Cinnamoyl valine bornyl ester and its preparation method and application

Abstract

The present invention discloses cinnamoyl valine borneol ester and its preparation method and application. The structural formula of the cinnamoyl valine borneol ester is shown in Formula I. The present invention first synthesizes Boc-Val-Borneol by esterification of borneol and Boc-protected L-valine, then removes the Boc protecting group, and then condenses with cinnamic acid amide to synthesize cinnamoyl valine borneol ester in high yield. The cinnamoyl valine borneol ester has good tyrosinase inhibition and anti-inflammatory activity; the raw materials and reagents used in the present invention are simple and easy to obtain, the reaction conditions are mild, the operation method is simple, the yield is excellent, and the product has broad application prospects in the treatment or prevention of skin pigmentation diseases and anti-inflammatory treatment.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical synthesis and biochemistry, and particularly relates to cinnamoyl valine bornyl ester and a preparation method and application thereof. Background Art

[0002] Under normal physiological conditions, melanin protects human skin from damage such as ultraviolet rays, toxic chemicals, and other environmental factors. However, excessive melanin production and abnormal accumulation can lead to hyperpigmentation disorders such as freckles, chloasma, age spots, pregnancy spots, café au lait spots, sun spots, moles, melanoma, and other hyperpigmented skin diseases, which severely affect the patient's appearance and cause significant mental stress. Skin pigmentation is closely related to melanin production. The pigment is formed in melanosomes within the basal layer of the epidermis. Tyrosine is first hydroxylated by tyrosinase to form dopa, which is then oxidized by tyrosinase to form dopaquinone. Subsequently, dopaquinone undergoes a series of reactions, primarily through natural oxidation, to ultimately produce melanin. Tyrosinase is a key enzyme in the biosynthesis of melanin, and inhibiting tyrosinase activity can effectively inhibit melanin production.

[0003] Currently, common tyrosinase inhibitors, such as kojic acid and ascorbic acid, suffer from poor stability and the potential for serious side effects. Therefore, the development of new, natural, non-toxic, and highly effective tyrosinase inhibitors is of great significance. Cinnamic acid and its derivatives exhibit significant inhibitory effects on tyrosinase and are potential tyrosinase inhibitors. The present invention provides a previously unreported cinnamoyl valine bornyl ester, which exhibits excellent tyrosinase inhibition and anti-inflammatory activity. Summary of the Invention

[0004] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a cinnamoyl valine bornyl ester.

[0005] Another object of the present invention is to provide a method for preparing the cinnamoyl valine-bornyl ester.

[0006] Another object of the present invention is to provide the application of the cinnamoyl valine bornyl ester.

[0007] The purpose of the present invention is achieved by the following technical solution: a cinnamoyl valine bornyl ester, whose structural formula is shown in Formula I:

[0008]

[0009] The preparation method of cinnamoyl valine bornyl ester comprises the following steps: dissolving borneol, Boc-L-valine (Boc-L-Val), and a catalyst in an organic solvent, reacting, and post-treating to obtain valine bornyl ester; then dissolving cinnamic acid, valine bornyl ester, and triethylamine in an organic solvent, stirring and reacting to obtain cinnamoyl valine bornyl ester; more preferably, the method comprises the following steps:

[0010] (1) Preparation of valine-borneol ester: Boc-L-valine and borneol were dissolved in an organic solvent under ice bath conditions to obtain a uniform solution; a catalyst was then added to the above solution, the reaction was stirred, and the reaction progress was monitored by TLC; after the reaction was completed, vacuum rotary evaporation and column chromatography were performed to obtain a colorless oil, namely Boc-valine-borneol ester (Boc-L-Val-Borneol);

[0011] (2) Removal of Boc protecting group: dissolve the Boc-valine borneol ester obtained in step (1) in an organic solvent, add hydrochloric acid, stir overnight, and monitor the reaction progress by TLC; after the reaction is complete, evaporate under reduced pressure to obtain a white solid, slurry with ether, filter, and vacuum dry to obtain white crystalline valine borneol ester hydrochloride;

[0012] (3) Preparation of cinnamoyl valine borneol ester: Cinnamic acid, valine borneol ester hydrochloride obtained in step (2) and 1-hydroxybenzotriazole (HOBT) are dissolved in an organic solvent under ice bath conditions, followed by dropwise addition of an acid binding agent. After the addition is complete, the mixture is stirred, and then a catalyst is added. The mixture is naturally warmed to room temperature and stirred. The reaction progress is monitored by TLC. After the reaction is complete, the reaction solution is washed with saturated brine, the organic phase is recovered, water is removed, filtered, the solvent is dried under reduced pressure, and a light yellow viscous solid is obtained by silica gel column chromatography. The solid is rotary evaporated and dried under vacuum to obtain cinnamoyl valine borneol ester.

