Indole derivative as well as preparation method and application thereof

Abstract

The invention provides an indole derivative as well as a preparation method and application thereof. The preparation method comprises the following steps: dissolving copper acetate and triethylamine in isopropanol, and stirring until copper acetate and triethylamine are completely dissolved to obtain a reaction medium; adding 5-bromoisatin and indole into the reaction medium, and fully stirring at room temperature until reactants are completely converted; and distilling to remove the reaction solvent to obtain a crude product, and purifying and recrystallizing to obtain solid powder of the high-purity indole derivative. The indole derivative has a remarkable inhibition effect on generation and transfer of tyrosinase and melanin; the antioxidant capacity of efficiently removing active oxygen free radicals in cells is achieved; the A549 cell is promoted to enter an apoptosis procedure by influencing the membrane potential of mitochondria, activating a related apoptosis signal channel, promoting the expression of apoptosis protein and other mechanisms, so that a new potential strategy and a research direction are provided for the apoptosis regulation and control of tumor cells, and the method has important research value and application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of organic compound synthesis, and in particular to an indole derivative and a preparation method and application thereof. Background Art

[0002] Tyrosinase (TYR), as an oxidase containing divalent copper ions, is widely distributed in natural organisms. It plays the role of a key rate-limiting enzyme in the process of melanin synthesis and plays a decisive role in regulating the rate of melanin production. The production of melanin is a complex biological process, which includes key links such as the production of melanosomes in melanocytes and the transport of melanin between cells. A proper amount of melanin has an important protective function for the skin and can effectively resist the damage caused by ultraviolet radiation to the skin. However, when melanin is overproduced, it will cause a series of related skin diseases such as pigmentation, which will bring many troubles and challenges to human skin health.

[0003] Lung cancer is one of the most common malignant tumors in the world. With the increase in the global population and the intensification of the aging trend, its incidence continues to rise, and it has become the type of tumor with the highest mortality rate. Non-small cell lung cancer (NSCLC) is a common malignant tumor of the lungs, accounting for about 80-85% of the total number of lung cancers. It is a tumor with significant heterogeneity and can be further subdivided into subtypes such as squamous cell carcinoma, adenocarcinoma and large cell carcinoma. Most patients are in the advanced stage when diagnosed. For NSCLC patients with locally advanced disease and good physical condition, although a disease-free survival period of 8 months can be achieved after a series of chemotherapy, radiotherapy and / or surgical treatment, the 5-year survival rate is still less than 15%.

[0004] Currently, there are some drugs on the market for the treatment of non-small cell lung cancer. Among them, the first-generation epidermal growth factor tyrosine kinase inhibitors (EGFR TKIs) include gefitinib and erlotinib; the second-generation EGFR TKIs include afatinib and dacomitinib; the third-generation EGFR TKIs are mainly osimertinib. However, almost all patients treated with these three types of drugs will inevitably develop acquired drug resistance. In order to prevent patients from falling into the dilemma of no drugs available after the occurrence of acquired drug resistance, the continuous development of new anti-tumor drugs has become a key issue that needs to be solved urgently. This is of vital significance to improving the treatment effect and prognosis of NSCLC patients, and also provides a new direction and impetus for promoting the development of tumor treatment. Summary of the invention

[0005] In order to solve the problems mentioned in the background technology, the present application provides an indole derivative and a preparation method and application thereof.

[0006] In the first aspect of the present invention, an indole derivative is provided, the chemical structure of which is:

[0007]

[0008] Furthermore, the indole derivative is a light brown solid powder.

[0009] The second aspect of the present invention provides a method for preparing an indole derivative, the preparation method comprising:

[0010] S1, preparing a reaction medium, dissolving copper acetate and triethylamine in isopropanol, and stirring until completely dissolved to obtain a reaction medium;

[0011] S2, preparing an indole derivative solution, adding 5-bromoisatin and indole to the reaction medium, and stirring well at room temperature until the reactants are completely converted;

[0012] S3, extracting the indole derivative powder, distilling off the reaction solvent to obtain a crude product, purifying and recrystallizing to obtain a high-purity solid powder of the indole derivative.

[0013] Further, step S1 includes the following:

[0014] S11, weigh 0.4 g, 2 mmol, of copper acetate and 0.2 g, 2 mmol, of triethylamine, and place them in a reaction bottle;

[0015] S12, add 10 ml of isopropanol to the reaction bottle and stir for 10 min to completely dissolve it to obtain a reaction medium.

[0016] Further, step S2 includes adding 4.52 g, 20 mmol, of 5-bromoisatin and 2.8 g, 24 mmol, of indole into a reaction bottle, stirring the mixture sufficiently at room temperature to allow a post-coupling reaction to occur, and monitoring the reaction progress by thin layer chromatography.

[0017] Further, the S3 step includes:

[0018] S31, performing reduced pressure distillation using a rotary evaporator to remove the isopropanol solvent in the reaction system, collecting the residue, and obtaining a crude product of the reactant;

[0019] S32, adding the crude product into a mixed solvent of petroleum ether and dichloromethane, stirring and dissolving the mixture sufficiently, and then recrystallizing the mixture to obtain a solid powder of an indole derivative.

[0020] The third aspect of the present invention provides an application of an indole derivative, which is an application of the indole derivative described in the first aspect or an indole derivative prepared by the method described in the second aspect as a tyrosinase inhibitor.

[0021] The fourth aspect of the present invention provides an application of an indole derivative, which is an application of the indole derivative described in the first aspect or an indole derivative prepared by the method described in the second aspect in inhibiting the production and / or transfer of melanin in cells.

[0022] The fifth aspect of the present invention provides an application of an indole derivative, namely, the application of the indole derivative described in the first aspect or the indole derivative prepared by the method described in the second aspect as an antioxidant.

[0023] The sixth aspect of the present invention provides an application of an indole derivative, namely, application of the indole derivative described in the first aspect or the indole derivative prepared by the method described in the second aspect in the treatment of lung cancer.

[0024] Compared with the prior art, the beneficial results of the present invention are:

[0025] (1) The present invention provides a novel indole derivative.

[0026] (2) The method for preparing the indole derivatives provided by the present invention is simple to operate, has high feasibility and repeatability, and makes it possible to prepare the indole derivatives on a large scale, thus laying a foundation for their industrial production and wide application, and ensuring the stability and consistency of product quality.

[0027] (3) When the indole derivatives provided by the present invention are used as tyrosinase inhibitors, they can effectively inhibit the activity of tyrosinase. They are of great significance in the fields of medicine and cosmetics. For example, in cosmetics, they can reduce the production of melanin and play a role in whitening the skin. In the field of medicine, they may be used to treat diseases related to abnormal tyrosinase activity, such as pigmentation diseases.

[0028] (4) The indole derivatives provided by the present invention have a regulatory effect on the production and transfer of melanin in cells. They can inhibit the excessive production of melanin or promote its reasonable distribution, which has potential application value in the treatment of skin diseases, the development of whitening products, and pigment-related biological research, and provides a new way and means to solve pigment-related problems.

[0029] (5) When used as antioxidants, the indole derivatives provided by the present invention can remove free radicals in cells and reduce the damage to cells caused by oxidative stress. This has a positive effect on the prevention and treatment of various oxidative stress-related diseases, such as cardiovascular diseases, neurodegenerative diseases, etc., and also has broad application prospects in the fields of food preservation and cosmetics anti-oxidation, which helps to extend the shelf life of products and protect the skin from oxidative damage.

[0030] (6) The indole derivatives provided by the present invention have potential application value in the treatment of lung cancer. They may inhibit the growth and proliferation of lung cancer cells or induce their apoptosis through a specific mechanism of action, providing new drug candidates or treatment strategies for the treatment of lung cancer. This is of great significance for improving the treatment effect and prognosis of lung cancer patients, and is expected to bring new breakthroughs and hopes for the treatment of lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings are included to provide a further understanding of the embodiments and are incorporated into and constitute a part of this specification. The accompanying drawings illustrate the embodiments and are used together with the description to explain the principles of the present invention. It will be easy to recognize other embodiments and many expected advantages of the embodiments because they become better understood by reference to the following detailed description. The elements of the drawings are not necessarily to scale with each other. The same reference numerals refer to corresponding similar parts.

