阅读说明：本技术 一种4-氟苄基哌啶类化合物在抑制酪氨酸酶活性中的应用 (Application of 4-fluorobenzyl piperidine compound in inhibiting tyrosinase activity ) 是由 王贯 胡秀英 刘丽 王方 李金� 于 2021-11-05 设计创作，主要内容包括：本发明涉及一种4-氟苄基哌啶类化合物在抑制酪氨酸酶活性中的应用,所涉及的应用为与酪氨酸酶活性有关的美白化妆品、黑色素瘤治疗。本发明解决的技术问题是发现了一种4-氟苄基哌啶类化合物具有抑制酪氨酸酶活性的作用。化合物的结构如下所示,化合物具有抑制酪氨酸酶活性的作用,能够应用于美白化妆品和抗黑色素治疗。(The invention relates to an application of a 4-fluorobenzyl piperidine compound in inhibiting tyrosinase activity, and relates to an application of a whitening cosmetic and melanoma treatment related to the tyrosinase activity. The invention solves the technical problem of finding that a 4-fluorobenzyl piperidine compound has the effect of inhibiting the activity of tyrosinase. The compound has the following structure, has tyrosinase activity inhibiting effect, and can be used in whitening cosmetics and cosmeticsAnd (4) performing anti-melanin treatment.)

1. The compound with the structural formula shown as the formula I has the effect of inhibiting the activity of tyrosinase, and is characterized in that: has tyrosinase activity inhibiting effect

2. Use according to claim 1, characterized in that: a pharmaceutical composition comprises a compound of formula I and a pharmaceutically acceptable salt for use in inhibiting tyrosinase activity.

3. Use according to claim 2, characterized in that: the application is a whitening cosmetic related to tyrosinase activity and anti-melanoma.

Technical Field

The invention relates to an application of a 4-fluorobenzyl piperidine compound in inhibiting tyrosinase activity, and relates to an application of a whitening cosmetic and melanoma treatment related to the tyrosinase activity.

Background

The color of human skin is determined by 4 chromophores in the body, namely carotenoids, hemoglobin, oxyhemoglobin, melanin. Among them, melanin is the most important component. Research has shown that the production, content, distribution and type of melanin have a significant influence on the color of human hair, eyes and skin, and the number of melanocytes, the activity of enzymes involved in synthesis and the transfer of melanosomes are important causes of pigmentation. Melanin in the human body can protect the skin from harmful effects caused by environmental factors such as ultraviolet radiation, oxidative stress, and the like. However, excessive accumulation of melanin can lead to the appearance of many pigmentation disorders, such as freckles, age spots, melanoderma, and even malignant melanoma. Malignant melanoma is one of the common malignant tumors and is also one of the most threatening skin cancers. Tyrosinase is the most important rate-limiting enzyme in melanin synthesis, and researches find that the tyrosinase is an effective strategy for reducing melanin generation by inhibiting the activity of tyrosinase to form whitening cosmetics and treating melanoma. Therefore, tyrosinase is a key target point for resisting pigment deposition and melanoma, and the tyrosinase inhibitor can be widely applied to the fields of medicines and cosmetics as a whitening cosmetic and a melanoma resisting medicine. Therefore, the search for new tyrosinase inhibitors with high efficiency and high selectivity has great significance in terms of anti-melanin deposition and anti-melanoma.

Disclosure of Invention

The invention relates to an application of a 4-fluorobenzyl piperidine compound in inhibiting tyrosinase activity.

The application of the invention is characterized in that: the application is food preservative with tyrosinase activity, whitening cosmetics, melanoma treatment and senile dementia treatment.

Description of the figures

Table 1 shows tyrosinase inhibitory activity test and antitumor cell proliferation activity results of the above compounds.

TABLE 1 tyrosinase inhibitory Activity assay of Compounds of formula I and results of antitumor cell proliferation Activity

Fig. 1 shows the effect of compound concentration on tyrosinase inhibitory activity, and data are expressed as mean inhibition (n-3).

FIG. 2 is a kinetic experiment of the compound on tyrosinase inhibition, and the inhibition type of the compound is judged to be non-competitive inhibition according to a Lineweaver-Burk double reciprocal diagram.

Detailed Description

The above object of the present invention is achieved by the following technical solutions:

the compound is a 4-fluorobenzyl piperidine compound, and the structure of the compound is shown as the following formula I:

the following examples are provided to further illustrate the embodiments of the present invention and are not intended to limit the scope of the present invention.