[0013] The borneol is preferably dextrorotatory borneol.

[0014] The ice bath condition in step (1) is preferably 0 to -5°C.

[0015] The Boc-L-valine and borneol in step (1) are mixed in a molar ratio of 1:1 to 1.5, preferably in a molar ratio of 1:1 to 1.2.

[0016] The organic solvent described in step (1) is preferably at least one of tetrahydrofuran and dichloromethane; more preferably dichloromethane.

[0017] The amount of the organic solvent used in step (1) is preferably calculated based on the total mass of Boc-L-valine and borneol (g): organic solvent volume (mL) = 0.2 to 0.25:1; more preferably, the total mass of Boc-L-valine and borneol (g): organic solvent volume (mL) = 0.15 to 0.21:1.

[0018] The catalyst described in step (1) is preferably a mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) and p-dimethylaminopyridine (DMAP); more preferably, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and p-dimethylaminopyridine are compounded in a molar ratio of 10:1.

[0019] The amount of the catalyst used in step (1) is preferably 1.5 to 1.6 times the molar amount of Boc-L-valine.

[0020] The stirring reaction time in step (1) is preferably 4 to 6 hours.

[0021] The developing solvent used for TLC in step (1) is preferably a solution obtained by mixing petroleum ether and ethyl acetate in a volume ratio of 100:1 to 5; more preferably a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 100:5.

[0022] The filler used in the column chromatography in step (1) is preferably silica gel with a size of 100 to 400 meshes; more preferably silica gel with a size of 300 to 400 meshes.

[0023] The column chromatography described in step (1) preferably comprises the following steps: loading a sample, eluting impurities with a petroleum ether solution, and eluting with a mobile phase to obtain a colorless oil.

[0024] The mobile phase is preferably a solution obtained by mixing petroleum ether and ethyl acetate in a volume ratio of 100:4-6; more preferably a solution obtained by mixing petroleum ether and ethyl acetate in a volume ratio of 100:5.

[0025] The organic solvent described in step (2) is preferably ethyl acetate.

[0026] The amount of the organic solvent is preferably calculated based on a mass volume ratio (g:mL) of Boc-valine borneol ester to organic solvent of 0.2-0.5:1; more preferably calculated based on a mass volume ratio (g:mL) of Boc-valine borneol ester to organic solvent of 0.3-0.4:1.

[0027] The concentration of the hydrochloric acid in step (2) is preferably 10 to 12 mol / L.

[0028] The amount of hydrochloric acid used in step (2) is preferably calculated based on a molar ratio of Boc-valine borneol ester to hydrochloric acid of 1:1.

[0029] The developing solvent used in the TLC described in step (2) is preferably a solution obtained by mixing petroleum ether and ethyl acetate in a volume ratio of 100:5-10; more preferably a solution obtained by mixing petroleum ether and ethyl acetate in a volume ratio of 100:10.

[0030] The ice bath condition in step (3) is preferably 0 to -5°C.

[0031] The amount of each reaction component in step (3) is preferably calculated according to the molar ratio of cinnamic acid: valine borneol ester hydrochloride: 1-hydroxybenzotriazole = 1:1:1-1.5; more preferably, it is calculated according to the molar ratio of cinnamic acid: valine borneol ester hydrochloride: 1-hydroxybenzotriazole = 1:1:1.2.

[0032] The organic solvent in step (3) is preferably at least one of N'N-dimethylformamide (DMF) and dichloromethane; more preferably dichloromethane.

[0033] The amount of the organic solvent used in step (3) is based on the ability to fully dissolve all the reaction components; preferably, the amount is 5 to 10 mL of organic solvent per 1 g of reaction components.

[0034] The acid binding agent described in step (3) is preferably triethylamine.

[0035] The amount of the acid-binding agent in step (3) is preferably calculated based on a molar ratio of 2 to 4:1 between the acid-binding agent and valine borneol ester hydrochloride; more preferably, the molar ratio of 3:1 between the acid-binding agent and valine borneol ester hydrochloride.