[0032] Figure 1 is a flow chart of a method for preparing an indole derivative according to one embodiment of the present invention;

[0033] Figure 2 This is a graph showing the experimental results of YM818 on monophenolase according to one embodiment of the present invention;

[0034] Figure 3 is a line graph of the inhibitory effect of YM818 on diphenolase according to one embodiment of the present invention;

[0035] Figure 4 is a diagram showing the experimental results of the inhibition mechanism and type of mTYR by YM818 according to one embodiment of the present invention;

[0036] Figure 5 is an experimental result diagram of the effect of YM818 on mTYR fluorescence emission according to one embodiment of the present invention;

[0037] Figure 6 is a molecular docking simulation diagram of YM818 and mTYR according to one embodiment of the present invention;

[0038] Figure 7 This is a microphotograph of a Masson-Fontana-stained B16F10 cell experiment showing the inhibitory effect of YM818 on melanin production according to an embodiment of the present invention;

[0039] Figure 8 It is an apparent graph and a bar graph showing the inhibitory effect of YM818 on the melanin content of B16F10 cells according to one embodiment of the present invention;

[0040] Fig. 9is the effect of YM818 on key proteins in intracellular melanin synthesis according to one embodiment of the present invention;

[0041] Fig.10 is the effect of YM818 on cellular melanin production under AZD5363 treatment according to one embodiment of the present invention;

[0042] Fig.11 This is a stereoscopic observation image of the melanin content on the surface of zebrafish by YM818 according to one embodiment of the present invention;

[0043] Fig.12 is the effect of YM818 on the key protein of zebrafish melanin production according to one embodiment of the present invention;

[0044] Fig.13 This is an experimental image taken with a Zeiss LSM 780 confocal microscope of YM818 inhibiting melanin transfer in HaCAT cells according to an embodiment of the present invention;

[0045] Fig.14 The change of melanin transfer of HaCAT cells detected by flow cytometry of YM818 inhibition of melanin transfer of HaCAT cells according to one embodiment of the present invention;

[0046] Fig.15 This is the effect of YM818 on melanin transfer protein in a co-culture system according to an embodiment of the present invention;

[0047] Fig.16 This is a graph showing the results of an experiment on the scavenging ability of YM818 on ABTS and DPPH free radicals according to one embodiment of the present invention;

[0048] Fig.17 is the effect of YM818 on reactive oxygen species in B16F10 cells according to one embodiment of the present invention;

[0049] Fig.18 This is a graph showing the experimental results of YM818 inhibiting A549 cell viability and colony formation ability according to one embodiment of the present invention;

[0050] Fig.19 The experimental images of the effect of YM818 on the morphology of A549 cells taken by a TE2000-U phase contrast microscope according to one embodiment of the present invention;

[0051] Fig. 20 This is the experimental result of the effect of YM818 on the migration and invasion ability of A549 cells according to one embodiment of the present invention;

[0052] Fig.21The effect of YM818 on the A549 cell cycle according to one embodiment of the present invention;

[0053] Fig. 22 This is a diagram showing the experimental results of YM818 inducing apoptosis of A549 cells according to one embodiment of the present invention;

[0054] Fig.23 This is a diagram showing the experimental results of YM818 inducing mitochondrial dysfunction in A549 cells according to an embodiment of the present invention. DETAILED DESCRIPTION

[0055] The technical solutions in the embodiments will be described clearly and completely below in conjunction with the drawings in the present invention, and similar component numbers in the drawings represent similar components. Obviously, the embodiments described below are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0056] It should be understood that when used in this specification and the appended claims, the terms "include" and "comprises" indicate the presence of described features, integers, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or combinations thereof.

[0057] It should also be understood that the terms used in this specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. As used in the specification of the present invention and the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" are intended to include plural forms.

[0058] In the first aspect, the present invention provides a flow chart of a method for preparing an indole derivative, such as Figure 1 As shown, the method includes:

[0059] S101, preparing a reaction medium, dissolving copper acetate and triethylamine in isopropanol, and stirring until completely dissolved to obtain a reaction medium.

[0060] In some specific embodiments, 0.4 g, 2 mmol, of copper acetate and 0.2 g, 2 mmol, of triethylamine were weighed and placed in a reaction bottle, 10 ml of isopropanol was added to the reaction bottle, and stirred for 10 min to completely dissolve them, thereby obtaining a reaction medium.

[0061] S102, preparing an indole derivative solution, adding 5-bromoisatin and indole to the reaction medium, and stirring well at room temperature until the reactants are completely converted.

[0062] In some specific embodiments, 4.52 g, 20 mmol, of 5-bromoisatin and 2.8 g, 24 mmol, of indole were added to a reaction bottle, and the mixture was stirred sufficiently at room temperature to allow a post-coupling reaction to occur. The reaction progress was monitored by thin layer chromatography.

[0063] S103, extracting the indole derivative powder, distilling off the reaction solvent to obtain a crude product, purifying and recrystallizing to obtain a high-purity solid powder of the indole derivative.

[0064] In some specific embodiments, a rotary evaporator is used for reduced pressure distillation to remove the isopropanol solvent in the reaction system, and the residue is collected to obtain a crude product of the reactant. The crude product is added to a mixed solvent of petroleum ether and dichloromethane, stirred and dissolved, and then recrystallized to obtain a solid powder of the indole derivative.

[0065] In the second aspect, an embodiment of the present invention provides an indole derivative, which is a light brown solid powder prepared by the preparation method described in the first aspect, and its full Chinese name is 3-hydroxy-5-bromo-(3-indolyl)-2-carbonyl indole, and its chemical structure is:

[0066]

[0067] Example 1

[0068] Accurately weigh copper acetate (Cu(OAC)2·H2O, 0.4 g, 2 mmol, 10 mol%) and triethylamine (0.2 g, 2 mmol, 10 mol%) in a glass bottle, add 10 mL of isopropanol, stir for 10 min to dissolve the drug components, accurately weigh 5-bromoindigo red (4.52 g, 20 mmol) and indole (2.8 g, 24 mmol) and add them to the glass bottle, stir well at room temperature, and track the reaction by thin layer plate tracking reaction (TLC) until the reaction of the raw materials is completed. The solvent is rotated out by vacuum distillation, and the residue is purified to obtain a light brown solid powder, i.e., the crude product YM818. Subsequently, a mixed solvent of petroleum ether and dichloromethane is added for recrystallization, and finally 0.318 g of light brown solid powder is obtained, the purity of which is above 93%, i.e., the indole derivative YM818. The molecular weight of YM818 is 364.99 [M + Na] + , the actual calculated molecular weight is 343.90[M] + The results of H NMR spectrum data are: 1H NMR (500MHz, DMSO-d6) δ11.04 (s, 1H), 10.50 (s, 1H), 7.44 (d, J = 8.1 Hz, 1H), 7.41-7.28 (m, 3H), 7.12 (s, 1H), 7.05 (t, J = 7.5 Hz, 1H), 6.90 (dd, J = 11.2, 8.1 Hz, 2H), 6.52 (s, 1H). The results of the nuclear magnetic carbon spectrum data are: 13 C NMR (126MHz, DMSO-d6) δ177.90,140.93,136.80,135.85,131.72,127.33,124. 67,123.58,121.18,120.01,118.66,114.67,113.33,111.76,111.62,74.93cm -1 The chemical structure of YM818 is shown below.

[0069]

[0070] The indole derivative YM818 prepared in Example 1 was used as a research object, and mushroom tyrosinase (mTYR), mouse melanoma cells B16F10, zebrafish, human immortalized epidermal keratinocytes HaCAT and co-cultured cells of B16F10-HaCAT cells were used as models to systematically study the inhibitory mechanism of YM818 on tyrosinase activity, melanin production and melanin transfer. Through in vitro enzymatic experiments, experiments on the effect of YM818 on cell melanin production, zebrafish experiments, experiments on the effect of YM818 on cell melanin transfer, experiments on the antioxidant activity of YM818, and experiments on YM818 inducing mitochondria-mediated apoptosis in A549 cell lines, its mechanism as a tyrosinase inhibitor and antioxidant, as well as its application in inhibiting intracellular melanin production and / or transfer and treating lung cancer were comprehensively and systematically analyzed.