Test example 1 half inhibition test of tyrosinase by the Compound

A0.5 mL centrifuge tube is selected as a reaction container, 0.1mL solution is set as a reaction system, 50 muL of sample solution (10 muM and 30 muM) and 25 muL of tyrosinase solution (1mM) with two concentrations are respectively added into the reaction system, after the reaction is carried out for 20 minutes at room temperature (25 ℃), 25 muL of substrate L-Tyrosine solution (2.5U/muL) is rapidly added, after the mixture is fully mixed, the change of Optical Density (OD) within 5 minutes is detected under the condition of the absorption wavelength of 492 nm. The experiment was repeated two more times in parallel and the average inhibition was calculated (n-3). The inhibition ratio was calculated by the following formula:

inhibition ratio%_vehicle-ΔOD_compound)/(ΔOD_vehicle-ΔOD_blank)*100％

Wherein, Δ OD_compoundChange in optical Density value, Δ OD, for addition of inhibitor_vehicleAs the change in optical density value, Δ OD, of the solvent control group_blankThe change value of the optical density value of the blank control group is shown.

The solvent control group was prepared by adding 50. mu.L of DMSO phosphate buffer, and the blank control group was prepared by adding 75. mu.L of DMSO phosphate buffer and 25. mu.L of 2.5U/. mu. L L-Tyrosine solution. According to the tyrosinase inhibition rate of the sample solution with different concentrations, a dose response curve of the sample and the tyrosinase is drawn, and the IC of the sample compound₅₀Values were calculated from dose response curves. The results are shown in Table 1 and FIG. 1. Kojic acid was used as a positive control drug.

As is apparent from table 1 and fig. 1, compound I has significantly stronger inhibitory activity against tyrosinase, which is about twice as strong as that of the positive control drug kojic acid.

Test example 2 inhibition mechanism of tyrosinase by the Compound

Sample solutions with the concentration of 30 mu mol/L and samples without samples are selected as two series, the dosage of the substrate L-tyrosine solution (39 nmol/L-1000 nmol/L) is changed in sequence, and the delta OD value of the enzyme catalysis initial velocity of tyrosinase is tested at each concentration. The reciprocal 1/[ S ] of the L-tyrosine concentration (. mu.mol/L) is plotted on the abscissa]Expressed as the initial speed Δ OD of the tyrosinase reaction on the ordinate_{Sample (A)}Is 1/V, a Lineweaver-Burk double reciprocal plot is drawn to calculate the Mie constant (K)_m) Maximum speed (V)_max) And inhibition constant (K)_i)。

The purpose of this experiment was to test the mechanism of the inhibition of tyrosinase by compound I of the present invention, and the results are shown in fig. 2. Experimental results show that the compound can obviously inhibit the activity of tyrosinase and is a non-competitive tyrosinase inhibitor.

Experimental example 3 anti-melanogenesis experiment of Compound

B16F10 cells were seeded into 24-well cell culture plates, 1X10⁵cells/well. After 24h of culture, 20. mu. mol/L of alpha-MSH, 20. mu. mol/L of Compound I or positiveThe control compound kojic acid. After further incubation for 48h under the above conditions, the cells were washed 2-3 times with PBS buffer, the medium contents were removed, and the melanin was dissolved with 1mol/L NaOH solution (200. mu.L). The solution was transferred to a 96-well plate, and the absorbance of the dissolved melanin solution was measured at 492nm with a microplate reader. Melanin in the cells of each experiment was optimized using a standard curve to normalize the intracellular melanin values. The above experiment was repeated three times and the average value was calculated. The result shows that the compound I can effectively inhibit the synthesis of melanin of melanoma cells B16F10 induced by alpha-MSH, and the compound I enables the content of melanin in the cells to be reduced by 65.43 percent under the concentration of 20 mu mol/L, which is better than the inhibition capability (42.56 percent) of the content of melanin of a positive control medicament kojic acid. Therefore, the compound I can effectively reduce the content of melanin in cells, and provides powerful evidence for the compound I as an effective component of a whitening cosmetic and a medicament for treating melanoma.

Experimental example 4 antiproliferative experiment of melanoma cells with Compound

We performed experiments on the antiproliferative activity of malignant melanoma cells with compound I, and the results are shown in table 1. After treatment of melanoma cells B16F10 with compound I, the median inhibitory concentration on malignant melanoma cells was determined, and the results indicated that compound I had antiproliferative activity on B16F10 cells.

In combination with the above, the compound I has the effect of inhibiting tyrosinase activity, can be applied to whitening cosmetics and anti-melanin treatment related to the tyrosinase activity, and has wide development space and application value.