[0036] The catalyst described in step (3) is preferably EDCI.

[0037] The amount of the catalyst in step (3) is preferably calculated based on a molar ratio of the catalyst to valine borneol ester hydrochloride of 1:1 to 1.5; more preferably, the molar ratio of the catalyst to valine borneol ester hydrochloride is 1:1.2.

[0038] The developing solvent used in the TLC described in step (3) is preferably a solution obtained by mixing petroleum ether and ethyl acetate in a volume ratio of 100:10-20; more preferably a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 100:20.

[0039] The stirring conditions during stirring after the dropwise addition in step (3) are preferably stirring at 300-600 r / min for 8-12 min; more preferably stirring at 500 rpm for 10 min.

[0040] The time required for the reaction to be complete in step (3) is preferably 12 to 18 hours.

[0041] The amount of saturated saline solution in step (3) is preferably calculated as follows: valine borneol hydrochloride: saturated saline solution = mass volume ratio (g: mL) of 0.02 to 0.04:1; more preferably, as follows: valine borneol hydrochloride: saturated saline solution = mass volume ratio (g: mL) of 0.025 to 0.03:1 (g: mL).

[0042] The number of washings in step (3) is preferably 2 to 4 times; more preferably 3 times.

[0043] The dehydration in step (3) is preferably carried out using at least one of anhydrous sodium sulfate and anhydrous magnesium sulfate; more preferably, anhydrous sodium sulfate is used for dehydration.

[0044] The time for removing water in step (3) is preferably 5 to 7 hours; more preferably 6 hours.

[0045] The eluent in the column chromatography described in step (3) is preferably a solution obtained by mixing petroleum ether and ethyl acetate in a volume ratio of 100:10.

[0046] The reaction in the present invention is carried out at room temperature unless the temperature is limited.

[0047] The room temperature is 10-35°C, preferably 24-28°C, and more preferably 24-26°C.

[0048] The stirring speed of the present invention is usually 300-800 rpm; stirring is for the smooth progress of the reaction.

[0049] Application of the cinnamoyl valine bornyl ester in the preparation of medicines or cosmetics for treating or preventing skin pigmentation diseases.

[0050] The skin pigmentation diseases include chloasma, freckles, age spots, sun spots, pregnancy spots, café au lait spots, post-inflammatory melanosis and genetic dark skin.

[0051] Application of the cinnamoyl valine bornyl ester in the preparation of anti-inflammatory drugs.

[0052] The present invention has the following advantages and effects compared to the prior art:

[0053] 1. The chemical synthesis method provided by the present invention can produce cinnamoyl valine borneol ester in high yield. This method uses readily available raw materials and reagents, relatively mild reaction conditions, a relatively simple operation method, and simple post-processing. Cinnamoyl valine borneol ester has broad application prospects in fields such as organic chemistry, medicine, and food.

[0054] 2. The cinnamoyl valine bornyl ester provided by the present invention has a stable structure, can significantly inhibit melanin synthesis and tyrosinase activity, and has excellent anti-inflammatory activity. It can be used as a drug and functional raw material for the treatment and prevention of hyperpigmentation diseases and added to whitening products such as cosmetics, and has wide application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 1 is a reaction equation diagram of Example 1 of the present invention.

[0056] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of the product of Example 1 of the present invention.

[0057] Figure 3 This is a high-resolution mass spectrum of the product of Example 1 of the present invention.

[0058] Figure 4 This is a diagram showing the effects of the test substances in each group of Application Example 1 on inhibiting tyrosinase activity. DETAILED DESCRIPTION

[0059] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0060] Unless otherwise specified, the methods in the embodiments and application examples are conventional methods, and the reagents used are conventional commercial reagents or reagents prepared according to conventional methods unless otherwise specified.