[0071] Application Example 1

[0072] In vitro enzymatic experiments included four experiments: the effect of YM818 on the monophenolase activity of mTYR, the effect of YM818 on the diphenolase activity of mTYR, the inhibition mechanism and type of YM818 on mTYR, and the interaction between YM818 and mTYR.

[0073] In the experiment of studying the inhibitory effect of YM818 on the activity of mTYR monophenolase, tyrosine was used as a substrate to determine the effect of YM818 on the activity of mTYR monophenolase. YM818 was dissolved in dimethyl sulfoxide (DMSO) solution to prepare a 30mmol / L mother solution for standby use, which was subsequently divided and diluted according to the experimental requirements. In the reaction system with a total volume of 3mL, 1.60mL of double distilled water, 750μL of 0.05mol / L phosphate buffer, 500μL of 1mg / ml tyrosine solution, 50μl of YM818 solution of different concentrations were added in sequence, and finally 100μL of 0.5mg / ml enzyme solution was added, mixed quickly, placed in a microplate reader, and the absorbance at 475nm was measured at a constant temperature of 37℃. After the reaction was completed, the curve graph simulated by GRAPHWIN software was obtained. The slope of the straight line was the steady-state enzyme activity, and the horizontal axis intercept was the reaction hysteresis time. The residual enzyme activity was plotted against the YM818 concentration, and the results were analyzed using the Statistical Package for Social Sciences (SPSS software) to obtain the IC value of YM818 for monophenolase. 50 value.

[0074] refer to Figure 2 , Figure 2 The experimental results of the inhibition of monophenolase by YM818 according to the present invention are shown in FIG. Figure 2 A represents the kinetic cycle diagram of the oxidation reaction of mTYR monophenolase catalyzed by YM818. Figure 2 1-6 in A correspond to YM818 concentrations of 0mmol / L, 0.1mmol / L, 0.2mmol / L, 0.3mmol / L, 0.4mmol / L, and 0.5mmol / L, respectively. Figure 2 B represents the steady-state tyrosinase activity diagram, Figure 2 C represents the hysteresis time diagram of the monophenolase catalytic reaction of YM818. Tyrosinase has monophenolase activity and catalyzes L-tyrosine (L-Tyr) to produce L-3,4-dihydroxyphenylalanine (L-DOPA) in melanin synthesis. Using L-Tyr as a substrate, the inhibitory effect of YM818 on the activity of mTYR monophenolase was determined. The results are shown in Figure 2 shown. Figure 2 A represents the kinetic curve of the oxidation reaction of mTYR monophenolase catalyzed by YM818. The product accumulation rate is slow at the beginning of the reaction, and the product accumulation increases rapidly after a certain period of reaction. As the concentration of YM818 increases, the activity of the steady-state enzyme gradually decreases (e.g. Figure 2 B). Figure 2C represents the hysteresis of the catalytic activity reaction time of monophenolase in the presence of YM818. As the concentration of YM818 increases, the hysteresis time of the catalytic reaction of monophenolase is prolonged. When the concentration of YM818 is 0.5mmol / L, the enzyme activity drops to 22.59% and the hysteresis time is 15.5min. The results show that YM818 has a significant inhibitory effect on monophenolase, and its mechanism of inhibiting the catalytic effect of the enzyme is related to prolonging the catalytic reaction hysteresis time of the enzyme and reducing the activity of monophenolase. The IC value of YM818 on monophenolase was calculated by SPSS software analysis. 50 The value is 0.261mmol / L.

[0075] In the experiment to study the inhibitory effect of YM818 on the activity of mTYR diphenolase, the effect of YM818 on the activity of mTYR diphenolase was determined using L-DOPA substrate. YM818 was dissolved in DMSO solution to prepare a 30mmol / L mother solution for standby use, and then diluted into solutions of different concentrations according to experimental needs. In a reaction system with a total volume of 3mL, 1.80mL of double distilled water, 750μL of 0.05mmol / L phosphate buffer, 300μL of 1mg / ml L-DOPA solution, 50μl of YM818 solution of different concentrations were added in sequence, and finally 100μL of 0.5mg / ml enzyme solution was added, mixed quickly, placed in an enzyme reader and the absorbance at 475nm was measured at a constant temperature of 37°C. The slope of the straight line measured at the end of the reaction is the steady-state enzyme activity. The relative residual enzyme activity corresponding to different YM818 concentrations was plotted, and the IC value of YM818 on diphenolase was obtained by analysis using SPSS software. 50 value.

[0076] Experimental results reference Figure 3 , Figure 3 The line graph shows the inhibitory effect of YM818 on diphenolase according to the present invention. Tyrosinase has diphenolase activity. Under the action of the enzyme, L-DOPA is oxidized to generate dopaquinone, and melanin is generated through a series of reactions. Figure 3 It can be seen that with the increase of YM818 concentration, the enzyme activity gradually decreased and showed a certain concentration-dependent effect. When the YM818 concentration was 0.5mmol / L, the enzyme activity dropped to 35.50%. The IC value of YM818 for inhibiting diphenolase activity was calculated by SPSS software. 50 It is 0.376mmol / L.

[0077] Further references Figure 4 , Figure 4The results of the mTYR inhibition mechanism and inhibition type experiment according to the present invention are shown. In the 3 mL activity test system, the mechanism of YM818 inhibiting the activity of mTYR diphenolase is further studied. The concentration of the substrate L-DOPA is fixed and unchanged, and the concentrations of the enzyme and YM818 are changed to measure the activity of mTYR diphenolase. The relative residual activity of the enzyme is plotted ( Figure 4 A), five straight lines passing through the origin were obtained. It can be seen that the slope of this group of straight lines decreases with the increase of YM818 concentration, indicating that the inhibitory effect of YM818 on mTYR activity is reversible. YM818 reduces the rate at which mTYR diphenolase catalyzes the substrate, but does not permanently denature and inactivate the enzyme. In the 3mL activity test system, the concentration of mTYR was fixed and unchanged, and the concentration of L-DOPA substrate and YM818 were changed to further study the type of inhibition of YM818 on mTYR diphenolase activity. The results are shown in Figure 4 As shown in B, five straight lines intersecting in the second quadrant were obtained by plotting the double reciprocal 1 / v against 1 / [S]. The results showed that YM818 could affect the maximum reaction rate (V m ), can also affect the Michaelis constant (K m ). Under the action of YM818, V m Reduce K m The value increases, indicating a mixed inhibition type. The slope and ordinate intercept are plotted against the concentration of YM818 again, and two straight lines are obtained ( Figure 4 C. Figure 4 D), calculate the inhibition constant (K I )=0.155mmol / L, the inhibition constant (K IS )=0.976mmol / L. K IS The value is much larger than K I The value of indicates that the affinity of YM818 to the tyrosinase-substrate complex is greater than that to free tyrosinase.

[0078] In the experiment to study the interaction between YM818 and mTYR, endogenous fluorescence quenching was used to measure the endogenous fluorescence intensity of mTYR under the action of different concentrations of YM818 using a Varian CaryEclipse fluorescence spectrophotometer. The parameters were set to an excitation light wavelength of 280nm, an emission light slit width of 10nm, and an excitation light slit width of 5nm, and the change in tyrosinase fluorescence intensity was detected in the range of 320-400nm. Before the experiment, the instrument was zeroed with 2mL of double distilled water, and a 0.2mg / mL tyrosinase solution was prepared with double distilled water. First, 2mL of the enzyme solution was placed in a cuvette with four sides transparent to light, and a 50mmol / L YM818 solution was prepared. 2μL of YM818 was added to the cuvette each time, and the solution in the cuvette was mixed and allowed to react for one minute, and then the fluorescence intensity of the solution was scanned and measured. The quenching rate of the compound and the fluorescent substance follows the Stern-Volomer curve equation: F0 / F=1+K SV [Q], where F0: endogenous fluorescence intensity without quencher, F: endogenous fluorescence intensity after quencher, [Q]: quencher concentration, KSV: quenching reaction constant. The Stern-Volmer curve of YM818 on mTYR fluorescence intensity is drawn according to the quenching reaction results to determine the type of fluorescence quenching mechanism. If the quenching constant K SV More than 100M -1 The equilibrium constant (K) of the reaction can be obtained by the Scatchard equation. A ) and binding constant (n): lg[(F0-F) / F] = lgK A +nlg[Q].

