are highly expressed in fibroblasts and keratinocytes, with RAR being more abundant in the latter. In human epidermis, RAR / RXR complexes are dominant in the basal layer, with RAR-gamma / RXR-alpha heterodimers being important signaling pathways in the skin. ATRA can down-regulate RAR-gamma expression and up-regulate RAR-beta expression, but does not affect RAR-alpha expression. Once in the nucleus of keratinocytes, ATRA can bind to RAR-gamma, which forms a heterodimer with RXR-alpha to exert the effects of retinoic acid in the tissue. Inhibition of RAR-dependent signaling in the basal cell layer can down-regulate keratinocyte differentiation, and this mechanism is related to the mechanism of keratinization abnormality diseases and ATRA regulation of keratinocyte differentiation.

[0005] In addition, studies suggest that RAR and RXR-dependent mechanisms are also involved in the process of skin aging. ATRA and RAR agonists can limit the damage of ultraviolet light to collagen fibers and increase the collagen content in photoaged skin. ATRA and RAR agonists can stimulate type I procollagen protein expression by down-regulating c-Jun protein and inhibiting the function of matrix metalloproteinases (MMPs).

[0006] Although ATRA has multiple effects on the skin, its short half-life and certain side effects limit its application. For example, ATRA has strong skin irritation when used topically, which can easily cause skin dryness, redness, stinging, and other problems. Such side effects are believed to be related to the release of pro-inflammatory factors (MCP-1, TNF-alpha, IL-1, IL-6, and IL-8), but the mechanism of causing side effects is not fully understood, so this poses a technical obstacle and challenge for the design of new ATRA derivatives (Mukherjee S, Date A, Patravale V, et al. Retinoids in the treatment of skin aging: an overview of clinical efficacy and safety [J]. Clinical interventions in aging, 2006, 1(4): 327.). Given the strong skin irritation of ATRA, the design and screening of new ATRA derivatives to reduce its side effects remains the focus of current ATRA drug research. SUMMARY

[0007] The present application aims to provide a trans-retinoic acid benzyl ester, which is a RARγ and RXR-α agonist, has less side effects and has the activity of retinoic acid, and a preparation method and application thereof.

[0008] The present application provides a trans-retinoic acid benzyl ester, which has the following structural formula:

[0009]

[0010] The present application also provides a preparation method of the trans-retinoic acid benzyl ester, which comprises the following steps:

[0011] The retinoic acid is mixed with a catalyst, an organic solvent, benzyl alcohol or a derivative thereof and a dehydrating agent, and then refluxed at 35-50°C for 8-20 hours, concentrated under reduced pressure, washed, filtered, and concentrated under reduced pressure to obtain the trans-retinoic acid benzyl ester.

[0012] The molar ratio of the retinoic acid, benzyl alcohol or a derivative thereof, the dehydrating agent and the catalyst is 1:1.3-1.7:1.3-1.7:0.3-0.6.

[0013] The catalyst is 4-dimethylaminopyridine (DMAP).

[0014] The derivative of the benzyl alcohol is one or more alkyl groups on the benzene ring of the benzyl alcohol.

[0015] The organic solvent is dichloromethane, and the ratio of the retinoic acid to the organic solvent is 1g:20-50ml.

[0016] The dehydrating agent is dicyclohexyl carbodiimide (DCC).

[0017] The washing comprises acid washing and alkali washing.

[0018] The acid washing is acid washing with 5%-30% citric acid, preferably with a concentration of 10%, and the alternative solvent is hydrochloric acid.

[0019] The alkali washing is alkali washing with 5%-30% sodium hydroxide, preferably with a concentration of 10%, and the alternative solvent is potassium hydroxide.

[0020] The present application also provides an application of the trans-retinoic acid benzyl ester in anti-aging and anti-wrinkle skin and personal care products.