[0061] Example 1

[0062] A preparation of cinnamoyl valine bornyl ester, such as Figure 1 As shown, the following steps are included:

[0063] (1) In a 100 mL flat-bottom flask, Boc-L-valine (Boc-L-Val, 2.17 g, 10 mmol) and borneol (Borneol, 1.85 g, 12 mmol) were added to a 100 mL flat-bottom flask under ice bath conditions. 20 mL of dichloromethane was added to dissolve the mixture. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 2.75 g, 15 mmol) and p-dimethylaminopyridine (DMAP, 0.183 g, 1.5 mmol) were then added to the solution. The mixture was stirred at room temperature for 4 h. The reaction progress was monitored by TLC (developing solvent: a mixture of petroleum ether and ethyl acetate in a volume ratio of 100:5). Phosphomolybdic acid was used for color development. Vacuum rotary evaporation and flash column chromatography (filled with 300-400 mesh silica gel) yielded 3.23 g of Boc-L-Val-Borneol as a colorless oil with a yield of 91.5%. In the column chromatography, impurities were first washed away with petroleum ether, and then eluted with a mobile phase; the mobile phase was a mixture of petroleum ether and ethyl acetate in a volume ratio of 100:5.

[0064] (2) Removal of the Boc protecting group: 3.23 g of Boc-L-Val-Borneol obtained in (1) was dissolved in 10 mL of ethyl acetate, and concentrated hydrochloric acid (12 M) in an amount equimolar to that of Boc-L-Val-Borneol was added. The mixture was stirred overnight and the reaction progress was monitored by TLC (developing solvent: a mixture of petroleum ether and ethyl acetate in a volume ratio of 100:10). The reaction was visualized with phosphomolybdic acid. After the reaction was complete, the solid was evaporated under reduced pressure to obtain a white solid. The solid was slurried with ether, filtered, and dried in vacuo to obtain 2.41 g of white crystalline valine borneol ester hydrochloride (L-Val-Borneol hydrochloride) with a yield of 90.5%.

[0065] (3) In a 100 mL flask, cinnamic acid (0.74 g, 5 mmol), 1-hydroxybenzotriazole (HOBT, 0.81 g, 6 mmol), and L-Val-Borneol hydrochloride (1.45 g, 5 mmol) were added under ice bath conditions and dissolved in 15 mL of dichloromethane. Triethylamine (15 mmol) was slowly added dropwise. After the addition was complete, the mixture was stirred for 10 min. EDCI (1.15 g, 6 mmol) was then added. The mixture was naturally warmed to room temperature and stirred overnight. The reaction was monitored by TLC (developing solvent: a mixture of petroleum ether and ethyl acetate in a volume ratio of 100:20). After the reaction was complete, the reactants were rinsed three times with saturated brine, each time with 50 mL of saturated brine. The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. Analyses were performed using a silica gel column with an average particle size of 37 μm. After loading, the sample was first washed with 100 mL of an eluent having a slightly smaller ratio (petroleum ether: ethyl acetate = volume ratio of 100:1) to wash out impurities with low polarity, and then gradient eluted with a solution obtained by mixing petroleum ether and ethyl acetate in a volume ratio of 100:10. The eluate was collected, rotary evaporated, and vacuum dried to obtain 1.26 g of cinnamoyl valine bornyl ester as white crystals with a yield of 65.72%.

[0066] (A) NMR images Figure 2Shown: 1H NMR (600MHz, Chloroform-d) δ = 0.86 (s, 3H), 0.90 (d, J = 13.6, 6H), 0.95-1.03 (m, 7H), 1.18-1.30 (m, 3H), 1.31-1.3 9(m,1H),1.64(s,1H),1.70(t,J=4.5,1H),1.77(tq,J=4.2,12.3,1H),1.95(ddd,J=4.5,9.5,13.5,1H),2.27(pd,J =4.6,6.9,1H),2.41(ddq,J=3.6,9.9,13.8,1H),4.12(q,J=7.1,1H),4.76(dd,J=4.6,8.6,1H),4.92(ddd,J=2.2,3 .5,9.9,1H),6.20(d,J=8.7,1H),6.48(d,J=15.6,1H),7.32-7.41(m,3H),7.49-7.54(m,2H),7.65(d,J=15.6,1H).

[0067] (B) Figure 2 and Figure 3 At the same time, the successful synthesis of cinnamoyl valine bornyl ester was demonstrated, and its structural formula is as follows:

[0068]

[0069] Example 2

[0070] (1) In a 100 mL flat-bottom flask, Boc-L-Val (2.17 g, 10 mmol) and d-borneol (1.54 g, 10 mmol) were added to a 100 mL flat-bottom flask under ice bath conditions, and 20 mL of dichloromethane was added to dissolve the mixture. EDCI (2.75 g, 15 mmol) and DMAP (0.183 g, 1.5 mmol) were then added to the solution. The mixture was stirred at room temperature for 4 h. The reaction progress was monitored by TLC, and the reaction was visualized with phosphomolybdic acid. Vacuum evaporation and flash column chromatography yielded 3.2 g of a colorless oily substance, Boc-L-Val-Borneol, with a yield of 90.7%. The mobile phase was a mixture of petroleum ether and ethyl acetate in a volume ratio of 100:5.