[0079] The Molecular Operating Environment software (MOE (2020) software) can be used to simulate the interaction mechanism between YM818 and mTYR and further study the interaction between YM818 and tyrosinase. The mTYR protein structure (PDB: 2Y9X) was downloaded from the Protein DataBank database (PDB database), and the MOE software was used to remove protein crystal water, original ligands, etc. The chemical structure of YM818 was drawn using ChemDraw software and energy was minimized. The area close to the copper ion active center was selected for simulated docking, highlighting the binding mode of YM818 and mTYR and the interacting amino acid residues, and the results were analyzed by the simulated docking diagram.

[0080] The experimental results are as follows Figure 5 and Figure 6 As shown, Figure 5 and Figure 6The experimental results of the effect of YM818 on the fluorescence emission of mTYR according to the present invention and the molecular docking simulation diagram are respectively shown. Tyrosinase contains tryptophan aromatic groups, which can be excited and emit endogenous fluorescence. The binding of the compound and the enzyme causes the endogenous fluorescence to be quenched, which can be used to explore the relationship between the interaction between the compound and the enzyme. Figure 5 A represents the fluorescence spectrum curve, Figure 5 Curves 1-8 in A represent YM818 concentrations of 0mmol / L, 0.05mmol / L, 0.1mmol / L, 0.15mmol / L, 0.2mmol / L, 0.25mmol / L, 0.3mmol / L, and 0.35mmol / L, and curve 9 represents the absorption peak of YM818; Figure 5 B represents the mTYR fluorescence quenching effect; Figure 5 C represents the Stern-Volmer curve, which is Figure 5 A and Figure 5 B shows that with the increase of YM818 concentration, the mTYR fluorescence intensity shows a regular peak weakening and a slight red shift phenomenon occurs, indicating that YM818 and mTYR have a coordinated effect and new substances are generated in the reaction. The Stern-Volmer curve showing a linear relationship is obtained by plotting F0 / F against the concentration of YM818 [I] ( Figure 5 C), Stern-Volmer equation: y = 12.33x + 1, R 2 =0.9683, K SV The value is 1.233×10 4 M -1 Because K SV More than 100M -1 , so the quenching type of YM818 on mTYR is static quenching. Plot lg[(F0-F) / F] against lg[I], the result is shown in Figure 5 D, fitting Scatchard equation is y = 1.247x-1.512, R 2 =0.9641. According to the equation, the equilibrium constant (K A ) is 3.27×105M -1 , the binding constant (n) was 1.247, indicating that YM818 could form a complex with an mTYR.

[0081] Figure 6 The results of the simulated docking of YM818 and mTYR. Figure 6 A represents the three-dimensional diagram of the interaction between YM818 and mTYR. Figure 6 B represents the interaction plane, Figure 6 C represents the interaction surface form diagram, Figure 6D represents a local 3D graph. It can be seen from the figure that YM818 has a good binding effect with TYR, and the binding energy of the interaction is -5.344 kcal / mol. Although YM818 cannot directly interact with the copper ion in the active center of mTYR, it can affect the activity of tyrosinase by forming hydrogen bonds and hydrophobic forces. The carbonyl and amino groups in YM818 can form hydrogen bonds with the amino acid residues Gln307 and Asp312 of the side chain donor, respectively, and the hydrogen bond lengths are YM818 can also interact hydrophobically with Lys376, Tyr311, Glu356, Asp357, Thr308 and Lys379 amino acid residues on the side chain of the active center, indicating that hydrogen bonds are formed between YM818 and mTYR, and hydrophobic forces are generated. The interaction between the two affects the structure of mTYR, thereby inhibiting the activity of the enzyme.

[0082] Application Example 2

[0083] The experiments on the effect of YM818 on cellular melanin production include the effect of YM818 on the melanin content and synthesis of B16F10 cells, the protein blotting (Western Blot) experiment to detect the expression level of key proteins in melanin production, and the effect of YM818 on melanin synthesis under the treatment of AKT inhibitor AZD5363.

[0084] In the study of the effect of YM818 on melanin synthesis in B16F10 cells, the Masson-Fontana melanin staining kit (Solarbio) was used for cytochemical staining observation. B16F10 cells were cultured in a six-well plate with a 24mm diameter sterile cell slide, and 30μmol / L and 60μmol / L YM818 were used to act on the cells for 48h, respectively, and the positive control was 60μmol / L arbutin. After the drug treatment, the old culture medium was removed and PBS (Phosphate Buffered Saline) was added to wash the cells three times. Add 4% polymethanol solution to fix the cells for 20min, wash with double distilled water, add the Fontana ammonia silver solution in the kit, and place in a 56°C incubator to react for 30min under light-proof conditions. Immerse in double distilled water and wash several times, add Hypo solution for 5 minutes, rinse with double distilled water for 3 minutes, add neutral red dye for re-staining for 5 minutes, wash with double distilled water, and finally add 95% ethanol and anhydrous ethanol for dehydration reaction. Use neutral gum to seal the cell slides and store them in a light-proof environment. Use a Leica upright fluorescence microscope to observe and take pictures.

[0085] Masson-Fontana melanin dye can reduce silver ammonia solution to generate metallic silver through chemical reaction, and the melanin particles appear obvious black after being stained. B16F10 cells were treated with 0μmol / L, 30μmol / L and 60μmol / L YM818 respectively, and stained after 48h. The coloration of melanin in B16F10 cells was observed and images were taken using an upright fluorescence microscope to obtain Figure 7 The micrographs of the B16F10 cell experiments shown in Figure 1. Figure 7 It can be seen that the number of melanin granules in the control group was larger and the staining was darker. After drug treatment, the coloring of the melanin granules in the cells became significantly lighter, and the inhibitory effect of the 60μmol / L YM818 treatment group was better, which was better than the same concentration (60μmol / L) of arbutin.

[0086] The cell density of cultured B16F10 cells was adjusted to 10 5 CFU / mL, add medium containing 0μmol / L, 30μmol / L, 40μmol / L, 50μmol / L, 60μmol / L of different concentrations of YM818, place in an incubator for 48h, wash with PBS, digest the cells and transfer to a 1.5mL centrifuge tube, add PBS to resuspend and wash. Centrifuge for 3min at 12000r / min and 4℃, then discard the supernatant, add 150μL of 1mol / L NaOH solution to the centrifuge tube to lyse the cell pellet, mix well and place in a dry and wet thermostat for 95℃ for 0.5h. After the solution is cooled to room temperature, transfer to a 96-well plate, and use an ELISA reader to detect the absorbance at 405nm. Set three replicates for each drug concentration. Use a standard melanin sample to determine the standard curve of melanin concentration, calculate the cell melanin content, and plot the relative melanin content against the YM818 concentration.

[0087] refer to Figure 8 , Figure 8 The graph and bar chart show the inhibitory effect of YM818 according to the present invention on melanin production in B16F10 cells. Figure 8 A represents the apparent image of B16F10 cell melanin lysate and melanin precipitation. Figure 8 B represents a bar graph of melanin content. B16F10 cells were treated with YM818 at concentrations of 0 μmol / L, 30 μmol / L, 40 μmol / L, 50 μmol / L and 60 μmol / L for 48 hours and then the changes in intracellular melanin content were detected. The results are shown in Figure 2. Figure 8 As shown in the melanin cleavage diagram ( Figure 8A) It can be observed that after YM818 treatment, the melanin content of B16F10 cells was significantly reduced, and the color of melanin became lighter. Figure 8 As shown in Figure B, it can be seen that under the action of YM818 at concentrations of 30μmol / L, 40μmol / L, 50μmol / L, and 60μmol / L, the intracellular melanin content was significantly reduced compared with the control group, and showed a concentration-dependent effect. In the 60μmol / L YM818 treatment group, the cellular melanin content decreased to 35.8% compared with the control group, indicating that YM818 can significantly inhibit the melanin production of B16F10 cells.