[0021] The synthetic reaction formula of the present application is as follows:

[0022]

[0023] Beneficial effects

[0024] The all-trans retinoic acid benzyl ester of the present application is a RARγ and RXR-α agonist, has less side effects and has the activity of retinoic acid, can promote the differentiation and exfoliation of epithelial cells, regulate and prevent keratinization and inhibit the secretion of sebum, and has good application value in anti-inflammatory and improvement of keratinization abnormality diseases BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 is a mass spectrum of the all-trans retinoic acid benzyl ester;

[0026] Figure 2 is a nuclear magnetic H spectrum of the all-trans retinoic acid benzyl ester;

[0027] Figure 3 is a dose response curve of the all-trans retinoic acid benzyl ester and RARγ and RXR-α receptors;

[0028] Figure 4 is the inhibitory effect of the all-trans retinoic acid benzyl ester on TGF-β induced MMP3 of human corneal fibroblasts. DETAILED DESCRIPTION

[0029] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not used to limit the scope of the present application. Furthermore, it should be understood that those skilled in the art can make various modifications or changes to the present application after reading the content taught by the present application, and these equivalent forms also fall within the scope of the appended claims of the present application.

[0030] Example 1

[0031] The present example provides a preparation method of the all-trans retinoic acid benzyl ester, comprising the following steps:

[0032] (1) 50 ml of dichloromethane and 2 g of retinoic acid were added into a 250 ml three-necked flask, and stirred for 10 min;

[0033] (2) 1.02 g of benzyl alcohol was added, and stirring was continued for 10 min;

[0034] (3) 0.24 g of DMAP was added, and stirring was continued for 0.5 h;

[0035] (4) the temperature was lowered to 0°C, and a DCC solution (2.0 g of DCC + 20 ml of DCM were uniformly mixed) was added dropwise;

[0036] (5) after the dropwise addition was completed;

[0037] (6) the temperature was raised to 35°C, and stirring was continued for 16 h;

[0038] (7) filtration to remove the solid;

[0039] (8) the filtrate was concentrated under reduced pressure to dryness to obtain a solid;

[0040] (9) 60 ml of ethyl acetate was added and stirred;

[0041] (10) 20 ml of 10% citric acid was added and stirred for 1 h, and separated;

[0042] (11) 20 ml of 10% sodium hydroxide solution was added to the organic phase and stirred for 1 h, and separated;

[0043] (12) 30 ml of purified water was added to the organic phase and stirred for 2 h, and separated;

[0044] (13) 30 ml of purified water was added to the organic phase again and stirred for 2 h, and separated;

[0045] (14) 20 g of anhydrous sodium sulfate was added to the organic phase and stirred for 4 h;

[0046] (15) filtration was performed, and the filtrate was concentrated under reduced pressure to dryness to obtain a yellow solid, which was all-trans retinyl phenylacetate.

[0047] Structure confirmation

[0048] (1) mass spectrum of all-trans retinyl phenylacetate

[0049] Test instrument: WATERS ZQ

[0050] Measured data:

[0051] Mass spectrum data table

[0052] Accurate mass determination Theoretical values [M+H]+ 391.5

[0053] Mass spectrum analysis: through mass spectrum analysis, the relative molecular weight of the compound was 390.5.

[0054] The mass spectrum is shown in Figure 1 .

[0055] (2) H spectrum of all-trans retinyl phenylacetate

[0056] 1H NMR (600 MHz, CDCl3): 7.20 (d, 4H), 6.20 (d, 3H), 6.06 (m, 2H), 5.75 (s, 1H), 5.08 (d, 3H), 4.61 (d, 2H), 2.32 (d, 3H), 1.92 (d, 3H), 1.64 (m, 4H), 1.39 (m, 3H), 0.96 (d, 6H);

[0057] The H spectrum is shown in Figure 2 .

[0058] Test Example 1: Affinity test of all-trans retinoic acid benzyl ester with RARγ / RXR-α receptor in vitro

[0059] The binding affinity of all-trans retinoic acid benzyl ester for RAR-γ and RXR-α was determined using time-resolved fluorescence spectroscopy (TR-FRET). Samples were prepared with dimethyl sulfoxide to create gradient concentrations. The in vitro detection kits used were the TR-FRET RXR-α coactivator assay kit (catalog number: Kit-0972) and the TR-FRETRARγ coactivator assay kit (catalog number: PV4411), and the detection was performed according to the instrument and kit instructions.