[0071] (2) Removal of the Boc protecting group: 3.2 g of Boc-L-Val-Borneol obtained in step (1) was dissolved in 10 mL of ethyl acetate, and an equimolar amount of concentrated hydrochloric acid (12 M) was added. The mixture was stirred overnight and the reaction progress was monitored by TLC. The reaction was visualized using phosphomolybdic acid. After the reaction was complete, the solid was evaporated under reduced pressure to obtain a white solid. The solid was slurried with ether, filtered, and dried under vacuum to obtain 2.29 g of white crystalline valine borneol ester hydrochloride (L-Val-Borneol hydrochloride) with a yield of 89.5%.

[0072] (3) In a 100 mL flask, cinnamic acid (0.74 g, 5 mmol), HOBT (0.81 g, 6 mmol), and L-Val-Borneol hydrochloride (1.45 g, 5 mmol) were added under ice bath conditions and dissolved in 15 mL of dichloromethane. Triethylamine (15 mmol) was slowly added dropwise. After the addition was complete, the mixture was stirred for 10 min. EDCI (1.15 g, 6 mmol) was then added. The mixture was naturally warmed to room temperature and stirred overnight. The reaction was monitored by TLC. After the reaction was complete, the reactants were rinsed three times with saturated brine, each time with 50 mL of saturated brine. The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. Analyses were performed using a silica gel column with an average particle size of 37 μm. After loading, the sample was first washed with 100 mL of an eluent with a slightly smaller ratio (petroleum ether: ethyl acetate = volume ratio 100:1) to wash out impurities with low polarity, and then gradient eluted with a solution obtained by mixing petroleum ether and ethyl acetate in a volume ratio of 100:10. The eluate was collected, rotary evaporated, and vacuum dried to obtain 1.11 g of cinnamoyl valine bornyl ester as white crystals with a yield of 57.91%.

[0073] Application Example 1: Application in Tyrosinase Inhibition

[0074] Mouse melanoma B16 cells (purchased from Beijing Kangwei Century Biotechnology Co., Ltd.) were inoculated into RPMI1640 medium (containing 10% newborn calf serum) and cultured at 37°C, 5% CO2 and saturated humidity. The cells grew adherently and were digested and passaged with trypsin solution. Cells in the logarithmic growth phase were used for experiments.

[0075] The cells were added to a 6-well culture plate at a density of 3 × 10 4 / mL mouse melanoma B16 cells 2mL / well, placed in a CO2 incubator and cultured for 24h. Set a gradient solution of cinnamoyl valine borneol ester (prepared by Example 1) with a concentration of 10-200μg / mL (10, 50, 100, 150, 200μg / mL, respectively), and the solvent is RPMI 1640 medium (containing 10% newborn calf serum, 0.1% DMSO); at the same time, set a kojic acid (500μg / mL) control group, a blank group RPMI 1640 medium (containing 10% newborn calf serum, 0.1% DMSO), a borneol-cinnamic acid combination group (concentration of 10-200μg / mL, borneol and cinnamic acid mass ratio of 1:1), and a borneol-valine combination group (concentration of 10-200μg / mL, borneol and valine mass ratio of 1:1); add 2mL of the solution to each well of a 6-well culture plate, and set up 3 parallels. After 24 hours of culture, the culture medium was aspirated, the cells were washed with PBS (0.01 mol / L, pH 7.4), then digested with trypsin for 30 seconds, centrifuged at 2000 rpm for 5 minutes, and the supernatant was discarded. 1% Triton X-100 (TritonX-100) was then added to the cell pellet, and then frozen at -80°C for 30 minutes. The cells were dissolved at room temperature, and after the cells were completely broken, they were centrifuged at 12000 rpm for 10 minutes and the supernatant was taken. After protein quantification and normalization, 90 μL of cell supernatant was mixed with 10 μL of 0.1% levodopa solution in a 96-well plate, incubated at 37°C for 2 hours, and the absorbance was measured at 490 nm on a microplate reader, and the tyrosinase activity was calculated:

[0076]