[0088] Continue to refer Fig. 9 , Fig. 9 The effect of YM818 according to the present invention on the key proteins of intracellular melanin synthesis is shown. Fig. 9 A represents the target protein band. Fig. 9 B. Fig. 9 C and Fig. 9 D represents the expression levels of TYR, TRP2, and MITF proteins, respectively. The changes in the expression levels of TYR, TRP2 (tyrosinase-related protein 2), and MITF (microphthalmia-related transcription factor) proteins in melanogenesis of B16F10 cells were determined by Western blot method after treatment with YM818 at different concentrations of 30 μmol / L, 40 μmol / L, 50 μmol / L, and 60 μmol / L for 48 h. Fig. 9 As shown in the figure, with the increase of YM818 concentration, TYR, TRP2, and MITF all showed a decreasing trend, among which TYR and MITF showed a concentration effect. At 50μmol / L and 60μmol / L concentrations, the inhibitory effects of TYR and TRP2 protein expression were more significant than those of the control group (P<0.01). There was no significant difference in TRP2 under the action of 30μmol / L YM818, and the expression of MITF protein changed significantly compared with the control group. The results showed that YM818 had an inhibitory effect on the expression of TYR, MITF, and TRP2, key proteins for melanogenesis in B16F10 cells.

[0089] refer to Fig.10 , Fig.10 The effect of YM818 on cell melanin production under AZD5363 treatment according to the present invention is shown. AZD5363 is an inhibitor of protein kinase B (AKT), which can inhibit AKT phosphorylation, and AKT phosphorylation can promote cell apoptosis. The expression changes of phosphorylated AKT protein (p-AKT protein) in B16F10 cells were examined by Western blot method. Fig.10 A represents the expression of p-AKT protein in B16F10 cells. Fig.10 B represents the melanin content and the appearance of the lysate. B16F10 cells were pretreated with 10 μmol / L AKT inhibitor AZD5363 for 1 hour, and then added with 60 μmol / L YM818 medium for 48 hours. Fig.10 A is the change in p-AKT protein expression after drug treatment. The 0.5 μmol / L AZD5363 treatment group significantly reduced the expression level of p-AKT protein compared with the control group. The experimental group treated with 0.5 μmol / L AZD5363 and 60 μmol / L YM818 simultaneously increased the expression level of p-AKT compared with the control group, and the upregulation level of the group treated with 60 μmol / L YM818 alone was higher than that of the group treated with both AZD5363 and YM818. Fig.10 B is the apparent graph of cellular melanin content and production, which shows that the melanin content in the inhibitor-treated group increased significantly, and the melanin production in the Y818-treated group was significantly reduced compared with the control group, the inhibitor group, and the inhibitor and YM818 co-treated group. It can be seen that YM818 can significantly inhibit the production of melanin in B16F10 cells, downregulate the expression of MITF protein by regulating and activating the AKT signaling pathway, and thus inhibit the production of B16F10 melanin.

[0090] Application Example 3

[0091] Reference in the experiment of observing the apparent content of zebrafish melanin under stereomicroscope Fig.11 , Fig.11 Figure 2 shows the stereoscopic observation images of the melanin content on the surface of zebrafish according to YM818. After zebrafish embryos were treated with YM818 solutions of different concentrations of 0μmol / L, 200μmol / L, 300μmol / L, 400μmol / L and 500μmol / L for 48h, the melanin deposition on the surface of the fish was photographed and observed under a stereoscope. Fig.11 It can be seen that a large amount of melanin is produced in the yolk sac, eyes and sides of the spine of the control group zebrafish. At the same time, the melanin granules are large in area and dark in color. Compared with the control group, the melanin granules in the yolk sac and tail of the zebrafish in the YM818 treatment group are significantly reduced, and the melanin color becomes lighter, indicating that YM818 has an inhibitory effect on the deposition of melanin on the surface of zebrafish.

[0092] Western blot was used to detect the expression changes of proteins related to melanin production in zebrafish. β-actin protein was used as an internal reference to study the effects of 0 μmol / L, 200 μmol / L, 300 μmol / L, 400 μmol / L and 500 μmol / L YM818 solution on the expression levels of TYR, TRP1, TRP2 and α-MSH in zebrafish after 48 hours. Fig.12 As shown, Fig.12 A is a representative image of the target protein band; Fig.12 B. Fig.12 C. Fig.12 D. Fig.12 E are relative protein expression levels. Under the action of YM818, the expression levels of TYR, TRP1 and α-MSH proteins were significantly downregulated compared with the control group, and showed a concentration effect, while the expression level of TRP2 was not affected.

[0093] Application Example 4

[0094] The experiments on the effect of YM818 on cellular melanin transfer included fluorescent microsphere phagocytosis experiment to detect the effect of YM818 on HaCAT cell melanin transfer, flow cytometry to detect the effect of YM818 on melanin transfer in co-cultured cell system, and Western Blot to detect the effect of YM818 on key proteins of melanin transfer.

[0095] In the experiment of observing the effect of YM818 on the phagocytosis of HaCAT cells under confocal microscope, the density of HaCAT cells was adjusted to 10 5 CFU / mL, transfer the cultured HaCAT cells to a 12-well plate containing a sterile slide, and place it in a cell culture incubator for 12 hours. When the cells are attached to the wall and the growth state is good and the growth density is appropriate, remove the original culture medium and continue to culture for 6 hours with YM818 (40, 60 μmol / L) or arbutin (100 μmol / L) containing 10 μmol / L Ser-Leu-Ile-Gly-Arg-Leu-NH2 (referred to as Sligrl). Remove the original culture medium and add the culture solution containing FluoSpheresTM carboxyl-modified microspheres (Thermo Fisher Scientific) (diameter 1 μm) at a concentration ratio of 100 / cell for co-culture for 16 hours. Wash three times with PBS, remove excess microspheres, and add 4% paraformaldehyde to fix for 15 minutes in a dark environment. Add DAPI staining solution on the cell area, turn the slide upside down and cover it on a glass slide to stain the cells. Be careful to avoid bubbles that affect image observation. Fix the slide with neutral gum, use a Zeiss LSM 780 confocal microscope to observe and shoot images, and use ZEN2.3.0 software to process and analyze images.

[0096] Fig.13 The experimental images taken with a Zeiss LSM 780 confocal microscope show that YM818 according to the present invention inhibits melanin transfer in HaCAT cells. Fig.13 A indicates the confocal magnification is 200X. Fig.13B indicates that the confocal magnification is 400X. Protease-activated receptor-2 (PAR-2) is also called protease-activated peptide. Sligrl-NH2 (Ser-Leu-Ile-Gly-Arg-Leu-NH2) is referred to as Sligrl. It has the effect of activating PAR-2 ​​and promoting melanin transfer. FluoSpheresTM carboxyl-modified microspheres are fluorescent microspheres with a diameter of 1 μm. In the phagocytosis experiment, the microspheres and HaCAT cells were co-cultured at a ratio of 100:1 to simulate the process of cell transfer of melanin particles. The distribution and density of the fluorescent microspheres in HaCAT cells were observed and scanned using a confocal microscope. Fig.13 As shown, the addition of 10 μmol / L sligrl stimulated HaCAT to transfer melanin, and the number of fluorescent microspheres increased significantly compared with the control group. In the 40 μmol / L and 60 μmol / L YM818 treatment groups, the number of fluorescent microspheres was less, showing a concentration effect, and the 60 μmol / L YM818 treatment group was better than the positive control 100 μmol / L arbutin treatment group. Therefore, the migration effect of HaCAT cells was significantly inhibited under the action of YM818.