[0060] A glutathione S-transferase (GST)-labeled human RARγ ligand-binding domain (RARγ-LBD) or RXR-α-LBD was added to the ligand test compound, followed by the addition of a mixture of luciferin coactivator peptide and terbium (Tb)-labeled anti-GST antibody. After incubation at room temperature, the TR-FRET 520:495 emission ratio was calculated, and the EC50 was determined based on the compound's dose-response curve.

[0061] like Figure 3 As shown, all-trans retinoic acid benzyl ester was determined to be an agonist of RARγ and RXR-α, with an EC50 of approximately 0.149 nM (95% confidence interval 0.034 nM–0.354 nM) for RARγ activation and approximately 10.77 nM (95% confidence interval 7.03 nM–15.68 nM) for RXR-α activation. It is known that all-trans retinoic acid has an EC50 of 19 nM for RXR-α and 0.22 nM for RARγ activation. All-trans retinoic acid benzyl ester exhibits slightly stronger agonistic effects on both RARγ and RXR-α than all-trans retinoic acid.

[0062] Test Example 2: In vitro experiment to investigate the effect of all-trans retinoic acid benzyl ester on the expression of matrix metalloproteinases.

[0063] All-trans retinoic acid can protect against photoaged skin by activating RARγ / RXR-α, regulating the expression level of matrix metalloproteinases (MMPs), and inhibiting skin collagen degradation. After receptor affinity testing, this experiment investigated the cellular activity of all-trans retinoic acid benzyl ester by measuring its effect on MMPs. Methods and results are as follows:

[0064] 1. Fibroblast culture and collagen gel construction

[0065] Human corneal fibroblasts were subcultured in MEM medium with 10% fetal bovine serum. After 6 passages, the cells were used in the study. The cells were digested with trypsin-EDTA and washed twice with serum-free MEM medium. The cells were resuspended in serum-free MEM medium and prepared into collagen gels according to the literature. The final concentration of the cells was 2 x 10 5 The cells were added to 24-well plates coated with 1% bovine serum albumin and incubated at 37°C for 1 hour. The plates were incubated at 37°C in a CO2incubator for 1 hour to solidify the collagen gels.

[0066] 2. Test substance addition and determination of matrix metalloproteinase expression levels

[0067] The test substances, all-trans retinyl benzoate (ATRABME), TGF-β and all-trans retinoic acid (ATRA) were prepared in serum-free MEM medium. The test substances were added to the collagen gels and the cells were incubated for 72 hours. The final concentration of TGF-β was 1 ng / mL and the final concentration of all-trans retinoic acid was 1 μM. The concentration of all-trans retinyl benzoate was 0.01 μM, 0.1 μM, 1 μM, 10 μM and 100 μM.

[0068] After the incubation, lysis buffer was added to the cells under ice bath conditions. SDS-polyacrylamide gel electrophoresis was performed on 10% gels. Immunoblot analysis of matrix metalloproteinase 3 (MMP-3) in the cells was performed according to the literature. The absorbance was measured at 560 nm after color development. One-way ANOVA was used to compare the absorbance between groups.

[0069] The results are shown in Figure 4 (ATRA is all-trans retinoic acid and ATRABME is all-trans retinyl benzoate of the present application. * indicates p < 0.05 compared with the TGF-β group and # indicates p < 0.05 compared with the ATRA group), and all-trans retinyl benzoate inhibited MMP-3 expression in a dose-dependent manner. The absorbance was significantly lower in the range of 1 μM to 10 μM of all-trans retinyl benzoate than in the case of all-trans retinoic acid (P < 0.05). This can be attributed to the difference in the affinity of the two substances for RXR-α and RARγ receptors. It is presumed that the strong inhibitory effect of all-trans retinyl benzoate on MMP-3 is at least partially due to the strong agonistic effect of all-trans retinyl benzoate on RXR-α and RARγ receptors.

Claims

1. The application of all-trans retinoic acid benzyl ester in the preparation of anti-aging skin care products, wherein the aging is skin aging caused by photoaging, and the structural formula of the all-trans retinoic acid benzyl ester is shown below: 。

Images