[0077] Test results such as Figure 4 As shown, the experimental results demonstrate that the synthesized cinnamoyl valine borneol ester inhibits tyrosinase activity in a concentration-dependent manner. Furthermore, cinnamoyl valine borneol ester exhibits a stronger inhibitory effect on tyrosinase activity than the combined effects of borneol-cinnamic acid and borneol-valine, indicating that the chemically bound cinnamoyl valine borneol ester exhibits greater activity than the combined effects of borneol, valine, and cinnamic acid, demonstrating a synergistic effect. Furthermore, at a concentration of 200 μg / mL, cinnamoyl valine borneol ester exhibited a higher tyrosinase inhibitory ability than the positive control, kojic acid.

[0078] Application Example 2: Experiment on the inhibition of zebrafish melanin synthesis by cinnamoyl valine bornyl ester

[0079] Wild-type AB zebrafish (purchased from the Wuhan Zebrafish Resource Center, China) were used for this experiment. Zebrafish were reared in accordance with DB32 / T 3979-2021, "Technical Conditions for Experimental Zebrafish Rearing." The night before the experiment, sexually mature zebrafish (6 to 12 months old) were selected and placed in a baffled spawning box with a 1:2 ratio of male to female, protected from light overnight. The next day, the light source was turned on, and the baffles of the spawning box were removed for mating and spawning. One hour after mating, the spawning status of the adult fish in each tank was checked, and eggs collected from at least three spawning tanks were pooled and selected for future use.

[0080] Under a stereomicroscope, normally developing embryos at 6 to 8 hpf (hpf = hours post fertilization) were randomly selected using a plastic pipette for use in the experiment. Six-well cell culture plates were used as experimental carriers, with 20 embryos added to each well. After draining the culture water with a plastic pipette, 3.0 mL of a prepared cinnamoyl valine-bornyl ester solution (100 to 1000 μg / mL) was added to each well of the six-well cell culture plates. Three replicates were set up, and the plates were covered and placed in a constant-temperature incubator at 28 ± 0.5°C. A solvent control group, a positive control group (arbutin, 1000 μg / mL), a borneol-valine combination group, and a borneol-cinnamic acid combination group (due to borneol's toxicity to embryos, the drug concentration in the combination groups did not exceed 500 μg / mL) were also set up. Three replicates were added to each well of the six-well culture plates.

[0081] Photographs were taken at 72 hpf. For each experimental group, at least three embryos were used for photography. The photographic parameters were fixed, the embryos were fixed upright, and the head of the embryo was focused on the downward position. The distribution area and number of melanin granules on the surface of each group of zebrafish were observed. The photographed images were analyzed using graphic analysis software (e.g., ImageJ). The optical density (OD) value was used to represent the melanin signal intensity of the zebrafish head, and the relative melanin content of the zebrafish was calculated:

[0082]

[0083] Where:

[0084] S0—average melanin content in the solution control group;

[0085] S1—average melanin content of the sample group.

[0086] Specific results are shown in Table 1. As shown, cinnamoyl valine borneol ester exhibits a strong, concentration-dependent inhibitory effect on melanin synthesis. Furthermore, at comparable concentrations, cinnamoyl valine borneol ester exhibited greater inhibitory activity against melanin synthesis than the combined effects of borneol-cinnamic acid and borneol-valine. This suggests that the chemically bound cinnamoyl valine borneol ester exhibits greater activity than the combined effects of borneol, valine, and cinnamic acid, demonstrating a synergistic effect. At a concentration of 1000 μg / mL, melanin production in zebrafish embryos was significantly reduced, surpassing that of the positive control, arbutin, demonstrating the significant potential of cinnamoyl valine borneol ester.

[0087] Table 1 Effect of cinnamoyl valine bornyl ester (CVB) on melanin synthesis

[0088]

[0089]

[0090] Application Example 3: Anti-inflammatory Application

[0091] Cinnamoyl valine bornyl ester reduces the number of inflammatory cell migration in zebrafish

[0092] The experiment used neutrophil transgenic zebrafish Tg (mpx: EGFP) purchased from the Wuhan Zebrafish Resource Center in China. The zebrafish breeding method was the same as that in Application Example 2. Zebrafish breeding and spawning preparation were carried out the day before the experiment, and the operation was the same as that in Application Example 2.