[0097] In the experiment of detecting the effect of YM818 on melanin transfer in the co-cultured cell system by flow cytometry:

[0098] (1) Construction of control group system: The control group is divided into single culture cells and co-culture cells. The single culture cell system is B16F10 cells stained with CFDA fluorescent labeling and HaCAT cells stained with incubated antibodies; the co-culture cells are single-stained HaCAT cells, single-stained B16F10 cells and cells without staining. B16F10 was cultured in two 6 cm culture dishes. When the cells were in good growth state and the growth density was appropriate, PBS buffer containing 2 mmol / L CFDA SE was added to one of the dishes and stained at 37°C in the dark for 40 minutes. The cells in the other dish were not labeled, and then the cells were transferred to a six-well plate for culture. HaCAT cells were also transferred to a six-well plate to construct the single culture or co-culture system of the control group. After the cells were cultured for 48 hours, the original DMEM medium was removed and washed three times with PBS solution. Trypsin was added to digest the cells for 2 minutes. The B16F10 cells in the single culture and co-culture cell systems were collected and transferred into centrifuge tubes. HaCAT cells in single culture and co-culture systems need to continue digestion for 8 minutes, collect cells and transfer to centrifuge tubes, and centrifuge at 300g for 3 minutes. The supernatant was removed by aspiration, and 500μL of 4% polymethanol fixative was added to the precipitate and fixed at 4°C for 20 minutes. The cells were incubated with 200μL of primary antibody (Pan-Keratin) at 4°C for 30 minutes, followed by incubation with 200μL of secondary antibody CoraLite594-conjugated GoatAnti-Mouse IgG (H+L) at 4°C for 30 minutes. The B16F10 cells digested and collected in the co-culture group were added to the treated HaCAT cells. The Fortessa (BD, USA) flow cytometer was used for on-machine detection and analysis.

[0099] (2) Experimental group reaction steps: B16F10 cells were stained with PBS containing 2mmol / L CFDA SE at 37℃ in the dark for 40min, and co-cultured for 12h at a ratio of B16F10 cells:HaCAT cells of 1:3. The cells were observed to be attached to the wall and growing well, and treated with YM818 or arbutin for 48h, rinsed three times with PBS, digested with trypsin for 2min, and rinsed three times with PBS to remove B16F10 cells in the system. The remaining HaCAT cells were incubated with antibodies and then detected and analyzed by flow cytometry. The number of double positive signals detected in HaCAT cells in the co-culture system is the number of B16F10 cell metastasis. If the number of CFDA SE positive cells in HaCAT cells decreases, it indicates that the drug has an inhibitory effect on the metastasis of B16F10 cells.

[0100] refer to Fig.14 , Fig.14The flow cytometry method for detecting the changes of melanin transfer of HaCAT cells by YM818 according to the present invention is shown. Fig.14 A is the negative control group, Fig.14 B is the experimental group, Fig.14 C is the level of melanin transferred by HaCAT cells. CFDA SE cell tracking dye can fluorescently label living cells. After entering the cells, it can be catalyzed by esterase to produce CFSE that emits strong green fluorescence. The fluorescence in the labeled cells is stable and uniform, and the adjacent cells are not stained. In the experiment, B16F10 was stained with CFDA SE, and the keratin in HaCAT cells was labeled with Pan-Keratin antibody. Then, the secondary antibody CoraLite594-conjugated Goat Anti-Mouse IgG (H+L) was used for incubation. After the incubation, the cells were stained with the coupled primary antibody to emit fluorescence. Fig.14 As shown, ae is the staining result of the control group, which respectively indicates the staining of B16F10 cells cultured alone, the staining of HaCAT cells cultured alone, the single staining of B16F10 or HaCAT in co-cultured cells, and the unstained co-cultured cells. The results show that the single cell staining effect is better, and the staining of co-cultured cells does not interfere with each other. fj is the result of the experimental group, which is the control group, 10μmol / L sligrl, and the YM818 (40 and 60μmol / L) and arbutin (100μmol / L) treatment groups containing 10μmol / L sligrl. In the experimental group, the proportion of HaCAT cells containing double positive signals in the control group was 13.5%, which increased to 18.6% after adding sligrl stimulation. The proportions of the 40 and 60μmol / L YM818 treatment groups dropped to 10.8% and 8.5%, respectively, and the proportion of double positive cells in the 100μmol / L arbutin treatment group dropped to 14.1%. The above results indicate that YM818 can significantly inhibit cell melanin transfer, and its effect is better than arbutin.

[0101] Continue to refer Fig.15 , Fig.15 The effect of YM818 of the present invention on melanin transfer protein in the co-culture system is shown. Fig.15 A is a representative image of the target protein band; Fig.15 B is the relative protein expression level. The expression of PAR-2 ​​protein in B16F10-HaCAT co-culture system cells after 48h treatment with different concentrations of YM818: 30 μmol / L, 40 μmol / L, 50 μmol / L and 60 μmol / L Fig.14As shown in the figure, compared with the control group, the expression of PAR-2 ​​protein in the four concentration treatment groups was significantly downregulated, showing a concentration effect. The results showed that YM818 could inhibit the effect of promoting black metastasis by reducing the expression of PAR-2 ​​protein in cells.

[0102] Application Example 5

[0103] The indole derivative YM818 prepared in Example 1 is used as an antioxidant. The antioxidant effect of YM818 includes an in vitro antioxidant test of YM818 and a flow cytometry test to detect the effect of YM818 on the intracellular ROS level.

[0104] In the in vitro antioxidant experiment, this study used the 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid free radical scavenging experiment (ABTS method) and the 1,1-diphenyl-2-trinitrophenylhydrazine free radical scavenging experiment (DPPH method) to detect the free radical scavenging ability of YM818. YM818 was prepared into 0mmol / L, 0.5mmol / L, 1.0mmol / L, 1.5mmol / L, 2.0mmol / L, and 2.5mmol / L solutions with different concentrations using DMSO solvent for standby use, and vitamin C was selected as the positive control sample. The method steps are as follows:

[0105] ABTS experiment: weigh 0.0768g ABTS and dissolve it in 20mL double distilled water, weigh 0.0134g potassium persulfate and dissolve it in 20mL double distilled water, mix the two solutions in equal proportions, place them at room temperature in the dark and let them react for 16 hours, use them as ABTS storage solution for standby, dilute them with PBS solution to ABTS working solution before the experiment, and measure the absorbance at 734nm to be 0.70±0.02, and store the solution in a light-proof environment. Take a 96-well plate, add 180μL ABTS solution and 20μL YM818 solution of different concentrations to each well, react at room temperature in the dark for 6min, put them in an ELISA reader and measure the absorbance of the reaction solution at 734nm. Double distilled water was used as the control group in the experiment, and three repeated experiments were set for each sample.

[0106] DPPH experiment: Use a balance to accurately weigh 14 mg of DPPH solid powder, dissolve it with 95% methanol and make it constant to 200 mL, and store the solution in a 4°C refrigerator in the dark as DPPH storage solution. The solution is prepared and used immediately. The experiment first uses methanol solution to adjust the concentration of the DPPH working stock solution so that its absorbance at 517 nm is between 0.7 and 0.8. After adjustment, add 1.5 mL of DPPH solution and 50 μL of YM818 of different concentrations to the centrifuge tube, mix well, and stand in the dark for 30 minutes. Use an enzyme marker to measure the absorbance of the reaction solution at 517 nm. The control group uses methanol solution, and three repeated experiments are set for each sample.

[0107] According to the absorbance of the reaction solution, the scavenging rate of YM818 on free radicals can be calculated, and the scavenging rate curve corresponding to the YM818 concentration can be drawn. The results are calculated based on different concentrations of YM818 in units of mmol / L. The antioxidant capacity of YM818 is calculated as follows: Antioxidant capacity (%) = (1-A1 / A0) × 100% In the formula, A0 is the absorbance value measured by the control group solution; A1 is the absorbance value measured by the drug treatment group solution.

[0108] Experimental results reference Fig.16 , Fig.16 The figure shows the experimental results of the ability of YM818 of the present invention to scavenge ABTS and DPPH free radicals. Fig.16 A represents the scavenging rate of YM818 on ABTS free radicals. Fig.16 B represents the scavenging rate of DPPH free radicals by YM818. The catalytic reaction of tyrosinase involves a variety of oxidation reactions. In the enzyme catalysis, most TYR inhibitors also have antioxidant effects. The chemical structure of YM818 contains carbonyl functional groups, nitrogen atom heterocycles and hydroxyl groups, which can serve as hydrogen donors for oxygen, thus having the effect of scavenging free radicals. The antioxidant capacity of YM818 was analyzed by ABTS and DPPH experiments. The results are as follows: Fig.16 As shown in the figure, YM818 has a significant antioxidant effect and can effectively remove ABTS ( Fig.16 A) and DPPH( Fig.16 B) Free radicals, IC 50 The values ​​were 0.67 and 1.69 mmol / L, respectively, which are comparable to vitamin C (0.59 and 1.93 mmol / L, respectively). The antioxidant capacity of YM818 and vitamin C is summarized in Table 1. In summary, YM818 has a strong antioxidant effect.