[0093] Normally developed zebrafish larvae at 3 dpf (dpf = days post fertilization) were randomly assigned to six-well plates, with 15 per well. The standard dilution water in the six-well plates was removed without harming the larvae. Then, 3 mL of an aqueous solution of cinnamoyl valine borneol ester containing 60 μg / mL of the modeling agent (sodium dodecyl sulfate) was quickly added to each well. The cinnamoyl valine borneol ester was solubilized with DMSO (the volume percentage of DMSO was <0.1%). After thorough mixing, the plates were covered and wrapped with aluminum foil. The cells were incubated in a 28.5±1.0°C incubator in the dark until the endpoint (a total incubation time of 18 hours). The experimental groups included a solvent control, a model control, a positive control (0.0625% dipotassium glycyrrhizate), a borneol group, a cinnamic acid group, a valine group, a borneol-valine combination group, and a borneol-cinnamic acid combination group.

[0094] After incubation, randomly select at least 12 zebrafish from those with normal phenotypes and behavior, fix them in 3% methylcellulose, and observe and photograph them under a fluorescence microscope. The zebrafish should be positioned with their heads facing left and their tails facing right, lying on their sides. All zebrafish images should be taken using the same instrument and environmental conditions, and the fish should maintain the same body position.

[0095] After taking the photos, use image analysis software to analyze the zebrafish images. The lateral skin surface area is selected as the quantitative region. The software analysis parameter is set to the number of neutrophils, with the number of neutrophils recorded as N. Ten valid data points are collected for each group.

[0096] According to the number of neutrophils N, the anti-inflammatory efficacy of the test substance (expressed as the neutrophil reduction rate) is calculated as follows:

[0097]

[0098] The experimental results are shown in Table 2. As can be seen from the table, the number of neutrophils in zebrafish treated with the modeling agent was greater than that in the normal control group, indicating successful modeling. Neutrophil counts in the positive control group and the cinnamoyl valine borneol ester group were significantly reduced, indicating a strong inhibitory effect on inflammation. Furthermore, the anti-inflammatory effect of cinnamoyl valine borneol ester was superior to that of borneol, cinnamic acid, valine, the combination of borneol and cinnamic acid, and the combination of borneol and valine. This suggests that the synthesized cinnamoyl valine borneol ester has a synergistic effect, enhancing the anti-inflammatory effects of borneol and cinnamic acid. Calculated using the zebrafish anti-inflammatory efficacy formula, the anti-inflammatory efficacy of cinnamoyl valine borneol ester was 83.72% ± 4.19%.

[0099] Table 2 Anti-inflammatory effect of cinnamoyl amino acid bornyl ester

[0100]

[0101] The above embodiments are preferred implementations of the present invention, but the implementation of the present invention is not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A cinnamoyl valine bornyl ester, characterized in that: The structural formula of the cinnamoyl valine bornyl ester is shown in Formula I:

2. The method for preparing cinnamoyl valine bornyl ester according to claim 1, wherein The method comprises the following steps: dissolving borneol, Boc-L-valine and a catalyst in an organic solvent, reacting and post-treating to obtain valine borneol ester; and then dissolving cinnamic acid, valine borneol ester and triethylamine in an organic solvent, stirring and reacting to obtain cinnamoyl valine borneol ester.

3. The preparation method of cinnamoyl valine bornyl ester according to claim 2, characterized in that The following steps are involved: (1) Preparation of borneol valine ester: Boc-L-valine and borneol were dissolved in an organic solvent under ice bath conditions to obtain a uniform solution; a catalyst was then added to the above solution, the reaction was stirred, and the reaction progress was monitored by TLC; after the reaction was completed, vacuum rotary evaporation and column chromatography were performed to obtain a colorless oil, namely Boc-valine borneol ester; (2) Removal of Boc protecting group: dissolve the Boc-valine borneol ester obtained in step (1) in an organic solvent, add hydrochloric acid, stir overnight, and monitor the reaction progress by TLC; after the reaction is complete, evaporate under reduced pressure to obtain a white solid, slurry with ether, filter, and vacuum dry to obtain white crystalline valine borneol ester hydrochloride; (3) Preparation of cinnamoyl valine borneol ester: Cinnamic acid, valine borneol ester hydrochloride obtained in step (2) and 1-hydroxybenzotriazole are dissolved in an organic solvent under ice bath conditions, followed by dropwise addition of an acid binding agent, stirring after completion of the dropwise addition, followed by addition of a catalyst, and the mixture is naturally heated to room temperature, stirred, and the reaction progress is monitored by TLC; after the reaction is complete, the reaction solution is washed with saturated brine, the organic phase is recovered, water is removed, filtered, the solvent is dried under reduced pressure, and a light yellow viscous solid is obtained by silica gel column chromatography, which is rotary evaporated and dried in vacuo to obtain cinnamoyl valine borneol ester.

4. The method for preparing cinnamoyl valine bornyl ester according to claim 3, wherein The following steps are involved: The Boc-L-valine described in step (1) and the borneol described are mixed in a molar ratio of 1:1 to 1.5; The amount of each reaction component in step (3) is calculated based on the molar ratio of cinnamic acid: valine borneol ester hydrochloride: 1-hydroxybenzotriazole = 1:1:1-1.

5.

5. The method for preparing cinnamoyl valine bornyl ester according to claim 3, wherein The following steps are involved: The ice bath condition in step (1) is 0 to -5°C; The stirring reaction time in step (1) is 4 to 6 hours; The column chromatography described in step (1) comprises the following steps: loading a sample, eluting impurities with a petroleum ether solution, and eluting with a mobile phase to obtain a colorless oil; The ice bath condition in step (3) is 0 to -5°C; After the dropwise addition in step (3), the stirring conditions are as follows: stirring at 300-600 r / min for 8-12 min; The time condition for the complete reaction in step (3) is 12 to 18 hours; The number of washing steps in step (3) is 2 to 4 times; The time for removing water in step (3) is 5 to 7 hours.

6. The method for preparing cinnamoyl valine bornyl ester according to claim 3, wherein The following steps are involved: The organic solvent in step (1) is at least one of tetrahydrofuran and dichloromethane; The catalyst described in step (1) is a mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and p-dimethylaminopyridine; The developing solvent used in the TLC described in step (1) is a solution obtained by mixing petroleum ether and ethyl acetate in a volume ratio of 100:1 to 5; The filler used in the column chromatography in step (1) is 100-400 mesh silica gel; The organic solvent described in step (2) is ethyl acetate; The developing solvent used in the TLC described in step (2) is a solution obtained by mixing petroleum ether and ethyl acetate in a volume ratio of 100:5-10; The organic solvent in step (3) is at least one of N'N-dimethylformamide and dichloromethane; The acid binding agent described in step (3) is triethylamine; The catalyst described in step (3) is EDCI; The developing solvent used in the TLC described in step (3) is a solution obtained by mixing petroleum ether and ethyl acetate in a volume ratio of 100:10-20; The amount of saturated salt water in step (3) is calculated as follows: valine bornyl ester hydrochloride: saturated salt water = mass volume ratio 0.02-0.04:1; The dehydration in step (3) is performed using at least one of anhydrous sodium sulfate and anhydrous magnesium sulfate; The eluent in the column chromatography described in step (3) is a solution obtained by mixing petroleum ether and ethyl acetate in a volume ratio of 100:

10.

7. The method for preparing cinnamoyl valine bornyl ester according to claim 6, wherein: The amount of the organic solvent in step (1) is calculated based on the total mass of Boc-L-valine and borneol: the volume of the organic solvent = 0.2 to 0.25:1; The amount of the organic solvent in step (2) is calculated based on a mass volume ratio of borneol valinate to organic solvent of 0.2 to 0.5:1; The amount of hydrochloric acid used in step (2) is calculated based on a molar ratio of borneol valinate to hydrochloric acid of 1:1; The amount of the organic solvent in step (3) is 5 to 10 mL per 1 g of the reaction components; The amount of the acid-binding agent in step (3) is calculated based on a molar ratio of 2 to 4:1 between the acid-binding agent and borneol valine hydrochloride; The amount of the catalyst used in step (3) is calculated based on a molar ratio of the catalyst to valine borneol ester hydrochloride of 1:1 to 1.

5.

8. Use of the cinnamoyl valine bornyl ester according to claim 1 in the preparation of medicines or cosmetics for treating or preventing skin pigmentation diseases.

9. The use according to claim 8, characterized in that: The skin pigmentation diseases include chloasma, freckles, age spots, sun spots, pregnancy spots, café au lait spots, post-inflammatory melanosis and genetic dark skin.

10. Use of the cinnamoyl valine bornyl ester according to claim 1 in the preparation of anti-inflammatory drugs.

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