[0109] Table 1 Antioxidant effects of YM818 and vitamin C

[0110]

[0111] In the experiment of detecting the effect of YM818 on the level of ROS in B16F10 cells by flow cytometry, B16F10 cells were cultured and the density was adjusted to 10 5CFU / mL, transfer the cells to a six-well plate, culture for 12 hours, remove the original culture medium after the cells adhere to the wall, add culture medium containing YM818 concentrations of 0μmol / L, 15μmol / L, 30μmol / L, 45μmol / L and 60μmol / L, respectively, and culture in a cell culture incubator for 48 hours. Digest the B16F10 cells and transfer them to a centrifuge tube, centrifuge at 2000rpm for 3 minutes, discard the supernatant, resuspend the cells with serum-free DMEM culture medium and wash three times. Use the active oxygen detection kit (Beyotime) to load the fluorescent probe DCFH-DA, the specific operation is as follows: dilute DCFH-DA to 10μmol / L at a ratio of 1:1000 with serum-free cell culture medium, collect the cells and transfer them to the diluted culture medium, place them at 37°C for 20 minutes, and invert the centrifuge tube every 5 minutes to fully mix and contact the probe and cells. After the reaction, the cells were centrifuged and the original culture medium was removed. The cells were resuspended and washed three times with serum-free culture medium to completely remove the DCFH-DA that had not entered the cells. The culture medium was then filtered with a 300-mesh filter and transferred to a flow tube (BD Company). The flow cytometer FC500 was used for on-machine detection, and the detection results were analyzed with WinMDI2.9 software.

[0112] Experimental results reference Fig.17 , Fig.17 The effect of YM818 on reactive oxygen species in B16F10 cells is shown, wherein: Fig.17 a- Fig.17e represents YM818 concentrations of 0 μmol / L, 15 μmol / L, 30 μmol / L, 45 μmol / L and 60 μmol / L, respectively. Under the action of reactive oxygen species in cells, the fluorescent probe DCFH-DA penetrates the cell membrane and is hydrolyzed to produce DCF, which can be oxidized to produce a fluorescent substance DCF. The ROS content in cells can be quantitatively determined by fluorescence detection using a flow cytometer. From the above analysis of the antioxidant capacity of YM818, it can be seen that YM818 has a high scavenging efficiency for free radicals. The experiment loaded the fluorescent probe and used a flow cytometer to detect the changes in the fluorescence signal in B16F10 cells. The fluorescence intensity is proportional to the level of ROS in the cells. After 48h of treatment of B16F10 cells with YM818 at different concentrations of 0μmol / L, 15μmol / L, 30μmol / L, 45μmol / L and 60μmol / L, the intracellular ROS level was detected. Compared with the control group, the intracellular ROS level of the YM818-treated group was reduced, and it showed a decreasing trend with the increase of YM818 concentration. In the 60μmol / L YM818-treated group, the ROS level was 26.4, which was 60% lower than the control (66.0). The results show that YM818 has strong antioxidant properties in cells and can significantly reduce the ROS level in B16F10 cells.

[0113] The results of enzymatic experiments showed that YM818 had an inhibitory effect on monophenolase IC 50 The value is 0.261mmol / L, and the IC 50 The value was 0.372mmol / L, and the inhibition of diphenolase was a reversible mixed inhibition type. The results of fluorescence quenching and MOE molecular docking showed that YM818 inhibited the activity of the enzyme through hydrogen bonds and hydrophobic forces. In terms of melanin production, YM818 can significantly reduce the melanin content and TYR activity of B16F10 cells, downregulate the expression of MITF by activating the AKT pathway, and further downregulate the expression of TYR family members, ultimately inhibiting the production of melanin. The results of zebrafish experiments showed that YM818 had an inhibitory effect on its melanin content and TYR activity, and inhibited the production of melanin by downregulating the expression of zebrafish TYR, TRP1 and α-MSH proteins. In terms of melanin transfer, YM818 had a significant inhibitory effect on the amount of melanin transfer and downregulated the expression of PAR-2, a key protein for melanin transfer. The results of antioxidant experiments showed that YM818 had a certain antioxidant capacity and could significantly reduce the level of intracellular ROS.

[0114] Application Example 6

[0115] The indole derivative YM818 prepared in Example 1 is used in the treatment of lung cancer. The experiment of YM818 inducing mitochondria-mediated apoptosis in A549 cell line includes experiments on YM818 inhibiting A549 cell viability and colony formation ability, the effect of YM818 on A549 cell morphology, the effect on cell migration and invasion ability, cell cycle, cell migration and mitochondrial dysfunction.

[0116] In the experiment of studying the ability of YM818 to inhibit the viability and colony formation of A549 cells, in order to test the effect of YM818 on the viability of human non-small cell lung cancer cell line A549, A549 cells were treated with different concentrations of YM818 for 24h and 48h, and the cell viability was detected by CCK-8 method. Fig.18 ,like Fig.18 As shown in A, YM818 inhibited the growth of A549 cells in a dose-dependent and concentration-dependent manner. After 24h and 48h of YM818 treatment, IC 50 The values ​​were 128.1μM and 101.0μM, respectively. After 48h of YM818 treatment, the number and diameter of A549 cell colonies decreased significantly (e.g. Fig.18 B).

[0117] In the experiment to study the effect of YM818 on the morphology of A549 cells, the cells were plated at 5×10 5 The cells were plated with DMSO and YM818 for 24 hours on the next day. A 10uL pipette tip was used to artificially scratch a cell wound in each well of the six-well plate, and then washed with PBS and cultured for another 24 hours. Finally, the attached cells were stained with crystal violet, and the artificially scratched cell wound was photographed with a phase contrast microscope (TE2000-U, Nikon, Tokyo, Japan, equipped with NIS-Element software). The photographic results refer to Fig.19 , Fig.19 The experimental images taken with a TE2000-U phase contrast microscope show the effect of YM818 on the morphology of A549 cells. Fig.19 A shows the effect of YM818 treatment on the morphology of A549 cells under an optical microscope. Fig.19 B shows the effect of YM818 treatment on the morphology of A549 cells under Giemsa staining. After 48 hours of treatment with different concentrations of YM818, the morphology of A549 cells changed significantly. Fig.19 As shown, the cells in the control group were spindle-shaped, plump and adherent, and the number of cells decreased significantly after drug treatment, the cells became curved and wrinkled, and a large number of suspended cells appeared in the high-concentration group.

[0118] In the experiment to study the effect of YM818 on the migration and invasion ability of A549 cells, a cell migration experiment was used to detect the invasion ability of the A549 cell line. After 48 hours, the cells on the top surface of the bottom of the upper chamber were wiped off with a cotton swab, and the cells that had migrated or invaded to the other side of the upper chamber were then fixed with paraformaldehyde for 30 minutes, then stained with 0.5% crystal violet for 15 minutes, and then washed three times with PBS and dried. Finally, using a phase contrast microscope, 5 areas were randomly selected to take pictures and count the cells that successfully migrated or invaded in the area. Experimental results reference Fig. 20 , Fig. 20 A represents the effect of YM818 on the migration ability of A549 cells. Fig. 20 B shows the effect of YM818 on the invasion ability of A549 cells. Fig. 20 C shows the effect of YM818 on the protein levels of β-catenin and MMP-2 in A549 cells. After the cells were scratched, compared with the cells in the control group, which gradually migrated to the middle area, almost no cells in the high-concentration drug treatment group migrated to the middle area, indicating that YM818 inhibited the migration ability of A549 cells (refer to Fig. 20 A). In the cell migration assay, as the concentration of YM818 increased, fewer and fewer cells were able to permeate through the bottom membrane (polycarbonate membrane) to reach the lower chamber, indicating that YM818 significantly inhibited the invasive ability of A549 cells (reference Fig. 20 B). Western blot results clearly showed that the downregulation of β-catenin and MMP-2 proteins was dose-dependent (ref. Fig. 20 C).

[0119] In the experiment to study the effect of YM818 on the A549 cell cycle, after YM818 treatment of A549 cells for 24 hours, the cells were collected and washed. The cells were fixed with 75% ethanol, then resuspended and treated with 5uL of 10mg / mL RNase at 37°C for 30min; after that, DNA was treated with 5uL of 10mg / mL PI at 4°C in the dark for 30min. Finally, a flow cytometer (FC500, Beckman Coulter) was used to detect the DNA content in the cells, and the data were output by ModFit LT 5.0 software. Experimental results refer to Fig.21 , Fig.21 A represents the results of treating A549 cells with different concentrations of YM818 for 24 h and measuring them by flow cytometry (FCM). Fig.21 A (above); the bar graph shows the quantitative results. Fig.21B shows the effect of YM818 on the expression of A549 cell cycle-related proteins. In order to further study the inhibitory effect of YM818 on the proliferation of A549 cells, the changes in the cell cycle after YM818 treatment were quantitatively detected by PI staining. The distribution of the cell cycle is shown in Figure 2. Fig.21 As shown in A, YM818 can induce cell cycle arrest in A549 cells at the G0 / G1 phase. Western blot results showed that the expression level of CDK2 protein was significantly reduced in a drug concentration-dependent manner ( Fig.21 B).

[0120] In the experiment of detecting cell apoptosis by Annexin V-FITC / PI double staining, cell apoptosis was detected by using the Annexin V-FITC / PI apoptosis detection kit, which was used according to the instructions and measured by flow cytometry. Cells were plated on a six-well plate and treated with YM818 for 48 hours, then digested with trypsin and resuspended in buffer. Subsequently, 5 μL of Annexin V-FITC and 5 μL of propidium iodide PI were added, incubated for 15 minutes, and apoptotic cells were detected by FC500 flow cytometer analysis.

[0121] The experimental results are as follows Fig. 22 As shown, Fig. 22 FIG. 4 shows the experimental results of YM818 inducing apoptosis of A549 cells according to an embodiment of the present invention, Fig. 22 A indicates the effect of YM818 on A549 cells observed after Hoechst 33258 staining. Fig. 22 B represents the cell apoptosis after Annexin V-FITC / PI staining analysis by flow cytometry, and the bar graph shows the cell apoptosis rate; Fig. 22 C shows the effect of YM818 on the expression of apoptosis-related proteins in A549 cells. Fig. 22 As shown in A, the A549 cells in the control group showed weak blue fluorescence, while the A549 cells in the YM818-treated group for 48 hours emitted dense strong blue light, indicating that YM818 may induce apoptosis by inhibiting the proliferation of A549. This conclusion was also confirmed by flow cytometry analysis. Fig. 22 As shown in B, with the increase of YM818 drug concentration, the apoptosis rate of A549 cells also increased. In order to reveal its molecular mechanism, we conducted a Western blot experiment, and the results are shown in Fig. 22As shown in Figure C, the ratio of Bax / Bcl-2 increased. In addition, the precursors of caspase-9, caspase-3, and poly(ADP-ribose) polymerase-1 (PARP-1) were cleaved, and the expression of the spliceosomes of these proteins showed an increasing trend. These results indicate that YM818 induces apoptosis of A549 cells through the mitochondrial pathway.

[0122] In the experiment of YM818-induced mitochondrial dysfunction in A549 cells, cells were plated in six-well plates for 12 hours and then treated with different concentrations of YM818, 50μmol / L, 100μmol / L, and 150μmol / L, for 48 hours. ROS analysis: The treated cells were collected, and DCFH-DA was added to resuspend the cells, incubated for 30 minutes under light-proof conditions, and finally the cells were collected and analyzed using an FC500 flow cytometer. On the other hand, JC-1 staining solution was added to the collected cells for one hour, and finally, the ΔΨm value was quantitatively analyzed and determined using an FC500 flow cytometer. Usually,

[0123] The red fluorescence content / green fluorescence content of 5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanineiodide(JC-1) reflects the change of ΔΨm value.

[0124] Experimental results reference Fig.23 , Fig.23 The figure shows the experimental results of YM818-induced mitochondrial dysfunction in A549 cells. Fig.23 A represents the fluorescence image of the changes in mitochondrial transmembrane potential in A549 cells after YM818 treatment. Fig.23 B shows the change of mitochondrial transmembrane potential in A549 cells after YM818 treatment. Fluorescent probe JC-1 can be used to detect the polarity of mitochondrial membrane and the participation of mitochondrial pathways. As shown in the figure, in the mitochondria of A549 cell line, the multimeric JC-1 in the control group showed bright red fluorescence, while in the YM818 treatment group, as the drug concentration increased, the red fluorescence gradually dimmed, and green fluorescence slowly appeared. The change from red fluorescence to green fluorescence indicates that with the treatment of YM818, the △Ψm of A549 cells gradually decreased (cell apoptosis signal).

[0125] Obviously, those skilled in the art can make various modifications and changes to the embodiments of the present invention without departing from the spirit and scope of the present invention. In this way, if these modifications and changes are within the scope of the claims of the present invention and their equivalents, the present invention is also intended to cover these modifications and changes. The word "comprising" does not exclude the presence of other elements or steps not listed in the claims. The simple fact that certain measures are recorded in mutually different dependent claims does not indicate that the combination of these measures cannot be used to profit. Any reference numerals in the claims should not be considered to limit the scope.

Claims

1. An indole derivative, characterized in that: The chemical structural formula of the indole derivative is as follows:

2. The indole derivative according to claim 1, characterized in that The indole derivative is a light brown solid powder.

3. A method for preparing an indole derivative, characterized in that: The preparation method comprises: S1, preparing a reaction medium, dissolving copper acetate and triethylamine in isopropanol, and stirring until completely dissolved to obtain the reaction medium; S2, preparing an indole derivative solution, adding 5-bromoisatin and indole to the reaction medium, and stirring at room temperature until the reactants are completely converted; S3, extracting the indole derivative powder, distilling off the reaction solvent to obtain a crude product, purifying and recrystallizing to obtain a high-purity solid powder of the indole derivative.

4. The method for preparing an indole derivative according to claim 3, characterized in that: The S1 step includes the following: S11, weigh 0.4 g, 2 mmol, of copper acetate and 0.2 g, 2 mmol, of triethylamine, and place them in a reaction bottle; S12, adding 10 ml of isopropanol to the reaction bottle, stirring for 10 min to completely dissolve it, to obtain the reaction medium.

5. The method for preparing an indole derivative according to claim 4, characterized in that: The S2 step includes adding 4.52 g, 20 mmol, of 5-bromoisatin and 2.8 g, 24 mmol, of indole into the reaction flask, stirring the mixture sufficiently at room temperature to allow a post-coupling reaction to occur, and monitoring the reaction progress by thin layer chromatography.

6. The method for preparing an indole derivative according to claim 3, characterized in that: The S3 step includes: S31, performing reduced pressure distillation using a rotary evaporator to remove the isopropanol solvent in the reaction system, collecting the residue, and obtaining the crude product of the reactant; S32, adding the crude product into a mixed solvent of petroleum ether and dichloromethane, stirring thoroughly to dissolve, and then recrystallizing to obtain a solid powder of the indole derivative.

7. An application of an indole derivative, characterized in that: Use of the indole derivative according to any one of claims 1 to 2 or the indole derivative prepared by the method for preparing the indole derivative according to any one of claims 4 to 6 as a tyrosinase inhibitor.

8. An application of an indole derivative, characterized in that: Use of the indole derivative according to any one of claims 1 to 2 or the indole derivative prepared by the method for preparing the indole derivative according to any one of claims 4 to 6 in inhibiting the production and / or transfer of melanin in cells.

9. An application of an indole derivative, characterized in that: Use of the indole derivative according to any one of claims 1 to 2 or the indole derivative prepared by the method for preparing the indole derivative according to any one of claims 4 to 6 as an antioxidant.

10. An application of an indole derivative, characterized in that: Use of the indole derivative according to any one of claims 1 to 2 or the indole derivative prepared by the method for preparing the indole derivative according to any one of claims 4 to 6 in the treatment of lung cancer.

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